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PMC

ACS Omega

PMC10720292

11-29-2023

10.1021/acsomega.3c07461

Study on Reaction Mechanism and Process Safety for Epoxidation

Cheng Chunsheng, Wei Zhenyun, Ming Xu, Hu Jie, Kong Rong

The reaction mechanism and process safety for epoxidation were investigated in this study. 1-(2-Chlorophenyl)-2-(4-fluorophenyl)-3-(1,2,4-triazole) propene (triazolene), a typical representative of high steric olefinic compounds, was chosen as the raw material. In addition, hydrogen peroxide was chosen as the oxygen source in the reaction. Online Raman spectroscopy combined with high-performance liquid chromatography (HPLC) was used for the process monitoring analysis. The results of this study indicated that the epoxidation process is exothermic, and the apparent reaction heat was 1340.0 kJ·kg–1 (measured by the mass of triazolene). The heat conversion rate was 39.7% immediately after hydrogen peroxide dosing to a triazolene and maleic anhydride mixture solution in chloroform. This result indicated that a considerable amount of heat is accumulated during the epoxidation reaction, which leads to a potential high safety concern. The study of the reaction mechanism showed that maleic anhydride reacts with hydrogen peroxide quickly to form maleic acid peroxide, which is controlled by hydrogen peroxide feeding, and the formed maleic acid peroxide further reacts with triazolenes slowly, which is a kinetically controlled reaction. Decomposition kinetics studies revealed that the temperatures corresponding to the time of maximum reaction rate for 8 and 24 h are TD24 = 89.9 °C and TD8 = 104.1 °C, respectively.

1IntroductionEpoxidation of alkenes is a very important oxidation reaction,1 which aims to form an epoxide compound by adding one atom of oxygen between the carbon atoms at both ends of the double bond of alkenes.2 The Epoxide compound is one of the most valuable compounds in the pharmaceutical and spice industries.3−5 In addition, it has an active ternary epoxy structure, which is prone to ring-opening reactions under various conditions, resulting in high-value-added chemical products and intermediates.6,7 At present, the traditional epoxidation methods are mostly used in industry, the halogen alcohol method and peroxide acid method are mainly used to prepare epoxide compounds.8 The halogen alcohol method9 was widely used in the early industrial preparation of Epoxide, but its synthesis process was complicated, and the separation and treatment of byproducts were difficult and then caused serious environmental pollution. Now, this method has been phased out in industrial production. The peroxide acid method10 is a relatively simple epoxidation method of olefins. Based on the different epoxidation methods, it can be classified into direct oxidation and indirect oxidation.11 In direct oxidation, the olefins react directly with organic peroxy acids, such as performic acid, peracetic acid, and peroxy benzoic acid. In indirect oxidation, organic peroxy acid is generated during the process and immediately participates in epoxidation with olefins. Because of the high price of organic peroxy acid and the existence of instability, it is easy to decompose, inconvenient to store, and involves other security risks. The indirect oxidation method is thus typically used for epoxidation in industry.A high concentration of hydrogen peroxide was commonly used as the oxidant in indirect epoxidation, and hydrogen peroxide is also unstable, heat, light, heavy metals, and other impurities will result in its decomposition, oxygen gas, and heat released.12−14 Under the influence of reactant activity, hydrogen peroxide and peroxy acid accumulate at different degrees during the reaction and decompose rapidly when heated.15 In addition, the epoxidation reaction itself is highly exothermic,16,17 once the temperature is out of control, extremely easy to cause reaction accidents.18−21 Therefore, the study on the safety of the indirect epoxidation process is helpful to avoid serious safety accidents in industrial processes and has important practical significance.In this paper, the synthesis of epoxiconazole was chosen as a typical indirect epoxidation of highly hindered substituted olefins. Epoxiconazole is a famous broad-spectrum fungicide commonly used in agricultural production22 and its effect inhibits synthesizing pathogenic ergosterol and hinders the pathogenic cell wall from forming.23 It is an effective preventative measure against leaf spots, mildew, and spotting for bananas, green onions, garlic, celery, kidney beans, melons, asparagus, peanuts, sugar beets, and other crops.24,25 The indirect epoxidation synthesis of fluconazole is carried out using triazolene as the raw material, using hydrogen peroxide and maleic anhydride to react to generate peroxy acid for epoxidation,26 as shown in Scheme 1.Scheme 1Chemical Reaction Showing the Formation of EpoxiconazoleThe safety of the indirect epoxidation process has not been reported. In view of the hazard of epoxidation, the process of synthesizing epoxiconazole was studied by online Raman combined with offline HPLC for the first time, clarifying the space-time rule and potential risk distribution of peroxide formation. Then the exothermic characteristics of the epoxidation process were studied by reaction calorimeter RC1, and the safety of the mixed system in different reaction stages was tested by TSU. Finally, the decomposition kinetics of the mixed system after the reaction was studied, and the relevant kinetic parameters were obtained. The change of reaction rate and conversion with time was simulated under adiabatic conditions. It provides the technical basis for final industrialization safety control.2Experiment2.1ReagentsHydrogen peroxide (50%), maleic anhydride (≥99%), sodium bisulfate (≥99%), and chloroform (≥99%) were all purchased from China Pharmaceutical Group Co., Ltd. 1-(2-Chlorophenyl)-2-(4-fluorophenyl)-3-(1,2,4-triazole) propene (95%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. All reagents were used without further purification.2.2High-Performance Liquid ChromatographyHigh-performance liquid chromatography (Waters1525); Agilent SB-C18 (150 mm × 4.6 mm (i.d),5 μm). Mobile phase: acetonitrile/methanol/water (volume ratio: 40:20:40); column temperature: 30 °C; flow rate: 1.0 mL/min; detection wavelength: 230 nm.2.3Automatic Reaction CalorimeterA reaction calorimeter (RC1e; Mettler Toledo, Zurich, Switzerland) was used to assess the exothermic conditions during the process. IControl software is used to analyze the test data processing, and can get the heat flow (Q), thermal conversion rate (X), heat transfer coefficient (U), adiabatic temperature rise (ΔTad), specific heat (Cp), and other thermodynamic information.2.4Thermal Screening UnitA thermal Screening Unit (TSU; HEL, United Kingdom) was used to study the thermal decomposition characteristics of the reactants. The test ball is constructed using Hastelloy with a volume of 10 mL. The temperature range was from 30 to 300 °C. The operating pressure range is 0–100 bar, the heating rate is 5 K/min, and the loading volume is 1–3 g.2.5Online Raman SpectroscopyAn Online Raman spectrometer (ReactRaman 785; Mettler Toledo) was used to monitor the reaction process. By comparing the disappearance rate of the significant absorption peak of the reactant and the formation rate of the significant absorption peak of the product, the existence of active intermediates in the reaction process is judged.27,28 The collection time of a single spectrogram is set as 15 s and the Raman exposure time as 1 s.2.6Differential Scanning CalorimetryA differential scanning calorimetry (HP DSC1, Mettler Toledo) was used for thermal safety study of a milligram sample to obtain the thermal decomposition status of the sample to be tested. The samples were contained in a high-pressure gold-plated crucible with a volume of 30 μL. The sample mass was 3.0 ± 0.2 mg and the temperature range was 30–350 °C. The heating rates selected in these experiments were 3, 5, and 8 K/min.2.7Synthesis MethodTriazolene chloroform solution (wt 20%) (134 g, 0.0855 mol, 1.0 equiv) and maleic anhydride (83.8 g, 0.8551 mol, 10.0 equiv) were added to a 500 mL reactor, and the mixture was cooled to 20 ± 1 °C after full dissolution. Sodium bisulfate (0.2 g, 0.0020 mol, 0.02 equiv) was added, and wt 50% hydrogen peroxide (35.0 g, 0.5130 mol, 6.0 equiv) was dropwise added in 2.5 h, and the temperature was kept at 20 ± 1 °C for 16–20 h after dropwise addition finished. Sampling and analysis to triazolene were less than 10% as the reaction end point. The NMR characterization29 of the product is shown in Figures S1 and S2.3Results and Discussion3.1Reaction MechanismThe C=C double bond changes from a one-dimensional linear moiety to a C–O–C two-dimensional plane in epoxidation. First, the acid anhydride reacts with hydrogen peroxide to form peroxy acid, and then, the peroxy acid reacts with alkene.30 The epoxide is obtained through a transition state. The reaction mechanism is shown in Scheme 2, which was further verified by online Raman spectroscopy combined with HPLC.Scheme 2Epoxidation Reaction Mechanism3.1.1Characteristic Peak IdentificationOnline Raman spectroscopy was used for analyzing chloroform, maleic anhydride, and hydrogen peroxide. The results of the Raman spectral analysis of each starting material are shown in Figure 1.Figure 1Raman spectra of the main raw materials.Spectral analysis revealed that the primary characteristic absorption peaks are at 260 cm–1 (Cl–C–Cl degeneracy bend), 366 cm–1 (Cl–C–Cl symmetrical bend), and 667 cm–1 (C–Cl symmetrical stretch) for chloroform;31,32 635 cm–1 (ring deformation), 869 cm–1 (C–C stretch), 1065 cm–1 (C–O stretch), 1592 cm–1 (C=C stretch), and 1849 cm–1 (C=O stretch) for maleic anhydride;33 and 881 cm–1 (O–O stretch) for hydrogen peroxide.343.1.2Process MonitoringCombined online Raman spectroscopy and HPLC analysis were adopted to monitor the reaction process in this study. The peaks at 1849, 881, and 1149 cm–1 were selected as the characteristic absorption peaks of maleic anhydride, hydrogen peroxide, and maleic acid peroxide,35 respectively. The concentration changes of the triazolene starting material and epoxiconazole during the reaction were analyzed using HPLC. The relative concentration changes of each component in the reaction are shown in Figure 2.Figure 2Reaction trend of each component. (Hydrogen peroxide is added in the dashed interval.)The results showed that the characteristic absorption peak of hydrogen peroxide at 881 cm–1 increased rapidly with hydrogen peroxide feeding, which is marked with a blue line (Figure 2). Conversely, the characteristic absorption peak of maleic anhydride at 1849 cm–1 showed a rapid decrease (black line). Furthermore, the characteristic absorption peak of maleic acid peroxide at 1149 cm–1 increased rapidly (red line). In the hydrogen peroxide feeding stage, maleic anhydride promptly reacts with hydrogen peroxide to generate maleic acid peroxide, which is a feeding control reaction. HPLC analysis showed that the conversion rate of triazolene was low, as indicated by the green line, and epoxiconazole was produced less, as indicated by the pink line in the hydrogen peroxide feeding stage. After the completion of hydrogen peroxide feeding, the conversion rate of triazolene and the generation of epoxiconazole gradually increased. Some solid epoxiconazoles were generated in the reaction system. The conversion of triazolene to epoxiconazole was related to the concentration of maleic acid peroxide in the system, and the reaction was kinetically controlled.3.2Exothermic Properties of the Epoxidation ReactionThe exothermic characteristics of the epoxidation process are shown in Figures 3 and 4.Figure 3Reaction heat flow rate curve.Figure 4Reaction heat flow rate curve in the early stage.Triazolene and maleic anhydride were added to chloroform to form a suspension solution, and 50% hydrogen peroxide was added dropwise when the temperature was decreased to 20.0 °C ± 1 °C. When hydrogen peroxide was added, the solid in the suspension solution gradually dissolved, and a chemical reaction occurred. The reaction was initially endothermic, with a maximum endothermic rate of 13.5 W·kg–1. After 30 min of hydrogen peroxide feeding, the apparent reaction became exothermic, gradually reaching the maximum heat release rate of 15.7 W·kg–1. The solid material precipitated out of the reaction medium as the feeding progressed; the heat release rate gradually decreased, and the average heat release rate throughout the dripping of hydrogen peroxide was 6.2 W·kg–1. After hydrogen peroxide feeding, the reaction heat conversion rate was 39.7%; this indicates considerable heat accumulation during the reaction process. After 30 min, the reaction heat release rate suddenly increased with accelerated solid precipitation. After maintaining for 11 h, the system exhibited no notable thermal effect.The results of the current study reveal that the specific heat capacity for the reactant liquid is 3.13 kJ·kg–1·K–1, the epoxidation reaction process was overall exothermic, and the total apparent heat release was 268.0 kJ·kg–1 (based on the mass of the triazolene chloroform solution). The adiabatic temperature rise was 45.6 K, reaction heat accumulation was 60.3%, and the maximum temperature of the synthesis reaction (MTSR) was 47.5 °C when the cooling system failed.3.3Process Safety3.3.1Thermal Stability Study during the ReactionThe stability and safety of the epoxidation reaction system were studied in different stages by a thermal stability test. We collected four samples denoted as a, b, c, and d. Sample a is collected at the end of the hydrogen peroxide feeding; b is the reaction mixture kept warm for 3 h; c is the reaction mixture kept warm for 6 h; and d is the reaction mixture collected at the end of the reaction. The results are shown in Figure 5.Figure 5Time–temperature–pressure curve for the TSU calorimetric test. (a) End of the hydrogen peroxide feeding; (b) reaction mixture kept warm for 3 h; (c) reaction mixture kept warm for 6 h; and (d) reaction mixture collected at the end of the reaction.The results show that the samples at each stage of the reaction are thermally unstable, and the temperature and pressure showed a sharp rise with more heat release at 35–50 °C for each sample. With the extension of the holding time, the maximum temperature and pressure rise rate gradually decreased (Figure 6). At the end of hydrogen peroxide feeding, the maximum temperature rise rate of sample a was 31 times more, and maximum pressure rise rate was 41 times more than that of sample d. A large amount of the maleic acid peroxide intermediate was generated in the reaction system during hydrogen peroxide feeding, resulting in the highest risk of decomposition. Therefore, risk control measures such as emergency quenching and overpressure relief must be established to avoid explosions in upscale tests and industrial applications of the epoxidation reaction.Figure 6Relationship between temperature and pressure rise rate in each stage of epoxidation.3.3.2Kinetics of Thermal DecompositionIn this study, considering the influence of the two important factors, conversion rate α and temperature T,36 and that these two parameters are independent of one another, the reaction rate equation can be expressed aswhere α is the conversion rate, t is the reaction time, and f(α) is the reaction mechanism function.The rate constant k is closely related to temperature T. Applying the Arrhenius equation, the following equation is obtained:where T is the temperature in Kelvin, t is the time in seconds, E is activation energy kJ·mol–1, A(α) is the pre-exponential factor with the unit s–1, and R is the universal gas constant with the unit kJ·mol–1·K–1.According to the reaction rate equation, the activation energy for the decomposition reaction is closely related to the reaction rate, conversion rate, and temperature. A differential scanning calorimeter was used to determine the variation trend of the solid–liquid self-decomposition reaction rate after the epoxidation reaction using different scanning rates, as shown in Figure 7.Figure 7Variation trend of the self-decomposition rate.The results indicate that the increased temperature rate was reduced by 2.7 times, the initial decomposition temperature was decreased by 19.3 °C, and the maximum self-decomposition rate was reduced by 2.5 times. Because of this, a higher rate of temperature increase indicates a higher initial detected decomposition temperature. Using Friedman’s equal conversion rate differential method,37 AKTS (Advanced Kinetics and Technology Solutions) software obtained the activation energy for the decomposition reaction. Figure 8 shows that the activation energy for the self-decomposition reaction from the sample was 44–104 kJ/mol. The fluctuation range was extensive, indicating that the decomposition process of the sample was more complex.38Figure 8Activation energy of the self-decomposition reaction of the feed liquid after epoxidation.3.3.3Decomposition Reaction SafetyUsing the thermokinetic results, the decomposition thermokinetics for the time taken to the maximum reaction rate under adiabatic conditions (TMRad)39 of the material solution after the epoxiconazole synthesis reaction were studied and analyzed. The results of the study are shown in Figure 9. Under adiabatic conditions, TD2 is the temperature at which the time to the maximum reaction rate for thermal decomposition is 2 h. Here, TD2 was 119.4 °C, while that at 4 h (TD4) was 110.2 °C, that at 8 h (TD8) was 100.8 °C, that at 24 h (TD24) was 87.2 °C, and that at 168 h (TD168) was 65.9. (the specific heat capacity for the sample was 3.13 kJ·kg–1·K–1, and the system Phi was 1.05).Figure 9TMRad curve of the self-decomposition reaction of epoxidation. (a) 0–14 h; (b) 0–210 h.TD8 and TD24 are the temperatures at which the time to the maximum reaction rate for material decomposition are 8 and 24 h under adiabatic conditions, which is critical for risk control in emergencies.40 Considering the relationship between time, temperature, and conversion rate for the decomposition reaction, the results for the decomposition mechanics study for TD8 and TD24 are shown in Figures 10 and 11, respectively.Figure 10Trend of self-decomposition reaction at TD8 = 100.8 °C.Figure 11Trend of self-decomposition reaction at TD24 = 87.2 °C.Under adiabatic conditions, when the sample was at 100.8 °C (TD8), the initial decomposition reaction rate, decomposition reaction conversion rate, and sample temperature slowly increased. At 4.4 h, the decomposition reaction conversion rate increased to 16.1% and the decomposition reaction rate significantly increased. The decomposition reaction rate reached its maximum at 8 h, and the decomposition reaction conversion rate increased to 83.8%. At 8.8 h, all of the materials were decomposed.Under adiabatic conditions, when the sample was at 87.2 °C (TD24), the initial decomposition reaction rate, decomposition reaction conversion rate, and sample temperature slowly increased. At 12 h, the decomposition reaction conversion rate increased to 13.9%, and the decomposition reaction rate significantly increased. The decomposition reaction rate reached its maximum at 24 h, and the decomposition reaction conversion rate increased to 88.5%. At 25.4 h, all of the materials were decomposed.4ConclusionsThe reaction mechanism and process safety for epoxidation of triazolenes and hydrogen peroxide as the oxygen source were studied herein, providing technical bases for their production, storage, and transportation. The results are summarized as follows:In the epoxidation of 1-(2-chlorophenyl)-2-(4-fluorophenyl)-3-(1,2,4-triazole) propene in the presence of maleic anhydride and with hydrogen peroxide as the oxygen source, maleic acid peroxide is produced first, and then, maleic acid peroxide reacts with triazolene to form the epoxiconazole. The former reaction is fast and is controlled by hydrogen peroxide feeding, while the latter is slow and is controlled by kinetics.The epoxidation process is complicated, involving solid maleic anhydride dissolution and epoxiconazole precipitation. The epoxidation process is exothermic, and the apparent reaction heat was 1340.0 kJ·kg–1, the adiabatic temperature rise was 45.60 K, and the maximum temperature of the synthesis reaction (MTSR) was 47.5 °C when the reaction runaway occurred.The reaction runaway occurred at 35–50 °C at different stages along the complete process with a significant release of heat and gas, thereby raising serious safety concerns. At the end of hydrogen peroxide feeding, the temperature and pressure increase rates of decomposition are at their maximum values. The decomposition kinetics study showed that the temperatures corresponding to the time of maximum reaction rate are 89.9 °C (TD24) and 104.1 °C (TD8). The maximum temperature of the epoxidation reaction process was 47.5 °C when the reaction runaway occurred, which exceeds the temperature at which the mixture decomposes violently at different stages of the reaction, thus posing a potential safety hazard.In the process of epoxidation reaction scale up and industrialization, control measures for emergency quenching and overpressure explosion relief should be established, and control measures should be started in a timely manner after the thermal runaway of the reaction system to avoid the violent decomposition of the materials leading to an accident.

PMC

Heliyon

PMC10241862

5-25-2023

10.1016/j.heliyon.2023.e16386

Antidiarrheal activities of methanolic crude extract and solvent fractions of the root of

Worku Solomon Ayenew, Tadesse Solomon Asmamaw, Abdelwuhab Mohammedbrhan, Asrie Assefa Belay

BackgroundIn Ethiopian traditional medicine, V. sinaiticum is one of the most often utilized medicinal herbs for the treatment of diarrhea. Therefore, this study was conducted to validate the use of the plant for the treatment of diarrhea in the traditional medical practice of Ethiopia.MethodsCastor oil-induced diarrhea, enteropooling, and intestinal motility test models in mice were used to evaluate the antidiarrheal properties of the 80% methanol crude extract and the solvent fractions of the root component of V. sinaiticum. The effects of the crude extract and the fractions on time for onset, frequency, weight, and water content of diarrheal feces, intestinal fluid accumulation, and intestinal transit of charcoal meal were evaluated and compared with the corresponding results in the negative control.ResultsThe crude extract (CE), aqueous fraction (AQF), and ethyl acetate fraction (EAF) at 400 mg/kg (p < 0.001) significantly delayed the onset of diarrhea. Besides, the CE and AQF at 200 and 400 mg/kg (p < 0.001) of the doses, and EAF at 200 (p < 0.01) and 400 mg/kg (p < 0.001) significantly decreased the frequency of diarrheal stools. Furthermore, CE, AQF, and EAF at their three serial doses (p < 0.001), significantly reduced the weights of the fresh diarrheal stools as compared to the negative control. The CE and AQF at 100 (p < 0.01), and 200 and 400 mg/kg (p < 0.001) of their doses and EAF at 200 (p < 0.01) and 400 mg/kg (p < 0.001) significantly decreased the fluid contents of diarrheal stools compared to the negative control. In the enteropooling test, the CE at 100 (p < 0.05), and 200 and 400 mg/kg (p < 0.001), AQF at 200 (P < 0.05) and 400 mg/kg (p < 0.01), and EAF at 200 (p < 0.01) and 400 mg/kg (p < 0.001) significantly decreased the weights of intestinal contents compared to the negative control. Additionally, the CE at 100 and 200 (p < 0.05) and 400 mg/kg (p < 0.001), AQF at 100 (p < 0.05), 200 (p < 0.01), and 400 mg/kg (p < 0.001) of the doses, and EAF at 400 mg/kg (p < 0.05), produced significant reductions in the volumes of intestinal contents. In the intestinal motility test model, the CE, AQF, and EAF at all their serial doses (p < 0.001), significantly suppressed the intestinal transit of charcoal meal and peristaltic index compared to the negative control.ConclusionOverall, the results of this study showed that the crude extract and the solvent fractions of the root parts of V. sinaiticum had considerable in vivo antidiarrheal activities. Besides, the crude extract, especially at 400 mg/kg, produced the highest effect followed by the aqueous fraction at the same dose. This might indicate that the bioactive compounds responsible for the effects are more of hydrophilic in nature. Moreover, the antidiarrheal index values were increased with the doses of the extract and the fractions, suggesting that the treatments might have dose-dependent antidiarrheal effects. Additionally, the extract was shown to be free of observable acute toxic effects. Thus, this study corroborates the use the root parts of V. sinaiticum to treat diarrhea in the traditional settings. Furthermore, the findings of this study are encouraging and may be used as the basis to conduct further studies in the area including chemical characterization and molecular based mechanism of actions of the plant for its confirmed antidiarrheal effects.

1IntroductionDiarrhea is defined as defecation of three or more loose or liquid stools in a day . It comes about as a result of an imbalance between the bowel's secretory and absorptive processes . Based on WHO criteria, it can be classified into three types: as acute, persistent, and chronic diarrhea . Acute diarrhea is the passage of stool with increased water content, volume, or frequency that lasts less than 2 weeks . Pathogens such as V. cholerae or E. coli, as well as rotavirus are common causes of acute watery diarrhea . Persistent diarrhea is defined as diarrhea that lasts at least 14 days and may include blood, and chronic if it lasts more than 4 weeks in duration. Children who are malnourished or who have other illnesses, such as AIDS, are more likely to have chronic diarrhea .Diarrhea is the second leading cause of pediatric mortality following pneumonia, with an estimated 688 million morbidities and 499,000 deaths globally among children under the age of five. Sub-Saharan African and South Asian countries account for 90% of all diarrhea related deaths in the world . A review study also reported that about 1.6 million people died from diarrheal diseases globally in 2017 and one-third of them were children under five years old . Evidence from demographic and health surveys of 34 sub-Saharan countries also reported that there was a significant clustering of diarrheal disease among under-five children across the communities and the overall prevalence of diarrhea in this age group was 15.3% . Furthermore, a meta-analysis study showed that in three east African countries, the prevalence of diarrheal diseases in children of less than five years of old was 27% from 2012 to 2017 and varied from 11% to 54% between different studies . Ethiopia, like sub-Saharan African nations, has a high level of morbidity and mortality due to acute diarrhea. The Ethiopian Demographic and Health Survey (EDHS) conducted in 2016 reported a 12.0% prevalence of diarrhea in the population .Many cases of sudden onset of diarrhea are self-limiting requiring no intervention. In severe cases, however, excessive fluid loss and electrolyte imbalance are the main concerns, especially in infants, children, and elderly patients, necessitating either nonpharmacologic treatments, such as oral rehydration therapy (ORT) and zinc supplements, or pharmacological treatments or both . Agents that suppress secretion and/or motility of the intestine are used in the symptomatic treatment of diarrhea. Of them, opioid drugs and their derivatives are being widely used in the management of diarrhea. Opioid drugs including diphenoxylate, loperamide, and difenoxin are commonly used opioids for this purpose. There are also many other drugs having antimotility or antisecretory effects on the intestine and used in treating diarrhea [12,13]. Antimicrobials are used for the treatment of infectious diarrhea and can reduce its severity and duration . The majority of the enteropathogens causing persistent diarrhea are treatable with antimicrobial drugs .The current drugs used for treatment of diarrhea have many problems, like drug resistance, drug-drug interaction, and adverse effects . Because of this, investigation of alternative medications derived from natural products is mandatory. Approximately 80% of the population in developing countries such as Ethiopia depends on traditional medicines for primary healthcare . In particular, the use of medicinal plants to treat gastrointestinal disorders such as diarrhea and dysentery occupied a major place in the traditional medicine of the Ethiopian community . There are a variety of medicinal plants used for the treatment of diarrheal diseases in Ethiopia. Verbascum sinaiticum, Cordia africana, Rumex nepalensis, Zehneria scabra, Verbena officinalis, Amaranthus caudatus, Calpurnia aurea, and Coffea arabica are some of the most commonly used medicinal plants .V. sinaiticum (ՙkutitina’ or ‘yeahiya jero’ in Amharic) is one of the medicinal plants used to treat diarrheal diseases [, , ]. The root and the leaf parts of the plant are used for the treatment of diarrheal diseases. The leaf part is crushed, homogenized in water and drunk. Similarly, the root is crushed and drunk with water or the juice of the root is taken orally . The herb is also utilized for the treatment of other ailments including hepatitis , mental illness, amnesia, tapeworm infestation, syphilis, gonorrhea, relapsing fever, rheumatic pain, elephantiasis, wound, and measles in Ethiopian traditional medicine . In addition, the plant has experimentally verified antibacterial, antitrypanosomal, hepatoprotective, and anti-proliferative activities [, , , ].V.sinaiticum is among the most commonly used medicinal plants to treat diarrhea in the traditional medicine of Ethiopia . However, the traditional claim of the plant for this use is not determined yet using scientific methods. Therefore, this study was conducted to validate the use of the plant to treat diarrhea in the traditional medical practice of Ethiopia. In addition, the findings of this study may initiate the research community in the field of pharmaceutical science to further investigate the chemical constituents of the plant for its antidiarrheal activity and their mechanism of action.2Materials and methods2.1Chemicals, drugs, and reagentsDistilled water, absolute methanol 99.9% (Hrego Chemical Ethiopia PLC), loperamide hydrochloride (Medochemie Ltd, Cyprus), atropine sulfate (Humanwell Pharmaceutical PLC, Ethiopia), castor oil (Amman Pharmaceutical Industries, Jordan), ethyl acetate (Alpha Chemika, India), activated charcoal (SD Fine Chemicals Limited, India), Tween 80 (Atlas Chemical Industries, India) were chemicals, drugs or reagents used. Additional lab reagents and chemicals were also used in the phytochemical screening test.2.2Instruments, apparatuses, and suppliesDigital electrical balance (Abron Exports, India), hot air oven (Medit Medizintechnik Vertriebs-GmbH, Germany), rotary evaporator (Yamato Scientific CO. Ltd., Japan), Whatman filter paper №1 (Schleicher & Schuell Microscience GmbH, Germany), surgical blade (SteriLance Medical Inc., China), oral gavage, gloves, gauze bandage, absorbent cotton, and syringes with needles were also used in this study.2.3Collection of the plant material and authenticationThe roots of V. sinaiticum were collected from Tara Gedam Monastery Forest which is located in South Gondar Zone, Amhara National Regional State, Ethiopia. In the mean while the plant specimen showing its full feature was collected for identification. Authentication of the plant specimen was done by a botanist at the Department of Biology, College of Natural and Computational Sciences, University of Gondar and the voucher number (SA01) was deposited there for future reference.2.4Extraction procedureThe collected roots were thoroughly washed with distilled water to remove dirt, soil, and any other foreign materials. The cleaned roots were chopped into smaller pieces manually and dried under shade at room temperature. The dried material was then grinded to coarse powder using mortar and pestle and extracted by cold maceration using 80% methanol as a solvent. Three flasks were taken and 650 g of the powder was soaked in a liter of 80% methanol in each flask and kept for 72 h at room temperature with ocassional shaking. Then the extract in each flask was filtered by using muslin cloth and Whatman grade №1 filter paper and the marc was re-extracted and filtered two times in the same fashion by using fresh 80% methanol. The methanol part of the filtrates was evaporated using a rotary evaporator set at 40 °C. The residue was then put in deep freezer at −20 °C and the aqueous portion was removed using a lyophilizer. The dried crude extract from each flask was combined and stored in a closed container and placed in a deep freezer until used for intended experiment.2.5FractionationSixty five g of 80% methanol crude extract was taken and successively fractionated using n-hexane, ethyl acetate, and distilled water. First, the crude extract was suspended in 390 ml of distilled water and an equal volume of n-hexane was added. The mixture was then shaken well in a separatory funnel and the n-hexane phase was separated. The aqueous residue was fractionated twice more using the same volume of n-hexane and separated similarly. The n-hexane portions from the three separate fractionation processes were combined. In the same fashion, the aqueous residue was fractionated in three rounds using 390 ml of ethyl acetate in each round and the ethyl acetate portions were combined. The n-hexane and ethyl acetate portions were concentrated by using a rotary evaporator. Then the ethyl acetate concentrate was stored in a tight container. But the n-hexane concentrate was found insignificant (all most null) and excluded from further consideration. The aqueous residue was also lyophilized and the dried aqueous fraction was stored in a tightly closed container. Finally, the dried ethyl acetate and aqueous fractions were placed in a deep freezer set at −20 °C until used.2.6Experimental animalsA total of 239 healthy Swiss albino mice of either sex weighing 20–30 g and aged 6–8 weeks were used in the study. The mice were bred under standard conditions. They were housed in plastic cages with softwood shavings as bedding, in the animal house of Department of Pharmacology, University of Gondar with a 12:12 dark-to-light period, at room temperature, and with free access to clean water and pelletized food ad libitum. All mice were acclimatized to the working laboratory environment one week prior to the experiment .2.7Animal grouping and dosingThe animals were randomly assigned to different groups for evaluation of the activities of the 80% methanol crude extract and the solvent fractions on castor oil-induced diarrhea, enteropooling, and charcoal meal transit models.In the evaluation of the effects of the crude extract, five groups each containing six mice were used for each model and dosed as follows.Group 1: received 10 ml/kg of 2% Tween 80 (negative control).Group 2: received 3 mg/kg of loperamide (in castor oil-induced diarrhea and enteropooling models) and 1 mg/kg of atropine (in gastrointestinal motility model) (positive control).Groups 3, 4, and 5: received 100, 200, and 400 mg/kg of 80% methanol extract.In each of the three models, eight groups each containing six mice were also used in the evaluation of the activities of the solvent fractions and received the treatments as follows.Group 1: received 10 ml/kg of 2% Tween 80 (negative control).Group 2: received 3 mg/kg of loperamide (in castor oil-induced diarrhea and enteropooling models) and 1 mg/kg of atropine (in gastrointestinal motility model) (positive control).Groups 3, 4, and 5: received 100, 200, and 400 mg/kg of the aqueous fraction, respectively.Groups 6, 7, and 8: treated with 100, 200, and 400 mg/kg of the ethyl acetate fraction, respectively.2.8Acute oral toxicity testThe acute toxicity of 80% methanol extract of V. sinaiticum roots was assessed according to OECD criteria for chemical testing . For the experiment, five female Swiss albino mice were chosen at random. First, a limit test dose of 2000 mg/kg body weight of the extract was administered to a single animal, which was then monitored for 24 h. Next, the limit dose was given to each of the remaining four animals because the first animal was still survived after a 24-h follow-up. The mice were closely observed for any signs of toxicity in the first 4 h, and then occasionally for the next 24 h. Thereafter, the mice were kept for up to 14 days with daily follow-up for the occurrence of any signs of morbidity or mortality.2.9Antidiarrheal activity determination2.9.1Castor oil-induced diarrhea in miceThis was done in accordance with the method employed by Shoba and Thomas . Thirty mice were divided into five groups at random, and each group was prevented access to food for 18 h. Each mouse was then put into a cage with a non-wetting paper sheet floored, which was changed every hour. Following this, each group received the crude extract, either of the fractions, standard drug, or the vehicle as narrated in the grouping and dosing section above. One hour after receiving the treatments, each animal in each group received 0.5 ml of castor oil orally. Following the castor oil delivery, each group's total number and weight of diarrheal drops were determined over the course of a 4-h observation period. The time for the onset of diarrhea in each animal was also determined as the interval between the castor oil delivery and the appearance of the first diarrheal feces. Then, the percentage inhibition of diarrhea from the negative control group was determined using the following formula.%inhibitionofdiarrhea=Meannumberofdiarrhealstoolsofnegativecontrol−treatedgroupMeannumberofdiarrhealstoolsofcontrolgroup×100This procedure was used in testing of the effect of the crude extract of the plant and the aqueous and ethyl acetate fractions of the crude extract.2.9.2Castor oil-induced enteropoolingThe effects of the 80% methanol extract and the solvent fractions on intraluminal fluid buildup were assessed using a method used by Sharma et al. . The mice were starved for 18 h and divided into groups. Then each group was housed in a cage, and given the treatment as described in the grouping and dosing section above. Each animal received 0.5 ml of castor oil an hour after receiving the vehicle (2% Tween 80), the crude extract, the aqueous or ethyl acetate fraction, and was sacrificed an hour later. The abdomen of each animal was then opened and the small intestine was ligated at the pyloric sphincter and the ileocecal junction and dissected out. Immediately after dissection, the intestine was weighed and its contents were collected by milking into a graduated tube and reweighed. The difference in the weights of the intestine before and after milking was noted. Then, using the following formulas, the percentage of decrease in intestinal secretion (in terms of weight and volume) was determined.%reductioninvolumeofintestinalcontent=MVICC−MVICTMVICC×100where, MVICC – mean volume of intestinal content (ml) of the negative control group, MVICT – mean volume of intestinal content (ml) of the treated group,%reductioninweightofintestinalcontent=MWICC−MWICTMWICC×100where, MWICC – mean weight of intestinal content (g) in the negative control, MWICT – mean weight of intestinal content (g) in the treated group.2.9.3Gastrointestinal motility testThe effects of the crude extract and the solvent fractions on gastrointestinal motility (transit) were examined in mice using the technique developed by Than et al. . The mice were chosen at random, fasted for 18 h, divided into groups, and subjected to the corresponding treatments as described above in the grouping and dosing section. After an hour of treatment, 0.5 ml of castor oil was administered orally to each mouse. Then, each animal received 1 ml of a 5% activated charcoal suspension in 2% Tween-80 orally, 1 h after castor oil administration. After 30 min of charcoal meal, each mouse was sacrificed and the small intestine was promptly dissected out from the pylorus to the caecum and placed lengthwise on a white paper sheet. Both the overall length of the intestine and the intestinal length traveled by the charcoal meal from the pylorus were measured. Finally, the peristaltic index (PI) and the percentage inhibition of the intestinal transit were calculated as follows for each animal.PeristalysisindexPI=IntestinallengthtravelledbythecharcoalmealTotallengthofthesmallintestine×100%inhibitionofintestinaltransit=Meanintestinallengthmovedbycharcoalincontrol−treatmentgroupMeanintestinallengthmovedbycharcoalincontrolgroup×1002.9.4In vivo antidiarrheal index (ADI)The in vivo anti-diarrheal index (ADI in vivo) was then expressed according to the formula developed by Than et al. .ADIinvivo=Dfreq×Gmeq×Pfreq3where, D freq is the delay in diarrheal onset (as % of control), G meq is the gut meal travel reduction (as % of control), and P freq is the reduction in the number of diarrheal stools (as % of control).Dfreq=Meantimeofonsetofdiarrheainthetreated−negtativecontrrolgroupMeantimeofonsetofdiarrheainthenegativecontrolgroup×1002.9.5Preliminary phytochemical screeningThe 80% methanol extract and the fractions were all subjected to qualitative phytochemical screening tests according to established testing protocols .2.10Ethical clearanceThe proposal of the study was presented to the Animal Ethics Review Committee of the Department of Pharmacology, University of Gondar, and an ethical approval letter was obtained from the Department of Pharmacology on behalf of the committee (Reference No. SoP 4/101/2013). Moreover, the animals were handed according to the guideline for the care and handling of laboratory animals .2.11Statistical analysisThe results were analyzed using SPSS software version 23 and expressed as mean ± standard error of the mean (SEM). The comparisons between group means were made using One-way Analysis of Variance (ANOVA) followed by Tukey HSD Post-hoc test. The differences between the group means were considered statistically significant at p-value < 0.05.3Results3.1Percentage yieldsA total of 203 g of dried crude extract was obtained from 1950 g of the coarse powder of the plant material. Accordingly, the percentage yield of the CE was 10.41%. From 65 g of the crude extract fractionated, 38 g (58.46%) and 16 g (24.62%) of AQF and EAF were obtained, respectively. The n-hexane concentrate was extremely small and beyond the sensitivity of the digital balance which was being used at the time.3.2Preliminary phytochemical screeningPreliminary phytochemical screening of the 80% methanol extract of the roots V. sinaiticum indicated the presence of anthraquinones, alkaloids, flavonoids, terpenoids, tannins, saponins, and phenols. However, steroids and glycosides were absent in 80% methanol crude extract of the plant. All of the phytochemicals detected in the methanolic extract were also detected in the AQF. Saponins were absent while others were similarly detected in the EAF (Table 1).Table 1Results of phytochemical screening test of the 80% methanolic extract and the solvent fractions.Table 1Secondary metaboliteCrude extractAQFEAFAnthraquinones+++Alkaloids+++Flavonoids+++Glycosides˗˗˗Tannins+++Terpenoids+++Saponins++˗Steroids˗˗˗Phenols+++AQF: aqueous fraction, EAF: ethyl acetate fraction, +present, ˗absent.3.3Acute oral toxicity testOver the course of the 14-day observation period, the limit dose, 2000 mg/kg, of the 80% methanol crude extract of V. sinaiticum roots did not result any significant toxicity or mortality. Furthermore, it was demonstrated that neither food nor liquid intake was decreased during the observation period.3.4The antidiarrheal activity of 80% methanol extract3.4.1Effects on castor oil-induced diarrheaThe crude extract of the plant significantly delayed the onset of diarrhea at 400 mg/kg (p < 0.001), relative to the negative control. The frequency of diarrheal drops was significantly decreased (p < 0.001) in groups received 200 and 400 mg/kg doses of the crude extract as compared to the negative control. The 100, 200, and 400 mg/kg of the extract treatments reduced diarrhea by 6.00, 41.8, and 50.76%, respectively. The three serial doses of the extract significantly reduced (p < 0.001) the weight of diarrheal stool compared to the negative control. Similarly, compared to the negative control, the water content of the fresh diarrheal stool was significantly decreased in groups received 100 (p < 0.01), 200, and 400 mg/kg (p < 0.001) doses of the extract. The effects of the highest dose of the crude extract were comparable with those of the standard drug in all parameters measured. The standard drug produced significant effects (p < 0.001) on the time for onset of diarrhea, frequency of diarrheal feces, and weights and water contents of the fresh diarrheal drops compared to the negative control (Table 2).Table 2Effect of 80% methanol extract of the root of V. sinaiticum on castor oil-induced diarrhea in mice.Table 2Group (treatment)Dose (mg/kg)Onset of diarrhea (min)Frequency of diarrheal stool% inhibition of diarrheaWeight of diarrheal stool (g)Water content of diarrheal stool (g)Group 1 (2% Tween 80)–61.50 ± 9.2211.17 ± 0.60–1.72 ± 0.050.82 ± 0.06Group 2 (Loperamide)3185.33 ± 19.29a3b3c14.67 ± 0.76a3b358.190.38 ± 0.05a3b30.23 ± 0.03a3Group 3 (CE)10072.17 ± 8.4310.50 ± 0.566.000.99 ± 0.09a30.43 ± 0.08a2Group 4 (CE)200120.50 ± 24.326.50 ± 0.43a3b341.810.60 ± 0.06a3b20.31 ± 0.07a3Group 5 (CE)400198.33 ± 8.12a3b3c15.50 ± 0.62a3b350.760.44 ± 0.05a3b30.30 ± 0.05a3Results are expressed as mean ± SEM (n = 6). acompared to Group 1(negative control), bcompared to Group 3, ccompared to Group 4, 1p < 0.05, 2p < 0.01, 3p < 0.001, CE: crude extract, Group 1: mice received 10 ml/kg of 2% Tween 80 in water and designated as negative control.3.4.2Effects on castor oil-induced enteropoolingIn the enteropooling assay, the 80% methanol extract of the root of V. sinaiticum demonstrated a significant reduction in the weight of intestinal contents at 100 (p < 0.05), 200 (p < 0.001), and 400 mg/kg (p < 0.001) of the doses. The percentage inhibitions in the weights of intestinal contents were found to be, 28.13, 45.31, and 43.75% at 100, 200, and 400 mg/kg doses of the extract in their order and 46.88% by the standard drug (Fig. 1(a) and (b)). The extract also significantly reduced the volume of intestinal contents at 100, 200 (p < 0.05), and 400 mg/kg (p < 0.001) doses as compared to the negative control. The percentage reductions in the volume of intestinal contents from that of the negative control were 35.37, 36.59, and 59.76%, at 100, 200, and 400 mg/kg doses of the crude extract, respectively. The standard drug produced 60.98% reduction in the volume of intestinal contents from the negative control (Fig. 1(c) and (d)).Fig. 1Effect of 80% methanol extract of the root of V. sinaiticum on castor oil-induced enteropooling in mice. (a) effect on weight of intestinal content, (b) % reduction in weight of intestinal content, (c) effect on volume of intestinal content, (d) % reduction in volume of intestinal content. Results are expressed as mean ± SEM (n = 6). acompared to Group 1(negative control), 1p < 0.05, 3p < 0.001. Group 1: mice received 10 ml/kg of 2% Tween 80 in distilled water (negative control), Group 2: mice treated with 3 mg/kg of loperamide (positive control), Group 3, 4, and 5: mice received 100, 200, and 400 mg/kg of the crude extract, respectively.Fig. 13.4.3Effects on intestinal motilityThe crude extract significantly reduced the intestinal transit of charcoal meal at all tested doses (p < 0.001) compared to the negative control. Compared to the lowest dose, the middle and the highest doses of the crude extract significantly reduced gastrointestinal transit of charcoal meal (p < 0.001) (Fig. 2 (a)). The three serial doses of the extract also showed significant reduction (p < 0.001) in peristaltic index compared to the negative control (Fig. 2 (b)). The percentage reductions in gastrointestinal transit were 44.52, 61.81, and 68.10% at 100, 200, and 400 mg/kg doses of the extract, respectively. The highest percentage reduction was produced by atropine, 70.03% (Fig. 2 (c)).Fig. 2Effect of 80% methanol extract of the root of V. sinaiticum on gastrointestinal transit in mice. (a) effect on length of small intestine moved by charcoal meal (intestinal transit), (b) effect on peristaltic index, (c) % reduction in intestinal transit. Results are expressed as mean ± SEM (n = 6). acompared to Group 1(negative control), bcompared to Group 3, ccompared to Group 4, 2p < 0.01, 3p < 0.001. Group 1: mice received 10 ml/kg of 2% Tween 80 in water (negative control), Group 2: mice treated with 1 mg/kg of atropine (positive control), Group 3, 4, and 5: mice received 100, 200, and 400 mg/kg of the crude extract, respectively.Fig. 2Fig. 3Diagrammatic representations of proposed antidiarrheal effects of the crude extract and the solvent fractions. *evidence from effects on castor oil-induced enteropooling, ♯evidence from effects on gastrointestinal motility, ⸸evidence from effects on castor oil-induced diarrhea.Fig. 33.4.4In vivo antidiarrheal index (ADI)The in vivo antidiarrheal indices of the crude extract were 16.67, 63.27, and 91.62 at the doses of 100, 200, and 400 mg/kg, respectively (Table 3).Table 3In vivo antidiarrheal index of 80% methanol extract of the root of V. sinaiticum.Table 3Group (treatment)Dose (mg/kg)Delay in onset of diarrhea (D freq)Reduction in intestinal length traveled by charcoal meal (G meq)Reduction in diarrheal stools (P freq)Antidiarrheal index (ADI)Group 1 (negative control)–––––Group 2 (positive control)–201.3570.0358.1993.62Group 3 (CE)10017.3544.526.0016.67Group 4 (CE)20095.9363.1641.8163.27Group 5 (CE)400222.4968.0950.7691.62Negative control: a group of mice that received 10 ml/kg of 2% Tween 80. Positive control: a group of mice received loperamide (3 mg/kg) in castor oil-induced diarrhea model and atropine (1 mg/kg) in gastrointestinal motility test model.3.5Antidiarrheal activities of the solvent fractions3.5.1Effects on castor oil-induced diarrheaThe AQF and EAF significantly delayed (p < 0.001) the onset of diarrhea at 400 mg/kg. Significant reductions in the frequency of diarrheal feces were produced by the AQF at 200 and 400 mg/kg (p < 0.001) and EAF at 200 (p < 0.01) and 400 mg/kg (p < 0.001) doses compared to the negative control. The percentage inhibitions of diarrhea were 8.57, 35.73, and 45.78% in groups treated with 100, 200, and 400 mg/kg of AQF and 8.57, 28.62, and 32.90% in groups received 100, 200, and 400 mg/kg of EAF, respectively. The highest percentage inhibition of diarrhea, 64.27%, was produced by loperamide. Furthermore, both the AQF and EAF at all of their serial doses (p < 0.001) showed significant reduction in the weight of fresh diarrheal stools compared to the negative control. Significant reductions on the fluid content of diarrheal stool were also produced by AQF at 100 (p < 0.01), 200, and 400 mg/kg (p < 0.001) and EAF at 200 (p < 0.01) and 400 mg/kg (p < 0.001) compared to the negative control. The highest doses of AQF and EAF showed comparable effects to the standard drug in all parameters measured in this model. The standard drug significantly (p < 0.001) delay the time of diarrhea onset and decreased the number, weight, and fluid content of diarrheal feces as compared to the negative control (Table 4).Table 4Effects of the solvent fractions of the crude extract of the root of V. sinaiticum on castor oil-induced diarrhea in mice.Table 4Group (treatment)Dose (mg/kg)Onset of diarrhea (min)Frequency of diarrheal stool% inhibition of diarrheaWeight of diarrheal stool (g)Fluid content of diarrheal stoolGroup 1 (2% Tween 80)–61.67 ± 4.9211.67 ± 0.49–1.70 ± 0.030.82 ± 0.06Group 2 (loperamide)3181.67 ± 19.15a34.17 ± 0.75a364.270.36 ± 0.04a30.21 ± 0.04a3Group 3 (AQF)10073.67 ± 4.58b310.67 ± 0.76b3d28.571.05 ± 0.09a3b30.50 ± 0.09a2b1Group 4 (AQF)200112.17 ± 23.31b17.50 ± 0.43a3b2c235.730.65 ± 0.05a3b2c30.40 ± 0.02a3Group 5 (AQF)400172.33 ± 7.25a3c36.33 ± 0.56a3c345.780.52 ± 0.05a3b30.40 ± 0.07a3Group 6 (EAF)10065.50 ± 5.47b310.67 ± 0.61b38.571.08 ± 0.06a3b30.65 ± 0.05b3Group 7 (EAF)200107.17 ± 23.31b28.33 ± 0.50a2b328.620.70 ± 0.05a3b3e30.45 ± 0.09a2Group 8 (EAF)400168.50 ± 8.25a3e3f17.83 ± 0.54a3b2e132.900.54 ± 0.06a3e30.36 ± 0.09a3e1Results are expressed as mean ± SEM (n = 6). acompared to Group 1(negative control), bcompared to Group 2 (positive control), ccompared to Group 3 (100 mg/kg AQF), dcompared to Group 4 (200 mg/kg AQF), ecompared to Group 6 (100 mg/kg EAF), fcompared to Group 7 (200 mg/kg EAF), 1p < 0.05, 2p < 0.01, 3p < 0.001, AQF: aqueous fraction, EAF: ethyl acetate fraction.3.5.2Effects on castor oil-induced enteropoolingThe AQF significantly reduced the weight of the intestinal contents at 200 (P < 0.05) and 400 mg/kg (p < 0.01) and the volume of the intestinal content at 100 (p < 0.05), 200 (p < 0.01), and 400 mg/kg (p < 0.001) of the doses, respectively, compared to the negative control. The percent reductions in the weight of intestinal contents were 26.98, 36.51, and 42.86% at 100, 200, and 400 mg/kg doses of this fraction, respectively. Similarly, this fraction produced 30.77, 42.31, and 50.00% reductions in the volume of intestinal contents at 100, 200, and 400 mg/kg of the doses, respectively. Significant reductions in the weight of the intestinal contents were also produced by 200 (p < 0.01) and 400 mg/kg (p < 0.001) doses of the EAF, while only 400 mg/kg of the fraction significantly decreased (p < 0.05) the volume of intestinal contents compared to the negative control. This fraction produced of 25.40, 36.51, and 42.86% reductions in the weight of intestinal contents at 100, 200, and 400 mg/kg of its doses, respectively. The fraction also produced 29.49, 39.74, and 48.72% reductions in the volume of intestinal contents at 100, 200, and 400 mg/kg of the doses, respectively. The standard drug, loperamide 3 mg/kg, showed a significant reduction in the weight (p < 0.01) and volume (p < 0.01) of intestinal fluid accumulation relative to the negative control. It decreased the weight and volume of intestinal fluid by 44.44 and 70.03%, respectively, relative to the negative control. There was no statistically significant difference between the effects of all doses of AQF and EAF. Similarly, there was no statistically significant difference between the effects of the standard drug and the solvent fractions on intestinal fluid accumulation (Table 5).Table 5Effects of the solvent fractions of the crude extract of the root of V. sinaiticum on castor oil-induced enteropooling in mice.Table 5Group (treatment)Dose (mg/kg)Weight of intestinal content (g)% reduction in weight of intestinal contentVolume of intestinal content (ml)% reduction in volume of intestinal contentGroup 1 (2% Tween 80)–0.63 ± 0.06–0.78 ± 0.04–Group 2 (loperamide)30.35 ± 0.02a244.440.32 ± 0.04a373.02Group 3 (AQF)1000.46 ± 0.0226.980.54 ± 0.07a130.77Group 4 (AQF)2000.40 ± 0.08a136.510.45 ± 0.09a242.31Group 5 (AQF)4000.36 ± 0.05a242.860.39 ± 0.03a350.00Group 6 (EAF)1000.47 ± 0.0525.400.55 ± 0.0929.49Group 7 (EAF)2000.40 ± 0.03a236.510.47 ± 0.1439.74Group 8 (EAF)4000.36 ± 0.02a342.860.40 ± 0.04a148.72Results are expressed as mean ± SEM (n = 6). acompared to Group 1(negative control), bcompared to Group 2 (positive control, 1p < 0.05, 2p < 0.01, 3p < 0.001, AQF: aqueous fraction, EAF: ethyl acetate fraction.3.5.3Effects on intestinal motilityAll the serial test doses of both of the solvent fractions of the root of V. sinaiticum significantly decreased (p < 0.001) the intestinal transit of charcoal and peristaltic index compared to the negative control. The 200 and 400 mg/kg doses of both fractions showed significant reductions (p < 0.001) in the intestinal transit of charcoal meal and peristaltic index compared to 100 mg/kg. The 100, 200, and 400 mg/kg doses of the AQF produced 40.05, 49.65, and 58.32% reductions in the gastrointestinal transit of the charcoal meal, respectively. The EAF inhibited intestinal transit of charcoal meal by 30.76, 49.69, and 59.11% at the doses of 100, 200, and 400 mg/kg, respectively. The standard drug, atropine 1 mg/kg, significantly reduced (p < 0.001) intestinal transit and peristaltic index compared to the negative control. The effect of the standard drug against gastrointestinal transit and peristaltic index was also significantly greater than those of the three serial doses of each fraction. The highest percent reduction in the gastrointestinal transit was produced by atropine, which was 69.59% (Table 6).Table 6Effect of the solvent fractions of the crude extract of the root of V. sinaiticum on gastrointestinal transit in mice.Table 6Group (treatment)Dose (mg/kg)Length of small intestine (cm)Length moved by the charcoal meal (cm)Peristaltic index% inhibition in intestinal transitGroup 1 (2% Tween 80)–60.62 ± 1.6945.32 ± 1.8474.62 ± 1.06–Group 2 (Atropine)160.05 ± 1.7713.78 ± 0.80a322.91 ± 0.98a369.59Group 3 (AQF)10058.40 ± 1.6027.17 ± 0.80a3b346.53 ± 0.48a3b340.05Group 4 (AQF)20055.85 ± 1.5122.82 ± 0.33a3b3c140.95 ± 0.79a3b3c349.65Group 5 (AQF)40059.37 ± 0.4918.89 ± 0.33a3b2c331.82 ± 0.61a3b3c3d358.32Group 6 (EAF)10061.07 ± 0.7931.38 ± 0.81a3b351.37 ± 0.95a3b330.76Group 7 (EAF)20056.13 ± 0.7022.80 ± 0.94a3b3e340.61 ± 1.58a3b3e349.69Group 8 (EAF)40058.35 ± 0.8518.53 ± 0.42a3b1e331.77 ± 0.71a3b3e3f359.11Results are expressed as mean ± SEM (n = 6). acompared to Group 1(negative control), bcompared to Group 2 (positive control), ccompared to Group 3 (100 mg/kg AQF), dcompared to Group 4 (200 mg/kg AQF), ecompared to Group 6 (100 mg/kg EAF), fcompared to Group 7 (200 mg/kg EAF), 1p < 0.05, 2p < 0.01, 3p < 0.001, AQF: aqueous fraction, EAF: ethyl acetate fraction.3.5.4In vivo antidiarrheal index (ADI)The in vivo antidiarrheal indices of AQF were 18.83, 52.57, and 78.25, while that of EAF were 11.79, 47.17, and 69.58 at the doses of 100, 200, and 400 mg/kg, respectively. The highest antidiarrheal index, 95.47%, was shown by atropine (Table 7).Table 7In vivo antidiarrheal index of the solvent fractions of the root of V. sinaiticum in mice.Table 7Group (treatment)Dose (mg)Delay in onset of diarrhea (D freq)Reduction in length traveled by charcoal meal (G meq)Reduction in diarrheal stools (P freq)Antidiarrheal Index (ADI)Group 1 (negative control)–––––Group 2 (positive control)–194.5869.5964.2795.47Group 3 (AQF)10019.4640.058.5718.83Group 4 (AQF)20081.8949.6535.7352.57Group 5 (AQF)400179.4458.3245.7878.25Group 6 (EAF)1006.2130.768.5711.79Group 7 (EAF)20073.7849.6928.6247.17Group 8 (EAF)400173.2359.1132.9069.58Negative control: a group of mice that received 10 ml/kg of 2% Tween 80. Positive control: group of mice received loperamide (3 mg/kg) in castor oil-induced diarrhea model and atropine (1 mg/kg) in gastrointestinal motility test model.4DiscussionAccording to published ethnobotanical study reports from Ethiopia [, , ], either the roots or leaves of V. sinaiticum are used to alleviate diarrheal diseases in the traditional medical practice. However, no prior investigation has been done to verify this assertion. Therefore, the reports were used as the foundation for the current experimental work. The antidiarrheal effects of various plants have been scientifically validated through analyzing their effects on different animal models. In light of this, castor oil-induced diarrhea, enteropooling, and gastrointestinal motility models in mice were used in this work to assess the antidiarrheal effects of the crude extract of the plant and the solvent fractions. The number and characteristics of the fecal outputs, time for the onset of diarrhea, intestinal transit ratio, and intestinal fluid accumulation are the commonly measured parameters in assessing the antidiarrheal effects of medicinal plants [, , ]. The models employed and the parameters considered in this investigation are consistent with those used in previously conducted similar studies.Due to their expanded polarity index, hydroalcoholic solvent combinations are often thought to provide good extraction yields. In general, hydroalcoholic co-solvents like 80% methanol appear to have the best solubility properties for initial extraction . Therefore, 80% methanol was preferred to extract the plant material. In addition, the 80% methanolic crude extract was fractionated with solvents of different polarities to get insight about the polarity of the phytochemical components of the plant.As shown in Tables 1, at least at the highest dose, the plant extract significantly delayed the onset of diarrhea and reduced the frequency, weight, and water contents of diarrheal drops in castor oil-treated mice in the 4-h observation period compared with the negative controls. Additionally, it was noted that an increase in the dose of the plant extract was accompanied by an increase in the percent reduction in the diarrheal outputs. Besides, the effect of the maximum dose of the crude extract was comparable to the effects of the standard treatment in all parameters examined. The significant delay in the onset of diarrhea caused by the highest dose of the extract, combined with an increasing pattern of percent inhibition in diarrheal episodes, suggests that the plant extract inhibits diarrhea more effectively at relatively higher doses. The percentage inhibition of diarrhea produced by the crude extract is comparable with the effects of the hydromethanolic root extract of Idigofera spicata and greater than that of Tetrastigma leucostaphylum leaves . However, the inhibitory effects of the extract is less than the extracts of other medicinal plants including Myrtus communis , Justicia schimperiana , and Ophiorrhiza rugosa .The results in castor oil-induced enteropooling model revealed that the 80% methanolic extract V. sinaiticum significantly reduced the weight and volume of intestinal contents at all the three serial doses in comparison to the vehicle. The percentage reductions in the weight and volume of intestinal contents were remarkably increased with the dose of the extract. The results demonstrated that the effect of the plant extract on percentage inhibition of castor oil-induced enteropooling is increased the the doses. Moreover, the results in this model revealed that the effect of the highest dose of the extract on intestinal fluid accumulation was found to be closer to and comparable with the inhibitory effect of loperamide. The findings in this model may indicate that the extract has a significant antisecretory effect and this contributes to its antidiarrheal effect noted in castor oil-induced diarrhea model.The reduction of gastrointestinal motility is one of the mechanisms by which antidiarrheal agents can act . The crude extract was found to decrease intestinal motility as shown by significant reduction (p < 0.001) in the intestinal transit of charcoal meal and peristaltic index compared to the negative control. Furthermore, the results in this model showed that the antimotility effect of the highest dose of the extract is comparable to that of the standard drug, atropine. This can be viewed, for example, in terms of the percentage reductions in gastrointestinal transit by 400 mg/kg of the extract and atropine, which were 68.10 and 70.03%, respectively. A decrease in the intestinal motility increases the stay of intestinal contents in the intestine and this might significantly increase the time for the absorption of water and electrolytes from the small intestine. This may in turn be attributed to the observed effects of the extract in castor oil-induced diarrhea and enteropooling models.Regarding the effect of the solvent fractions against castor oil-induced diarrhea, the AQF and EAF significantly delayed (p < 0.001) the onset of diarrhea at 400 mg/kg. Additionally, the middle (p < 0.01) and the highest doses (p < 0.001) of the fractions significantly reduced the frequency of diarrheal feces compared to the negative control. The antidiarrheal effects of AQF and EAF were further shown by the progressive percentage inhibitions of diarrhea with increasing doses. Furthermore, the fractions were shown to produce significant reductions in the weights of diarrheal stools as compared to the negative control. Besides, they also significantly decreased the fluid contents of diarrheal stools at all the serial doses compared to the negative control. Overall, the results in this model indicated that the AQF and EAF had significant activities against castor oil-induced diarrhea.The effects of the solvent fractions against castor oil-induced enteropooling were also assessed, and the results (Table 5) revealed that the fractions produced remarkable effects against intestinal fluid accumulation. Both fractions produced increasing reductions in the weight and volume of intestinal contents with increasing doses. Their effects were further elaborated by the progressive increments in the percentage reductions of the weight and volume of intestinal contents in the treated groups. The effects of AQF and EAF determined in this model could be attributed to their antidiarrheal effects demonstrated in the castor oil-induced diarrhea model.In the testing of the effects on intestinal transit of charcoal, all the serial doses of the fractions significantly decreased (p < 0.001) the intestinal transit of charcoal and peristaltic index compared to the negative control. The fractions were found to produce remarkable and consistent effects with increasing doses, as shown by a corresponding reduction in the mean intestinal length traveled by the charcoal meal and peristaltic index and an increasing percentage inhibition in intestinal transit. Therefore, the results in this model are in support of the effects of the fractions against castor oil-induced diarrhea.The ADI value often provides a more reliable measurement of the effectiveness of extracts in treating diarrhea . The ADI values increased with the doses of the crude extract and the fractions, suggesting that the crude extract and the solvent fractions caused dose-dependent antidiarrheal effects. Additionally, the crude extract exhibited the highest ADI value across all the test treatments at the corresponding doses, showing that it might have higher antidiarrheal activity than the solvent fractions. Regarding viewing the antidiarrheal effect in consideration of the ADI value, the result of this study is not consistent with a result reported by Ayalew et al. which shows that the highest antidiarrheal activity might be produced by the chloroform fraction, whereas the highest activity was produced by the methanolic crude extract followed by the aqueous fraction in this study. The results are also differed from those of a study on the antidiarrheal effects of various solvent extracts of Tetrastigma leucostaphylum which reported that the percentage inhibition of diarrhea and antidiarrheal index values produced by the various organic solvents are generally greater than resulted by the methanolic extract .The pathophysiologic mechanisms that cause diarrhea include altered intestinal motility that results in a shorter intestinal transit time, increased luminal osmolality and electrolyte release, and decreased electrolyte absorption [46,47]. Castor oil has been commonly employed to induce diarrhea in antidiarrheal activity studies because it releases ricinoleic acid, a metabolite that causes diarrhea, upon metabolism in the gut . Ricinoleic acid causes diarrhea by irritating the GI mucosa and promoting the release of prostaglandin, which in turn accelerates gut motility and electrolyte secretion and lowers electrolyte absorption from the small intestine and colon . In light of this, the effects of the crude extract and the solvent fractions against diarrhea may be the result of actions that counteract the activities of this metabolite to cause the pathophysiologic changes leading to diarrhea. It has been demonstrated that both the crude extract and the fractions significantly reduced the accumulation of fluid in the intestine. This suggests that they may promote water and electrolyte absorption and/or decrease the secretion. This in turn may lessen the overload and distension of the intestine. As a result, the intestinal motility may be decreased and this gives more time for the absorption of its contents. Hence, relatively lower water contents and frequency of diarrheal stools and longer onset of time for diarrhea episodes in groups received treatments may be due to underlying activities of the treatments to decrease secretion and/or promote the absorption of intestinal contents. This notion is compatible with the literature-presented mechanism of action of loperamide for its antidiarrheal effect . Reduced motility and secretion of the intestine may be also through atropine activities .The phytochemicals identified in the screening test may be principally responsible for the antidiarrheal effects of the extract and its solvent fractions. Anthraquinones, alkaloids, flavonoids, terpenoids, tannins, saponins (not in EAF), and phenols were identified in the extract and the fractions. The contents are similar to those of other plants having scientifically verified antidiarrheal activities, with some variations, including Zehneria scabra , Justicia schimperiana , Indigofera spicata , and Ophiorrhiza rugosa . Literature reports revealed that alkaloids and terpenoids (especially the monoterpenoid group) have antispasmodic activity , saponins suppress ilium contraction , flavonoids inhibit intestinal contractions and possess antispasmodic activity [52,54], tannins have an antispasmodic and muscle relaxant effect, and phenols reduce intestinal secretion and transit and have an astringent action . All of these activities could be the undelaying mechanisms for the antidiarrheal effects of the test treatments of the study. These chemical components may therefore be responsible for the antidiarrheal properties of the plant extract and the solvent fractions. Regarding the phytochemical test results, there is some discrepancy with a previous report on the phytochemical contents of the methanolic root extract of the plant . This difference may be attributed to seasonal variations in the collection of the plant material and/or differences in geographical area from which the plant material was collected.Furthermore, the CE of the plant was determined to be safe because no considerable signs of toxicity were noted from the acute toxicity test. This shows that the plant may not have observable toxicity in short-term use, even at doses higher than those utilized in the three antidiarrheal models of this investigation. This supports that the plant is most probably safe in short-course usage in traditional settings as well.This study did not include quantitative phytochemical determination and chemical characterization works. In addition, the study did not determine the possible chronic toxicities of the medicinal plant in the long run. These issues are considered the limitations of this study, and we recommend additional investigation on the plant to address these issues.5ConclusionOverall, the results of this study showed that the crude extract and the solvent fractions of the root parts of V. sinaiticum had considerable in vivo antidiarrheal activities. The crude extract, especially at 400 mg/kg, produced the highest effect followed by the aqueous fraction at the same dose. This might indicate that the bioactive compounds responsible for the effects are more of hydrophilic in nature. Moreover, the antidiarrheal index values were increased with the doses of the extract and the fractions, suggesting that the treatments might have dose-dependent antidiarrheal effects. Additionally, the extract was shown to be free of observable acute toxic effects. Thus, this study corroborates the use of the root parts V. sinaiticum to treat diarrhea in the traditional settings. Furthermore, the findings of this study are encouraging and may be used as the basis to conduct further studies in the area including chemical characterization and molecular-based mechanism of actions of the plant for its confirmed antidiarrheal effects.Author contributionsAll authors took part in the title suggestion, proposal development, and report writing processes. SAW carried out the lab works, data organization, and analysis. ABA prepared the manuscript. Finally the manuscript was reviewed by SAT, and some changes were made in response to his feedback. Furthermore, all authors are responsible for the accuracy and originality of this work.FundingSome financial support was received from 10.13039/501100007861University of Gondar, Ethiopia and used to purchase laboratory chemicals and supplies used in the study.Data availabilityThe data of this article can be obtained upon request.Additional informationNo additional information is available pertaining to this article.Declaration of competing interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

ACS Omega

PMC10357566

07-05-2023

10.1021/acsomega.3c02968

Metronidazole and Ketoprofen-Loaded Mesoporous Magnesium Carbonate for Rapid Treatment of Acute Periodontitis

Yu Zhaohan, Xiong Yan, Fan Menglin, Li Jiyao, Liang Kunneng

In the clinical pharmacological treatment of acute periodontitis, local periodontal administration is expected to be preferable to systemic administration. However, the action of the active medicine component is hindered and diminished by the limitation of drug solubility, which does not provide timely relief of the enormous pain being suffered by patients. This study aimed to develop a mesoporous magnesium carbonate (MMC) medicine loading system consisting of MMC, metronidazole (MET), and ketoprofen (KET), which was noted as MET-KET@MMC. A solvent evaporation process was utilized to load MET and KET in MMC. Scanning electron microscopy, nitrogen sorption, thermogravimetric analysis, and X-ray diffraction were performed on the MET-KET@MMC. The rapid drug release properties were also investigated through the drug release curve. The rapid antiseptic property against Porphyromonas gingivalis (P. gingivalis) and the rapid anti-inflammatory property (within 1 min) were analyzed in vitro. The cytotoxicity of MET-KET@MMC was tested in direct contact with human gingival cells and human oral keratinocytes. Crystallizations of MET and KET were completely suppressed in MMC. As compared to crystalline MET and KET, MMC induced higher apparent solubility and rapid drug release, resulting in 8.76 times and 3.43 times higher release percentages of the drugs, respectively. Over 70.11% of MET and 85.97% of KET were released from MMC within 1 min, resisting bacteria and reducing inflammation. MET-KET@MMC nanoparticles enhanced the solubility of drugs and possess rapid antimicrobial and anti-inflammatory properties. The MET-KET@MMC is a promising candidate for the pharmacotherapy of acute periodontitis with drugs, highlighting a significant clinical potential of MMC-based immediate drug release systems.

IntroductionEpidemiologically, periodontitis is among the most worldwide epidemic, afflicting 70% of the adult population older than 65 years in the US.1 Periodontitis originates from oral pathogens, for instance, Porphyromonas gingivalis (P. gingivalis), and pathogen-associated inflammation, which cause collateral tissue damage as well as clinical attachment loss.2 Patients with acute periodontitis, in particular, may experience severe pain, periodontal abscesses, or even systemic complications.3 One of the main reasons patients seek help from their dentists is to manage periodontal infection or severe pain; hence, appropriate antimicrobial and anti-inflammatory agents were utilized to combat acute infection and sharp inflammation as an adjunct strategy to the surgical approach.4,5Antibiotics and nonsteroidal anti-inflammatory drugs (NSAIDs) are routinely utilized to assist in the medication of periodontal diseases.6 For instance, metronidazole [MET, 1-(2hydroxyethyl)-2-methyl-5-nitroimidazole] is one of the primary antipathogen agents to treat acute periodontitis.7 Unfortunately, the antibiotics abuse in the medical domain has posed the risk of leading to antibiotic-resistant bacterial species.8 In addition, orally administered antibiotics have low concentrations in gingival crevicular fluid and may lead to antibiotic resistance in bacteria.9 Ibuprofen (IBU) is frequently prescribed as an analgesic in dental treatment due to its efficacy in alleviating mild to moderate pain.4 Ketoprofen (KET) exhibits superior efficacy compared to IBU in the management of topical pain at therapeutic doses, while also possessing a more favorable benefit–risk profile.10 Nevertheless, all NSAIDs increase the risk of potentially fatal bleeding and heart attacks or strokes when the drugs are given systemically.11Consequently, local drug delivery has been verified to have the ability to considerably improve medication concentration at the target site while decreasing adverse effects, treatment expense, and drug dosage.12 In recent years, many underdeveloped local periodontal materials have been proven to be effective.13 Low-solubility drugs exhibit delayed attainment of loading dose and inadequate local tissue concentration for the therapeutic efficacy within a short timeframe.13 Approximately 90% of agents in study and 40% of commercial agents are poorly soluble.14 The low solubility of these agents is, accordingly, among the primary causes of their low bioavailability.15 Besides, most recent trials on topical periodontal administration have focused on extended-release materials, which are ineffective for treating acute periodontitis.16 To date, there is still an unresolved problem with the poor water solubility of topical drugs used for periodontal administration. Additionally, there has been no investigation into the rapid effects of topical periodontal medications.Various solutions have been proposed to tackle this issue, including formulations of crystalline salts, reductions in active pharmaceutical ingredient (API) particle size, and co–ground combinations.17 Nevertheless, the effectiveness of formulation techniques is determined by the chemical composition of the agents as well as actual manufacturing issues.18 For example, the reduction of API particle size may lead to static charge accumulation, resulting in difficulties in handling certain medications.18 Yao and co-workers loaded 5-fluorouracil into azobenzene-functionalized interfacial cross-linked reverse micelles, relying on the high permeability of small molecules for drug delivery. Although this strategy enhanced the local drug accumulation, it failed to consider the poor solubility of the insoluble drug itself.19 However, recent studies have revealed that the crystallization of drugs could be restrained when they are embarked into mesoporous holes with pore diameters, ranging from 2 to 50 nm.20 Mesoporous magnesium carbonate (MMC) has recently been synthesized, exhibiting a narrow distribution of pore sizes and a wide superficial area.21,22 The amorphous forms of several poorly water-soluble compounds have been stabilized by loading them into MMC, resulting in increased solubilities and faster dissolution rates.23−25 This implies that MMC could fundamentally alter the solubility characteristics of the drug, converting crystalline low water-soluble medications into amorphous water-soluble medications.26 MMC improves solubility by increasing the amount of insoluble drug dissolved. Drug solubilization enables a rapid increase in the local drug concentration in a short period of time, resulting in rapid release.26,27 Therefore, the utilization of MMC is anticipated to serve as a highly efficacious strategy in enhancing the bioavailability of poorly water-soluble medications for treating acute periodontitis.28 Nonetheless, there have been no studies on MMC as a topical treatment for acute periodontitis.Herein, a MET-KET@MMC drug immediate-release system was established through a simple solvent evaporation method, using the commonly used oral antibiotic MET and KET. The MET and KET in MMC were rapidly released to achieve effective drug concentrations within 1 min, providing antibacterial and anti-inflammatory effects. Our work has led to the first use of the immediate-release carrier MMC in the field of pharmacological treatment of acute periodontitis.Results and DiscussionIn this study, MET and KET were loaded into MMC simultaneously for the first time. Both MET and KET have small molecular weights and could therefore access the pores of MMC.22,23,25 After dissolving in ethanol, the drug molecules free in solution were adsorbed into the pores of the MMC surface.22 As shown in Figure 1A, MET, KET, and MET-KET@MMC were white powder. Scanning electron microscopy (SEM) of MET, KET, MMC, and MET-KET@MMC is shown in Figure 1B. The surface of MMC had a concave and convex porous morphology. No major morphological changes were noted after loading drugs. Figure 1C illustrates the distribution of Mg, C, N, and O elements, thus indicating the incorporation of MET and KET. The results of thermogravimetric analysis (TGA) are shown in Figure 1D. The decomposition of the unloaded MMC took place at approximately 380 °C. At lower temperatures, crystallographic MET and KET decomposed almost completely (280 °C for MET and 350 °C for KET). The TGA curves of MET-KET@MMC indicate that no discernible mass loss occurred at the temperatures where crystalline MET and KET would typically decompose. The TGA profiles revealed a significant disparity in weight loss between MMC and MET-KET@MMC, which corresponded to the loading of both MET and KET (Figure 1D). Since MMC tends to absorb water, the quality decreased as the water in MMC and MET-KET@MMC evaporated in the range of 100–200 °C. Notably, the decomposition temperature of drug-loaded MMC was higher than that of unloaded MMC. The temperature elevation in this process can be accounted for by the Kelvin equation, which states that within the pores of MMC, the boiling point of the liquid will increase due to a rise in vapor pressure.23,29 This appearance has previously been noticed in the mesoporous drug carrier.23,29 In the differential scanning calorimetry (DSC) pattern, the absence of peaks corresponding to MET (at 160 °C) and KET (at 95 °C) indicates that drugs were incorporated in an amorphous state (Figure 1E). Previous attempts to incorporate additional APIs into the MMC resulted in drug crystallization within the pores of carrier materials.23 In contrast, the X-ray diffraction (XRD) of this experiment indicates that neither 10% wt of MET nor KET showed crystallization, demonstrating that there was no crystallization outside the pores of MMC (Figure 1F). The lack of the peaks of crystalline drugs indicated that the MET and KET incorporated into MMC were amorphous.25 Without MMC, the drug molecules in solution precipitated as crystals when the solvent evaporated. In contrast, after the drug molecules were absorbed into the pores of MMC, the mesoporous structure had an area-bound effect when the solvent was evaporated. This effect stopped the nucleation and crystal growth of identical molecules, which kept the drug in an amorphous form.30 The absence of crystalline drug peaks suggests that the MET and KET incorporated into MMC were amorphous.25 In the Fourier transform infrared spectroscopy (FTIR) spectra (Figure 1G), the absorbance band at ∼3440 cm–1 corresponded to adsorbed water. Bands at ∼1400 cm–1 correspond to the carbonate group. The overlapping absorption bands from MET and KET (1541 and 1697 cm–1) in MET-KET@MMC indicated that MET and KET were loaded into MMC.31 The FTIR spectra of MET-KET@MMC samples did not exhibit any new absorption bands, except for those observed in the absorption spectra of free KET and MET samples, indicating that the adsorption of MET and KET onto the pore walls was physical rather than chemical.23 The Brunauer–Emmett–Teller (BET) surface areas and pore volumes are presented in Table 1. The surface area and pore volume were reduced after drugs were loaded. This supports the results of XRD and DSC, indicating that MET and KET had actually entered the mesoporous structure of MMC. From the foregoing, we could consider that MET and KET were successfully loaded into MMC. The crystallizations of MET and KET were completely suppressed by the mesoporous structure of MMC.32Figure 1(A) Morphology of MET, KET, MMC, and MET-KET@MMC. (B) SEM images of MET, KET, MMC, and MET-KET@MMC. (C) Elemental mapping of MET-KET@MMC. (D) Normalized mass TGA of MET, KET, MMC, and MET-KET@MMC. (E) DSC curves for MET, KET, MMC, and MET-KET@MMC. (F) XRD patterns for MET, KET, MMC, and MET-KET@MMC. (G) FTIR transmittance spectra for MET, KET, MMC, and [email protected] 1SSA and Pore Volume of Unloaded and MET-KET@MMC [email protected] m2/g103.2685 m2/gpore volume0.83 cm3/g0.21 cm3/gDissolution is the rate-limiting process of absorption and consequently the limiting step of bioavailability.33 The rapid dissolution of the drug allows for its absorption in the body at the fastest possible rate, thus allowing the drug to react in a short time, which is significant for the relief of the sufferings of patients. Albeit short-term, quick release offers the benefit of the immediate therapeutic effect.34 It is imperative to investigate the capacity of MMC for prompt co-delivery of both drugs. Figure 2A illustrates the dissolution curves of crystallography. Figure 2A shows the dissolution profiles of MET, KET, and MET-KET@MMC in phosphate buffer saline (PBS). According to the concentration released from MET-KET@MMC, the entrapment efficiency and drug loading of MET and KET were 88.33% ± 0.99 and 7.36% ± 0.08, and 94.82% ± 1.23 and 7.90% ± 0.10, respectively (Table 2). Evidently, the dissolution kinetics of amorphous MET and KET in MMC were superior to those of their crystalline counterparts. During the initial minute, the release of MET from MMC was 8.76-fold higher than that from crystalline MET; similarly, the release of KET from MMC was 3.43-fold higher than that from crystalline KET (Figure 2A). Apparently, within the MMC, the dissolution efficiencies of amorphous MET and KET were higher than those of the free crystalline substances. Over 70.11% of MET and 85.97% of KET were released from MMC within 1 min (Figure 2B–E). After 2 h, the final concentrations of MET and KET released from MMC were 8.37 and 9.46 μg/mL. For a comprehensive assessment of the impact of the carrier on drug release, we recorded and presented 5 time points of the drug concentration (as shown in Figure 2B–E). The concentrations of MET and KET released from MMC were significantly higher than those of crystalline MET and KET in all 5 time points (P < 0.0001). The observed rapid release and dissolution of the drugs within the carrier pore structure do not affect the ultimate dissolved drug levels. The final concentrations of MET and KET were 8.37 and 9.46 μg/mL, while the final concentrations of MET and KET in MMC were elevated to 58.21 and 42.37 μg/mL, respectively. It can be observed that both drugs were released rapidly from MMC. The input amounts in PBS of crystalline MET and KET were basically the same as the actual drug loading in MMC, while more drugs were released in MMC during the same time. Crystalline drugs were dissolved in alcohol and turned into a free state (Figure 2G). The drug molecules were subsequently adsorbed into the mesoporous structure of the MMC. The crystalline propagation was blocked by the confinement of the mesoporous structure; hence, the drugs were stabilized in their amorphous forms, which is attributed to the interactions between APIs and pore walls, alterations in nucleation mechanisms, and kinetics within mesopores.23,32,35 The mechanisms for the increased solubility are as follows: when the amorphous molecules in MMC were in contact with water molecules, they could diffuse directly into the aqueous solution because there was no process to overcome the intermolecular forces from the crystal structure.21 Thus, the insolvable drugs in MMC could be rapidly dissolved in water (Figure 2G); according to the Noyes–Whitney equation, the rate of dissolution is directly proportional to the surface of particles and the solubility of their respective solvents. Therefore, it is a viable approach to enhance drug solubility through the expansion of surface area. In the present study, MET and KET were absorbed in the surface pores of MMC in their non-crystalline states, which expanded the contact area with water. The solubilities of MET and KET were boosted, and the drug concentrations elevated rapidly within a short time.Figure 2Drug release properties. (A–F) Dissolution profiles for MET, KET, and MET-KET@MMC in PBS. (G) Possible mechanism of the solubilization of MMC for insoluble crystalline drugs. The crystalline drug was dissolved in alcohol, allowing the drug molecules to infiltrate into the mesoporous structure of the MMC. The mesoporous structure maintained the drug molecules in the non-crystalline state. Mean ± SD is shown (n = 3). **** represents P < 0.0001.Table 2Entrapment and Drug-Loading Efficiencies entrapment ratedrug-loading rateMET88.33% ± 0.997.36% ± 0.08KET94.82% ± 1.237.90% ± 0.10Although MMC has been considered safe, we evaluated the cytotoxicity of MET-KET@MMC. As shown in Figure 3A, the live/dead staining of human gingival cells (hPDLCs) confirmed a non-toxic effect of MET-KET@MMC. The live cells of MET-KET@MMC were morphologically comparable to other groups. Figure 3B,C demonstrates that the viability of hPDLCs and human oral keratinocytes (HOKs) exposed to MET-KET@MMC (1 mg/mL) did not exhibit a significant decrease in comparison to the control cells (P < 0.0001). MMC has been classified as “Generally Recognized as Safe” (GRAS) by the FDA. One study reported that MMC preparations administered orally to male rats did not show toxicity to rats.20 Furthermore, in this study, the clinical application scenario for MMC was a periodontal rinse. Residual MMC was readily removed by the mouthwash, and there was little physical harm from the low cytotoxicity of MMC.Figure 3Cell viability of hPDLCs cultured in studied samples after 24 h and compared to growth media only. (A) Live/dead staining of hPDLCs. (B,C) CCK-8 assay of hPDLCs and HOKs. Viability of samples containing MMC presented acceptable cytotoxicity compared with the control group (1000 μg/mL) (mean ± SD, n = 6) (P < 0.05). n.s. indicates no significant difference.In the pathology of periodontitis, periodontal pathogens and cellular immune responses are two major factors.36P. g LPS has been used to induce immune responses in a number of studies around periodontal diseases.13,37 hPDLCs in periodontal ligaments recognize pathogenic factors and play a significant role in the innate immune response.38 Hence, P. g LPS was utilized to simulate the inflammatory state of hPDLCs infected with P. gingivalis in this study.39 Connective tissue injury and alveolar bone loss are induced by the pro-inflammatory cytokines IL-6 and IL-8.40 Therefore, we assessed the level of cellular inflammation by evaluating IL-6 and IL-8. The translational level and protein quantification, which were separately measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) after 24 h of culturing with P. g LPS, are shown in Figure 4. After being stimulated by P. g LPS, IL-6 levels increased (∼3-fold at the transcriptional level and ∼3.24-fold regarding protein production) compared to control cells (Figure 4A,C). Relative to the MET-KET group, the expression levels of IL-6 and IL-8 were reduced in the MET-KET@MMC group (Figure 4B,D). The expression of IL-6 and IL-8 was considerably lower in the MET-KET@MMC group compared to the MET-KET group because of the higher KET concentration (P < 0.0001). This indicated that the anti-inflammatory agents released from MET-KET@MMC in only 1 min suppressed LPS-mediated cytokine production in hPDLCs. There was no statistically significant difference observed between the groups treated with P. g LPS and MET-KET. These results indicated that the inflammatory gene expression and the protein level could be significantly reduced by KET, which was rapidly released from MMC in 1 min, while the drug released from crystalline MET-KET in 1 min had no significant anti-inflammatory effect (P < 0.0001).Figure 4Anti-inflammatory effects of MET-KET and MET-KET@MMC on (A,B) cytokine and (C,D) protein expression of IL-6 and IL-8 in hPDLCs and HOKs stimulated with 10μg/mL P. g LPS. Mean ± SD is shown (mean ± SD, n = 3). The drug released from MET-KET@MMC in 1 min has been able to take an anti-inflammatory effect. **** represents P < 0.0001.The spread plate method proved that the antibiotic loaded in MMC was sufficient to kill P. gingivalis. The antibiotic constituents released from crystalline MET within 1 min exhibited a negligible killing effect against P. gingivalis (Figure 5A). The time–kill curves demonstrated the rapid efficacy of MET-KET@MMC against periodontal pathogens, demonstrating that the antibiotic agents released from MET-KET@MMC in just 1 min are sufficient to eradicate pathogens (Figure 5B). In contrast, a drastic decrement in microbe colonies appeared in the MET-KET@MMC group, where almost 100% of P. gingivalis were killed (Table 3). Damaged bacteria were observed on the titanium disc incubated with MET-KET@MMC, while no remarkable difference in bacterial form was found between the titanium disc incubated with MMC, MET-KET, and the control titanium disc (Figure 5C). The immediate killing effects were verified by live/dead staining (Figure 5D). P. gingivalis in the control group, MMC group, and the MET group displayed obvious green fluorescence. In contrast, 96.21% of dead bacteria (red fluorescence) was observed in the MET-KET@MMC group, indicating that MET released within 1 m resulted in rapid bacterial killing. These consequences were due to the rapid release of MET from MMC. In this study, a high concentration of MET was released from MMC in a short time, while crystalline MET only possessed a little antimicrobial property on account of its poor solubility. The rapid antibiotics process corresponds to the clinical treatment principle of using a large dose of antibiotics as the infection appears.22 Additionally, a previous study has demonstrated that MMC possesses antimicrobial properties attributed to the generation of reactive oxygen species, direct contact with microorganisms, and its alkaline effect.40 From these results, the drug released from MMC in 1 min showed antibiotic bacteriostasis against P. gingivalis, while the APIs released from crystalline drugs had no antibacterial effect due to the lower medicine concentration, according to the release curve and antiseptic test results. Given the above, MMC nanoparticles possess a wide field of application prospects in the treatment of acute periodontitis as well as various acute microbial infections.Figure 5Rapid antibacterial ability of MET-KET@MMC within 1 min. (A) BHI and agar plates of each group. The BHI of the MET-KET@MMC group is clarified, and there is no colony present on the agar plate. (B) Growth curves of MMC, MET-KET, MET-KET@MMC, and the control group without antibacterial agent. The antibiotic released from MET-KET@MMC in 1 min prevented the growth of P. gingivalis. (C) SEM micrographs of titanium discs incubated with studied samples after 2 d. The P. gingivalis were in normal shape in the MMC group and MET-KET group compared with the control group, while damaged bacteria could be observed on the titanium discs in the MET-KET@MMC group. (D) Live/dead staining images. The green fluorescence represented the live germs, while the red one denoted the dead. The MET-KET@MMC group shows the strongest red fluorescent signal.Table 3Antibacterial Efficiencies antibacterial rateacontrol1.48% ± 0.01aMMC3.14% ± 0.01aMET-KET5.35% ± 0.02aMET-KET@MMC100% ± 0.00baDissimilar letters (a and b) indicate significantly different values (mean ± SD, n = 3, P < 0.05).Our study demonstrated that MMC released both drugs rapidly and enabled drug concentrations to reach effective antibacterial and anti-inflammatory levels within brief periods. MET-KET@MMC could be applied in injectable pastes or flushes to rinse swollen gums and periodontal pockets in patients with acute periodontitis. The rapid release of MET and KET from MMC would provide a rapid bactericidal and anti-inflammatory effect, thus relieving the pain of patients and eliminating swelling. Furthermore, MMC is not specific for loading drugs and can be loaded with various insoluble small molecules such as IBU, tolfenamic acid, and rimonabant.23,25 It is conceivable that the use of MMC loaded with insoluble broad-spectrum antibiotics would be beneficial in the treatment of various acute infections. Therefore, MMC is anticipated to be used as a direct topical delivery agent for the treatment of skin infections and bone infections. In view of the intricate and diversified microenvironment of periodontal microbial communities, the effects of MMC as a fast-release carrier on multi-microbial or clinically relevant animal models are worth further proving. In summary, MMC, as an efficient fast-release drug carrier, could load diverse insoluble agents. If further developed, this technology is expected to be applied to a variety of acute dental infections for rapid relief of patient suffering.ConclusionsIn this study, we innovatively prepared a dual-functional topical rapid-release drug formulation, MET-KET@MMC, with antibacterial and anti-inflammatory functions, which was expected for the treatment of acute periodontitis. MET-KET@MMC possessed a faster release and dissolution compared to the dissolution of crystalline MET and KET. The rapid killing efficacy against periodontitis-related pathogens and rapid anti-inflammatory properties have also been confirmed in vitro, which would be beneficial to patients suffering from acute periodontitis. MET-KET@MMC did not show cell toxicity toward hPDLCs and HOKs. This work paves the way for further investigations of MMC as a pharmaceutical carrier in topical formulations targeting different types of acute oral infections and inflammation.Materials and MethodsSynthesis of MMCMMC was prepared by a combination of solvothermal synthesis, as described previously.25 In brief, 3 g of MgO (Sigma-Aldrich Inc., St. Louis, MO, USA) was put into 45 mL of CH3OH (Sigma-Aldrich Inc., St. Louis, MO, USA) with stirring at 500 rpm under 4 bar CO2 for 4 d at room temperature. Subsequently, air pressure was released, and the 5 mL of suspension in the reaction was slowly dropped to 250 mL of ethyl acetate at 25 °C. The resulting suspension was then continuously stirred until all of the solvents had evaporated in a well-ventilated area, leaving only a dried powder. To remove any residual organic groups formed during the reaction, the powder was heated to 250 °C for 30 min at a temperature ramp rate of 1 °C/min. The MMC was stored in a dry environment.25Drug-Loading ProcedureMET and KET (Sigma-Aldrich Inc., St. Louis, MO, USA) were incorporated into MMC via a simple solvent evaporation method to obtain MET-KET@MMC. Specifically, 25 g of MET and 25 g of KET were dissolved in 500 mL of ethanol, followed by the addition of 200 g of MMC into the solution. The mixture was subjected to orbital shaking at 500 rpm for 2 day at a temperature of 25 °C to facilitate drug diffusion into the porous structure of MMC. Subsequently, the solution was subjected to heating at 80 °C in an oven for alcohol evaporation and blended to achieve homogeneity.25Characterization of MET-KET@MMCSEM and Energy-Dispersive SpectroscopyMET, KET, and MET-KET@MMC were examined via SEM equipped with energy-dispersive spectroscopy mapping (Inspect F50, FEI, USA). Images were recorded with an acceleration voltage of 3 kV using the in-lens detector.41Differential Scanning CalorimetryDSC was performed with a DSC instrument (Netzsch, GER). Specimens of 3.5–5.5 mg were frozen at −20 °C and subsequently heated to 270 °C (3 °C min–1) under a N2 atmosphere. The temperature elevation process was accompanied by the recording of heat flow.Thermal Gravimetric AnalysisThe mass of 15 mg of samples was recorded as they were heated from 30 to 600 °C at a rate of 3 °C/min under a nitrogen atmosphere using a TGA instrument (Mettler Toledo TGA/SDTA851e).X-Ray DiffractionXRD was performed after MMC and MET-KET@MMC had been placed on a silicon holder (Ultima IV, Rigaku, Japan). The patterns were collected in the 2θ range from 5° to 80° at a rate of 0.02 °/s. Standard cards for MET and KET were obtained from Jade software.Fourier Transform Infrared SpectroscopySamples were ground with potassium bromide and pressed to slices.42 FTIR analyses were carried out using Fourier transform infrared spectroscopy (NICOLET is 50, Thermo Scientific, USA) from 350 to 4400 cm–1 at 25 °C.43Nitrogen Sorption AnalysisAt first, samples got degassed under vacuum for 12 h at 90 °C. The SSA was determined using the BET method. The total pore capacity was determined through single-point adsorption (P/P0 ≈ 1). The ASAP 2020 (Micromeritics) was employed for all computations.44In Vitro Drug Release Measurement10 mg of MET and 10 mg of KET were dissolved in a vessel containing 1500 mL of PBS at 37 °C with a stirring rate of 100 rpm. 100 mg of MET-KET@MMC was dissolved in another vessel containing 1500 mL of PBS under the same condition.25 As the same volume of fresh PBS was blended into the medium at the same moment, 0.5 mL aliquots were collected from each vessel and filtrated via a 0.2 m nylon filter (Servicebio, China).25 The liquid samples were measured by a Nanodrop 2000 spectrometer (ThermoScientific, Waltham, MA, USA). Absorbance measurements were conducted at 320 nm (MET) and 255 nm (KET).42 The concentrations of MET and KET were obtained via standard curves corresponding to the known dissolved MET and KET values. The measurements were carried out three times, and the standard and mean concentration values were also computed. The entrapment efficiency and drug loading of MET and KET were determined by the following calculationsCell CulturehPDLCs were obtained from healthy human premolar periodontal ligaments. Following extraction, the premolars were rinsed five times using PBS. Peripheral tissues of the middle third surface of tooth roots were scraped off and digested using collagenase type I (1 mg/mL, HyClone, UT, USA) at 37 °C for 0.5 h. The tissues were cultured in α-MEM medium (Gibco, Grand Island, NY, USA) with 10% fetal calf serum (Gibco, Grand Island, NY, USA), penicillin (100 U/mL, Servicebio, China), and streptomycin (100 g/mL, Servicebio, China). hPDLCs were passaged by trypsin after the cells had adhered and spread across the plate wall, and the cultures used for our experiments were those between passages 3 and 7.45,46 Besides, HOKs were grown for 24 h.In Vitro Cytotoxicity/Viability AssayCell viability was accessed using a cell counting kit-8 (CCK-8, Servicebio, China) assay.47 Cytotoxicity was evaluated in hPDLCs. hPDLCs and HOKs were incubated for 1 d and then inoculated in treated 96-well plates with medium containing MET, KET, MMC, and MET-KET@MMC (1 mg/mL) for 24 h, respectively. The seeding density was 5000 cells/well in 100 μL. According to the directions, cell viability was measured every 2 d. The absorbance was measured at 450 nm against a medium-only baseline.The following formula was used to calculate cell viabilityEvaluation of Anti-inflammation ActivitiesIn order to verify the solubilization capacity of MMC, all the samples were dropped in solution (α-MEM medium) for 1 min under the same conditions, and only the solution was collected after being filtrated through a 0.2 mm cylinder filter for the follow-up experiments. To be consistent with the practical drug concentration of MET-KET@MMC, MET and KET (MET-KET) were added to solutions in the same mass. As previously described, ultrapure lipopolysaccharide from P. gingivalis (P. g LPS) (Invivogen, San Diego, USA) in vitro, P. gingivalis was employed to emulate conditions of inflammation: 1 × 106 hPDLCs per well were cultivated in a 25 mm2 culture dish. After 24 h serum starvation, hPDLCs were irritated with P. g LPS (10 μg/mL) for 1 d.45 Then, the hPDLCs were rinsed with normal saline and incubated separately in the culture medium mentioned above. The hPDLCs that were incubated with pure α-MEM medium subsequently were referred t as a negative control group.Enzyme-Linked Immunosorbent AssayHuman IL-6 and IL-8 ELISA kits (Jiangsu Meimian Industrial Co., Ltd) were employed to measure the levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) in the culture supernatants according to the manufacturer’s instructions.45Quantitative Reverse Transcription PCR (RT-qPCR)After a 24 h incubation, TRIzol reagent (Invitrogen) was used to extract mRNA according to the instructions.45,46 The RNA was reverse-transcribed into cDNA using the Rayscript cDNA Synthesis KIT (GENEray, GK8030, Shanghai, China) and subsequently employed in RT-PCR reactions with SYBR Green (GENEray, GK8030, Shanghai, China). Table 4 describes the primers of cytokines. Reactions were carried out on an ABI7500 apparatus (Applied Biosystems Inc., USA). Before being compared with the control, the level of mRNA to be tested was standardized to the level of GAPDH. Relative game expression levels were quantitated using the ΔΔCt method.48Table 4Primers of Cytokinescytokinesprimer sequenceIL-6 FF: TGCAATAACCACCCCTGACCIL-6 RR: GTGCCCATGCTACATTTGCCIL-8 FF: TTTTGCCAAGGAGTGCTAAAGAIL-8 RR: AACCCTCTGCACCCAGTTTTCGADPH FF: CCAGAACATCATCCCTGCCTGADPH RR: CCTGCTTCACCACCTTCTTGEvaluation of Antibacterial ActivityBacteria CultureP. gingivalis (ATCC33277, China) was cultivated in brain–heart infusion broth medium (BHI, Difco, Sparks, MA, USA) with hemin (5 mg/mL) and vitamin K1 (1 mg/mL) at 37 °C under anaerobic conditions (80% N2, 10% H2, and 10% CO2) in 96-well plates for 2 d. The amount of P. gingivalis was estimated by the absorbance of cultured germs at 600 nm utilizing a microplate reader (Multiskan Go, Thermo Scientific), corresponding to 1 × 108 bacteria/mL. The P. gingivalis was diluted to 3 × 107 colony-forming units (CFUs)/mL before use.22Spread Plate MethodThe P. gingivalis suspension mentioned above was incubated with BHI containing MMC (1 mg/mL), MET-KET (1 mg/mL within 1 min), and MET-KET@MMC (1 mg/mL within 1 min) separately. 100 μL of 10-fold serial dilutions from the mixed suspension was spread onto the BHI agar plates mentioned above, and the plates were incubated for 2 d in order to count the visible numbers of CFUs. The number of CFUs was recorded, the number of CFUs was determined, and the antimicrobial efficiency was computed using the equation belowwhere NC is the number of the control group and NE is the number of the experimental groups.49SEMThe bacteria suspension mentioned above was cultivated with sterile titanium discs in a 24-well plate for 2 d. BHI with MMC (1 mg/mL), MET-KET (1 mg/mL within 1 min), and MET-KET@MMC (1 mg/mL, within 1 min) were added into the plates and incubated for another 2 d anaerobically.50 After the incubation, the titanium discs were placed in a 2% glutaraldehyde–cacodylate-buffered fixative solution, dehydrated in graded alcohol, and critical-point dried. The occlusal section was sputter-coated with gold palladium.41 The SEM mentioned above was used to obtain all images of P. gingivalis.Time Kill AssayMMC (1 mg/mL), MET-KET (1 mg/mL within 1 min), and MET-KET@MMC (1 mg/mL, within 1 min) were added into BHI separately, and then, the plates were cultured anaerobically at 37 °C. Growth of P. gingivalis was monitored using a microplate reader every 2 h for 2 d.51Live/Dead Staining of BacteriaP. gingivalis suspensions mentioned above were cultivated in confocal dishes with different kinds of BHI at 1 mg/mL MMC (1 mg/mL), MET-KET, and MET-KET@MMC (1 mg/mL within 1 min), separately. After 48 h, the culture mediums were extracted, and the bacteria were stained by a Baclight Live/dead bacterial viability kit (Servicebio, China) in accordance with the instructions.52 A confocal laser scanning microscope (Olympus FV1000, Japan) was put to use to capture the fluorescence image.53Statistical AnalysesAll data were presented as means ± standard deviations (SD). The IBM SPSS (IBM Corp., New York, NY, USA) was used to analyze the data set. A one-way analysis of variance and Student’s t-test were performed to assess the significant effects of the variables. The means of each group were compared by Tukey’s multiple comparison test. Statistical significance was determined by P < 0.05.

PMC

Quantitative Imaging in Medicine and Surgery

PMC10784106

1-03-2024

10.21037/qims-23-291

Mono+ algorithm assessment of the diagnostic value of dual-energy CT for high-risk factors for colorectal cancer: a preliminary study

Chen Jun-Fan, Yang Jing, Chen Wei-Juan, Wei Xin, Yu Xiang-Ling, Huang Dou-Dou, Deng Hao, Luo Yin-Deng, Liu Xin-Jie

BackgroundRisk factors for colorectal cancer (CRC) affect the way patients are subsequently treated and their prognosis. Dual-energy computerized tomography (DECT) is an advanced imaging technique that enables the quantitative evaluation of lesions. This study aimed to evaluate the quality of DECT images based on the Mono+ algorithm in CRC, and based on this, to assess the value of DECT in the diagnosis of CRC risk factors.MethodsThis prospective study was performed from 2021 to 2023. A dual-phase DECT protocol was established for consecutive patients with primary CRC. The signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), overall image quality, lesion delineation, and image noise of the dual-phase DECT images were assessed. Next, the optimal energy-level image was selected to analyze the iodine concentration (IC), normalized iodine concentration (NIC), effective atomic number, electron density, dual-energy index (DEI), and slope of the energy spectrum curve within the tumor for the high- and low-risk CRC groups. A multifactor binary logistic regression analysis was used to construct a differential diagnostic regression model for high- and low-risk CRC, receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) was calculated to assess the diagnostic value of the model.ResultsA total of 74 patients were enrolled in this study, of whom 41 had high-risk factors and 33 had low-risk factors. The SNR and CNR were best at 40 keV virtual monoenergetic imaging (VMI) based on the Mono+ algorithm (VMI+) (SNR 8.79±1.27, P<0.001; CNR 14.89±1.77, P=0.027). The overall image quality and lesion contours were best at 60 keV VMI+ and 40 keV VMI+, respectively (P=0.001). Among all the DECT parameters, the arterial phase (AP)-IC, NIC, DEI, energy spectrum curve, and venous phase-NIC differed significantly between the two groups. The AP-IC was the optimal DECT parameter for predicting high- and low-risk CRC with AUC, sensitivity, specificity, and cut-off values of 0.96, 97.06%, 87.80%, and 2.94, respectively, and the 95% confidence interval (CI) of the AUC was 0.88–0.99. Integrating the clinical factors and DECT parameters, the AUC, sensitivity, specificity, and predictive accuracy of the model were 0.99, 100.00%, 92.68%, and 94.67%, respectively, and the 95% CI of the AUC was 0.93–1.00.ConclusionsThe DECT parameters based on 40 keV noise-optimized VMI+ reconstruction images depicted the CRC tumors best, and the clinical DECT model may have significant implications for the preoperative prediction of high-risk factors in CRC patients.

IntroductionColorectal cancer (CRC) accounts for approximately 10% of diagnosed cancers and cancer-related deaths worldwide each year, has the second highest mortality rate, with a prognosis influenced by tumor node metastasis (TNM) stage and other high-risk factors, and has 5-year survival rates ranging from 10% to 90% (1-3). High-risk factors for CRC include lymph node metastasis, extramural vascular invasion (EMVI), peripheral nerve invasion (PNI), high-grade tumors (including poorly differentiated adenocarcinomas and undifferentiated carcinomas, both less than 50% gland formation) T4 stage, and tumor deposits (4,5). For high-risk patients, the recommended duration of adjuvant therapy is 6 months if FOLFOX is chosen, while for the low-risk group, the recommended duration of adjuvant therapy is 3 months if the CapeOX chemotherapy regimen is chosen . In addition, patients with stage-II high-risk colon cancer who undergo routine standard management may have a worse prognosis than patients with stage-III low-risk colon cancer . Thus, screening the high-risk factors for CRC is important for the selection and timing of adjuvant chemotherapy regimens.Histopathology is the gold standard for the diagnosis of high-risk factor CRC. However, biopsy is invasive, and the results can be falsely negative due to the site and depth of the tumor. Thus, an accurate, objective and non-invasive preoperative method for evaluating high-risk factors for CRC is needed.Computerized tomography (CT) is one of the most important preoperative examinations for CRC. Compared to magnetic resonance imaging (MRI), CT has a faster imaging time and a better ability to detect distant metastases , but conventional CT provides only limited information to accurately assess high-risk factors, such as EMVI, lymph node metastasis, and tumor deposits.Dual-energy computerized tomography (DECT), which uses both high and low energies to achieve material decomposition and material classification, has the ability to assess the biological behavior of tumors by CT (9,10). Current applications of DECT in CRC include determining the nature of the lymph nodes, pathological staging, and histological grading (11,12). A recent study demonstrated the diagnostic value of DECT for the EMVI of rectal cancer . These previous studies focused on only one high-risk factor for CRC, but in clinical settings, patients may actually have one or more high-risk factors, and the preoperative predictive value of DECT for these patients remains unknown.Recently, the quantitative parameters of DECT have been a major advantage in tumor imaging, and the introduction of the Mono+ algorithm has pushed this to new heights. Mono+ is an algorithm that uses frequency division to superimpose the low spatial frequencies of a 40 keV image with the high spatial frequencies of a 70 keV image to improve the image noise at low keV in virtual monoenergetic imaging (VMI) mode, thus improving the contrast-to-noise ratio (CNR) of the image . Compared to standard linear reconstruction, VMI based on the Mono+ algorithm (VMI+) reduces image noise while improving iodine attenuation, especially at low kilo-electron volt (keV), which has been reported to improve the reliability and diagnostic accuracy of the DECT quantitative size measurements used to assess colorectal liver metastases (CRLMs) . Compared to conventional VMI, VMI+ has proven to be superior in qualitative and quantitative image analysis of cutaneous malignant melanomas, abdominal malignant lymphomas, and CRLMs (16-18). However, the quality of the qualitative and quantitative images based on VMI+ of CRC is unknown.This study had two main objectives. First, it sought to assess the optimal energy level for the qualitative and quantitative analysis of CRC based on the Mono+ algorithm. Second, it sought to analyze the tumor quantitative DECT parameters based on the optimal image and constructed a regression model with clinical factors to evaluate the diagnostic value for high-risk CRC. We present this article in accordance with the STROBE reporting checklist (available at prospective study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University approved the study and waived the requirement for informed consent because the study did not adversely affect the rights and health of the subjects.Between April 2021 to January 2023, the clinical and imaging data of 265 patients diagnosed with CRC were prospectively analyzed at The Second Affiliated Hospital of Chongqing Medical University. Sample size calculations were based on a small sample pre-experiment [venous phase normalized iodine concentration (VP-NIC) of 40.25±7.29 vs. 46.23±7.47 for the high- and low-risk groups] with 90% power at a two-sided alpha level of 0.05. Based on these results, about 34 cases were needed in each of the high- and low-risk groups. Ultimately, 74 patients (a total of 75 tumors) were included in this study, including 41 in the high-risk group and 33 in the low-risk group (Figure 1).Figure 1Flow chart showing the inclusion and exclusion criteria and the final grouping of patients. CT, computed tomography; ROI, region of interest.Based on the pathology of biopsy samples or postoperative lesion samples, the patients in this study were allocated to the high-risk group if they had ≥1 of the following risk factors: lymph node metastasis, EMVI, PNI, high-grade tumors, stage T4, stage M1, and tumor deposits. While the patients were allocated to the low-risk group if they had no risk factors.To be eligible for inclusion in this study, the patients had to meet the following inclusion criteria: (I) have histologically confirmed colon and rectal adenocarcinoma; (II) have no history of pelvic surgery; and (III) not have undergone preoperative chemoradiotherapy before the dual-energy CT examination. Patients were excluded from the study if they met any of the following exclusion criteria: (I) had poor quality images that affected diagnosis; (II) had not undergone surgery or a histological examination within two weeks of the CT examination; (III) had benign lesions or atypical colorectal adenocarcinoma based on the pathological findings; and/or (IV) had tumors that were too small (i.e., <20 mm) to draw regions of interest (ROIs).DECT acquisitionPatients were asked not to take drugs containing heavy metals, such as barium, for a week before the CT scan, and were asked to fast 12 hours before the CT examination and drink 600–1,000 mL of water 10 minutes before the examination. All the examinations were performed on a dual-energy CT system (Drive, Siemens Healthcare, Germany). Two contrast-enhanced CT scans were performed from the apex of the diaphragm to the inferior margin of the pubic symphysis in dual-energy spectral CT imaging mode. The scan parameters were as follows: tube voltages: 100 and 140 kV; tube current: 350 mA; pitch: 1; and gantry rotation time: 0.5 s. The contrast agent (Ultravist, 370 mg/mL, Schering, Berlin, Germany; dose: 1.5 mL/kg, flow rate: 3 mL/s) was injected through an elbow vein using a double-head power injector. The scan was threshold triggered, with the arterial phase (AP) automatically starting after a delay of 15 s when the abdominal aortic threshold reached 120 HU, and with the VP automatically starting after a delay of 17 s.DECT image reconstructionAll the DECT data sets were postprocessed on a commercially available three-dimensional (3D) multimodality workstation (syngo.via, version VA30A, Siemens) using a dedicated soft tissue convolution kernel (Qr40, Siemens) and an iterative reconstruction technique (ADMIRE, Siemens; strength level, 3). Standard linear mixed images were automatically reconstructed using a mixing factor of 0.6 (M0.6, containing 60% 100 keV low tube voltage, and 40% 140 keV high tube voltage) . Given the low attenuation of iodine concentration (IC) at energy levels above 100 keV in previous studies, for the image quality evaluation part of this study, the energy-level range was set at 40–100 keV in 10 keV increments .Quantitative image analysisThe quantitative image analysis was performed by a radiologist with three years of experience in abdominal radiodiagnosis on a Siemens syngo MMWP VE36A workstation. The location of the tumor was confirmed according to the histopathological results. Three circular ROIs with areas of 20–30 mm2 were placed in the largest axial image of the tumor, avoiding necrotic foci and larger vessels, and the final value was the average of these three ROIs. Defining fat standard deviation (SD) as image noise, three circular ROIs with an area of 100 mm2 were placed in the subcutaneous fat on the same slice of the outlined tumor, and the final value was averaged over the three ROIs. Based on previous studies, the signal-to-noise ratio (SNR) and CNR were calculated as follows : SNR=HU(lesion)SD(fat) CNR=[HU(lesion)−HU(fat)]SD(fat) Qualitative image analysisThe qualitative image analysis was performed by two radiologists with three and four years of experience in abdominal radiology, respectively. The observers were only aware of the patients’ clinical information and were not aware of the image reconstruction algorithm. The standard linear hybrid images (M0.6) and noise-optimized VMI+ reconstructed images were rated randomly, and only one random energy level was assessed during each round of evaluation. Based on a five-point Likert scale, the following quality criteria were assessed: overall image quality (where 1 = poor overall image quality and 5 = excellent overall image quality), lesion delineation (where 1 = unable to exclude lesions and 5 = excellent lesion margins), and image noise (where 1 = very pronounced image noise and 5 = barely detectable image noise).Imaging analysisAll the images were individually analyzed by two radiologists with more than 3 years of experience each in abdominal radiology who outlined the ROIs based on the histological examination as a reference. Duplex-enhanced images with a slice thickness of 1 mm were imported into the workstation and opened in dual-energy mode to automatically obtain virtual single-energy images, iodine-based material decomposition images, and effective atomic number images in the range of 40–150 keV. The localization of the lesions and outlining of the ROIs were performed with reference to 60 keV VMI+ on a 40 keV VMI+ single-energy image. Three ROIs of 20–30 mm2 were placed in the largest axis image of the tumor, avoiding large vessels and necrotic lesions, and selecting areas of uniform and distinct enhancement. The final value was the average of these three ROIs. The previously outlined ROIs were then copied to the iodine-based material breakdown image and the effective atomic number image. The final IC, NIC, effective atomic number, electron cloud density, dual-energy index (DEI), and slope of the energy spectrum in the AP and VP were recorded; the NIC of the tumor was obtained by normalizing the IC of the tumor to the IC of the aorta or iliac artery at the same level. The differential iodine concentration (DIC) in the arteriovenous phase was calculated using the following formula: DIC = (AP – VP) IC. The dual-energy curve slope value (λHU) was calculated from the CT values of the 50 and 140 keV virtual monochrome images {λHU = [CT50 value (50 keV) – CT140 value (140 keV)]/[140–50]}.Statistical analysisThe statistical analysis was performed using MedCalc Version 19.2 (Ostende, Belgium) and GraphPad Prism Version 9.4.1 (San Diego, California, USA). The intraclass correlation coefficient (ICC) was used to evaluate the interobserver agreement of the measured parameters (ICC 0.6), and all the measurements are summarized in Table 4. The final values of the DECT parameters of the high- and low-risk groups in the AP and VP are shown in Table 5. Patients in the high-risk group had significantly higher AP-IC (3.09±0.17 vs. 2.80±0.14 mg/mL, P<0.001), AP-NIC (22.30±4.77 vs. 19.65±2.84, P=0.005), AP-DEI [(21.72±1.75)×10–3 vs. (19.85±2.05)×10–3, P<0.001], AP-λHU (1.49±0.19 vs. 1.39±0.19, P=0.029), DIC (0.75±0.24 vs. 0.35±0.22, P<0.001), and VP-NIC (41.03±7.09 vs. 47.13±7.19, P<0.001) than those in the low-risk group, while the differences in the other DECT parameters were not statistically significant (Table 5). Figures 4,5 show the images of low- and high-risk CRC.Table 4Results of the intragroup correlation coefficients measured by two observers in the high- and low-risk groups in the arterial and venous phasesParametersICNICZRhoDEIλHUArterial phase ICC0.820.660.920.840.880.88 95% confidence interval0.73–0.880.51–0.770.87–0.950.75–0.890.82–0.930.81–0.92Venous phase ICC0.730.710.910.750.840.87 95% confidence interval0.60–0.820.58–0.810.86–0.940.63–0.830.76–0.900.80–0.91IC, iodine concentration; NIC, normalized iodine concentration; Z, effective atom number; Rho, electron density; DEI, dual–energy index; λHU, dual-energy curve slope value; ICC, intragroup correlation coefficient.Table 5Results of the DECT parameter measurements in the high- and low-risk groups of patients with CRC in the arterial and venous phasesDECT parametersHigh-risk group (n=41)Low-risk group (n=34)t/Z valueP valueArterial phase IC (mg/mL)3.09±0.172.80±0.146.77 (Z)<0.001 NIC (%)22.30±4.7719.65±2.842.79 (Z)0.005 Z8.86±0.168.80±0.16−1.59 (t)0.116 Rho41.20±6.8941.35±6.210.28 (Z)0.782 DEI (×10–3)21.72±1.7519.85±2.053.77 (Z)<0.001 λHU1.49±0.191.39±0.19−2.23 (t)0.029Venous phase IC (mg/mL)2.35±0.272.45±0.241.72 (t)0.090 NIC (%)41.03±7.0947.13±7.19−3.71 (Z)<0.001 Z8.64±0.188.67±0.170.71 (t)0.480 Rho40.75±5.4339.09±5.200.97 (Z)0.330 DEI (×10–3)16.85±2.6517.28±2.420.72 (t)0.470 λHU1.24±0.201.23±0.19−0.17 (t)0.867 DIC (mg/mL)0.75±0.240.35±0.22−7.35 (t)<0.001Data are shown as the mean ± SD. CRC, colorectal cancer; DECT, dual-energy computed tomography; IC, iodine concentration; NIC, normalized iodine concentration; Z, effective atom number; Rho, electron density; DEI, dual-energy index; λHU, dual-energy curve slope value; DIC, differential iodine concentration in the arteriovenous phase.Figure 4An 80-year-old woman with rectal cancer pathologically confirmed as a moderately highly differentiated adenocarcinoma without metastasis. (A) Images with an optimal SNR and CNR at 40 keV VMI+ in the arterial phase; (B) DECT images of IC in the arterial phase; (C) images of the effective atomic number and electron density in the arterial phase; (D) images of 40 keV VMI+ in the venous phase; (E) DECT images of IC in the venous phase; (F) images of the effective atomic number and electron density in the venous phase. SNR, signal-to-noise; CNR, contrast-to-noise ratio; VMI+, virtual monoenergetic imaging based on Mono+ algorithm; DECT, dual-energy computed tomography; IC, iodine concentration.Figure 5A 70-year-old man with rectal cancer, pathologically confirmed as a moderately differentiated adenocarcinoma but with six lymph node metastases. (A) Images with optimal SNR and CNR at 40 keV VMI+ in the arterial phase; (B) DECT images of IC in the arterial phase; (C) images of the effective atomic number and electron density in the arterial phase; (D) images of 40 keV VMI+ in the venous phase; (E) DECT images of IC in the venous phase; (F) images of the effective atomic number and electron density in the venous phase. SNR, signal-to-noise; CNR, contrast-to-noise ratio; VMI+, virtual monoenergetic imaging based on Mono+ algorithm; DECT, dual-energy computed tomography; IC, iodine concentration.In descending order, the areas under the curve (AUCs) for each parameter were AP-IC: 0.96, DIC: 0.89, AP-DEI: 0.75, VP-NIC: 0.75, AP-NIC: 0.69, and AP-λHU: 0.64 (Table 6; Figure 6). Parameters with P values <0.1 in the one-way binary logistic regression analysis were included in the multifactor binary logistic regression, and the final regression model obtained was as follows: logit (P) = 139.64 – 42.36 × AP-IC – 0.83 × AP-NIC – 4.26 × DIC – 560.57 × AP – DEI – 0.21 × VP-NIC – 2.03 × AP-λHU – 0.58 × CEA – 23.42 × histological grade. The AUC, sensitivity, specificity, and predictive accuracy of the model were 0.99, 100.00%, 92.68%, and 94.67%, respectively, and the AUC had a 95% CI of 0.93–1.00.Table 6Diagnostic efficacy of the DECT quantitative parametersParametersAP-ICAP-NIC (%)AP-DEIAP-λHUDICVP-NIC (%)AUC0.960.690.750.640.890.7595% CI for AUC0.88–0.990.57–0.790.64–0.850.52–0.750.80–0.950.64–0.84Cut-off point2.9421.5219.501.350.4841.50Sensitivity (%)97.0685.2952.9052.9485.2976.47Specificity (%)87.8060.9885.3778.0582.9368.29DECT, dual-energy computed tomography; AP-IC, concentrations of iodine in the arterial phase within the region of interest; AP-NIC, concentrations of normalized iodine in the arterial phase within the region of interest; AP-DEI, dual-energy index in the arterial phase within the region of interest; AP-λHU, dual-energy curve slope value in the arterial phase within the region of interest; DIC, differential iodine concentration in the arteriovenous phase; VP-NIC, concentrations of normalized iodine in the venous phase within the region of interest; AUC, area under the curve; CI, confidence interval.Figure 6Diagnostic efficiency of the DECT parameters and the predictive model in differentiating between high- and low-risk factors for CRC. AP, arterial phase; DE, dual-energy; NIC, normalized iodine concentration; IC, iodine concentration; VP, venous phase; λHU, dual-energy curve slope value; DIC, differential iodine concentration in the arteriovenous phase; AUC, area under the curve; DECT, dual-energy computed tomography.DiscussionThe preoperative prediction of risk factors for CRC is important because it not only influences the survival time of patients but also informs individual treatment plans. In this study, we first evaluated the optimal DECT image of CRC, and we then investigated whether the quantitative parameters of DECT could help in the preoperative selection of CRC patients with high-risk factors and constructed a comprehensive diagnostic model.Previous studies have shown that noise-optimized low-keV VMI+ series reconstructed images significantly reduce image noise while improving tumor saliency compared to conventional VMI series reconstructed images and standard linear hybrid reconstructed series images (16,21,22). The accuracy of DECT for tumor diagnosis has been directly enhanced by the improved contrast and lesion delineation with VMI+. One study found 40 keV VMI+ had significantly higher sensitivity and diagnostic accuracy for detecting CRC liver metastases than standard linear mixed reconstruction (90.6% vs. 80.6%, and 89.1% vs. 81.3%, respectively) . Lee et al. found that compared to standard 40 keV VMI, deep learning-based 40 keV VMI+ had better image quality and a higher detection rate of hypoenhancing hepatic metastasis, which shows that the strengths of VMI+ series reconstructed images could be replicated in the field of deep learning.In our quantitative image analysis, we also found that the 40 keV VMI+ series exhibited the highest tumor attenuation and higher SNR and CNR than the standard linear mixed M0.6 image series, and the results were consistent with those of previous studies . However, the overall image quality analysis in our study showed that the 60 keV VMI+ series scored the highest, indicating that tumor attenuation and noise balanced out at 60 keV VMI+, which is generally consistent with the findings of Lenga et al. . Thus, we further compared the 40 and 60 keV VMI+ CRC images. Notably, the blood supply vessels inside or beside the tumors were easier to identify in the 40 keV VMI+ images than the 60 keV VMI+ images; this finding enabled us to place the ROIs’ more accurately in the tumor, avoiding the interference of larger blood vessels. Thus, considering the advantages of both the 40 and 60 keV VMI+ images, we recommend using the 60 keV VMI+ series images as a lesion reference and then outlining the ROI on the 40 keV VMI+ series image to provide a more accurate delineation of the lesion for DECT quantitative analysis.To the best of our knowledge, the risk factors for CRC include lymph node metastasis, EMVI, PNI, high-grade tumors, and T4 stage. In an analysis of the prognosis of T2N0M0 CRC patients, lymphovascular permeation, perineural invasion, and poor differentiation were found to be risk factors for a poor prognosis . Another prognostic analysis of lymph node-negative CRC found that perineural infiltration, EMVI, and T4 staging were independent prognostic factors, and these patients would benefit from adjuvant or more aggressive treatment . Notably, a recent study demonstrated that high preoperative CEA levels and the presence of vascular cancer emboli were risk factors for lymph node metastasis, which suggests that the risk factors for CRC may be interrelated and coexist . Thus, in our study, we allocated patients with one or more risk factors to the high-risk group to provide a more objective and comprehensive preoperative predictive analysis of risk factors for CRC and evaluated the association between DECT quantitative parameters and high-risk factors in CRC tumors.According to the results, AP-IC, AP-NIC, AP-DEI, AP-λHU, and DEI were significantly higher in the high-risk group than the low-risk group. During tumor progression, tumor cells secrete vascular endothelial factors that increase the permeability of tumor blood vessels, leading to a higher intratissue microvascular density (MVD) and microvascular permeability in high-risk patients. IC, which is positively correlated with blood volume and permeability, directly reflects the angiogenesis and blood supply of the tumor tissue (27,28). Thus, it was not surprising that the AP-IC and AP-NIC values were significantly higher in the high-risk group than the low-risk group, and the results support those of Luo et al. .Additionally, due to the higher X-ray attenuation capacity of iodine compared to soft tissues, the higher the tumor tissue uptake of iodine intake, the greater the attenuation to X-rays and the higher the DEI and AP-λHU value, so the AP-DEI and AP-λHU were also significantly higher in the high-risk group than the low-risk group. However, as we noted that the VP-NIC in the high-risk group was lower than that in the low-risk group (41.59±7.30 vs. 46.56±7.75), we also analyzed the DIC between the two groups in the arteriovenous phase, and the results showed that the DIC was significantly higher in the high-risk group than the low-risk group (0.73±0.25 vs. 0.36±0.22), which may be due to the higher MVD and vascular endothelial growth factor (VEGF) expression in the high-risk group.The high expression of VEGF could increase the local vascular permeability and cause a higher clearance rate of the contrast agent (30,31). Thus, it may be that more contrast agents were retained in the tumor blood vessels in the low-risk group in the VP, resulting in the higher VP-NIC in the low-risk group than in the high-risk group. Nevertheless, there were no significant differences between the two groups in terms of electron density and effective atomic number, and the results support those of Qiu et al. . However, Zhang et al. found that the normalized effective atomic number value of metastatic sentinel lymph nodes in breast cancer patients was significantly larger than that of non-metastatic lymph nodes. This may be related to the different ways of outlining the ROI and the differences in the settings of the DECT scanning parameters, as well as the different tumor types. Thus, there is a need for a standardized dual-energy scanning protocol for different type of tumors.Of all the DECT parameters, AP-IC had the highest AUC. This is probably because IC is a direct reflection of the vascular enhancement of the tumor tissue and was influenced by other factors, such as NIC. This result supports that of Zou et al. . Further, we combined all the DECT parameters with significant differences and CEA and histological grade to establish a clinical DECT model and found that the AUC, sensitivity, specificity, and prediction accuracy of the regression model were 0.99, 100.00%, 92.68%, and 94.67%, respectively. The predictive accuracy of the clinical DECT model was significantly improved compared to the DECT parameter alone, indicating that the combined model may have greater value in high-risk factor predictions. The results were in line with some artificial intelligence (AI) CRC studies (35,36).Li et al. constructed a machine learning-based CT model that combined radiomics and clinical features that could predict early the presence of metachronous liver metastases in patients by measuring the primary lesions of CRC . The model had and AUC of 0.79±0.08 . Zhao et al. confirmed the excellent predictive efficacy of deep learning-based imagingomics models for lymph node metastasis in CRC . The models constructed in these studies have good predictive efficacy for subgroups with high-risk factors for CRC; however, they were based on conventional CT images of the arteriovenous phase. We speculated that the iodograms and effective atomic number maps derived from DECT may provide potential information, and the AI models based on these maps may perform well in studies related to high-risk prediction and the prognosis of CRC patients.Liver metastasis is one of the most important factors in the survival and prognosis of CRC patients. The early diagnosis of CRLMs implies a smaller lesion size, which is an indication for minimally invasive surgery . Compared to traditional open surgery, robotic-assisted minimally invasive surgery not only allows for the simultaneous resection of CRLMs and primary CRC, but also reduces intraoperative bleeding, postoperative complication rates, and postoperative hospital stays, while shortening the learning curve due to enhanced 3D full-high definition (HD) vision and wristed instruments (38,39). One study showed that laparoscopic ultrasound has better sensitivity than MRI for CRLMs, especially for lesions located in the liver dome , which may be due to the abnormal MRI signal in the liver dome. As far as we know, relatively few studies of DECT on CRLM have been conducted compared to studies of MRI. We believe that DECT will play an important role in the development of surgical protocols for CRLM and in the assessment of efficacy following chemotherapy as the benefits of its quantitative and qualitative analysis become better understood.This study had several limitations. First, it was limited to adenocarcinoma CRC, and special histologic types, such as mucinous adenocarcinoma, were not included. In addition, while one of the principles of ROI outlining is to match the biopsy results, ROI outlining is also based on the maximum cross-sectional area of the tumor, so the ROI outlining and biopsy results may not completely match. Finally, the sample size in this study was relatively small, and no further subgroup analysis was performed based on risk stratification. It will be interesting for us to explore the predictive performance of DECT for the risk stratification of CRC in the future.ConclusionsIn conclusion, our study demonstrated that 40 VMI+ images were optimal for CRC DECT quantitative evaluation. Additionally, the AP-IC, AP-NIC, AP-DEI, AP-λHU, and DEI DECT parameters reflected the biological behavior of high-risk CRC tumors; thus, these DECT parameters can serve as additional tools for the potential prediction of high-risk factors in CRC. The diagnostic performance of the composite model constructed by combining DECT parameters with CEA and histological grading appears to be at a clinically acceptable level, allowing a more accurate prediction of high-risk factors in each patient and assisting in clinical decision-making.SupplementaryThe article’s supplementary files as10.21037/qims-23-29110.21037/qims-23-291

PMC

Clinical and Experimental Reproductive Medicine

38035589

PMC10914497

3-01-2024

10.5653/cerm.2023.06009

Criteria for implementing artificial intelligence systems in reproductive medicine

Güell Enric

This review article discusses the integration of artificial intelligence (AI) in assisted reproductive technology and provides key concepts to consider when introducing AI systems into reproductive medicine practices. The article highlights the various applications of AI in reproductive medicine and discusses whether to use commercial or in-house AI systems. This review also provides criteria for implementing new AI systems in the laboratory and discusses the factors that should be considered when introducing AI in the laboratory, including the user interface, scalability, training, support, follow-up, cost, ethics, and data quality. The article emphasises the importance of ethical considerations, data quality, and continuous algorithm updates to ensure the accuracy and safety of AI systems.

IntroductionArtificial intelligence (AI) algorithms have become ubiquitous in our lives, and the field of assisted reproductive technology (ART) is no exception. In recent years, increasingly many publications in scientific journals and conferences have highlighted the various applications of AI in reproductive medicine [1,2]. These applications span a wide range of areas within the field of reproductive medicine [3-5]. As embryologists, as well as physicians, we have the duty to keep abreast of the existing technologies, and above all, their function and results, before accepting the incorporation of any new tool in clinical practice. The present work aims to provide key concepts to be taken into consideration when considering integrating AI systems into reproductive medicine practices.Artificial intelligence in assisted reproductive technologyAmong the numerous published algorithms, we can find predictive models for embryo transfer outcomes on day 2/3 and blastocyst stage [7,8], sperm selection by image recognition correlated with fertilization and blastocyst formation , prediction of obtaining spermatozoa from testicular biopsies , non-invasive oocyte scoring on two-dimensional images , cytoplasmic recognition of the zygote , morphokinetic automated annotation of the embryo [13-15], automated blastocyst quality assessment , embryo implantation potential via morphokinetic biomarkers , euploidy prediction using metabolic footprint analysis , ranking for embryo selection [19-25], blastocoel collapse and its relationship with degeneration and aneuploidy , morphokinetics and clinical features for the prediction of euploidy , prediction of aneuploidy or mosaicism using only patients’ clinical characteristics , tracking of menstrual cycles and prediction of the fertile window , control of culture conditions and quality control of embryologist performance [25,30], intrauterine insemination success , computer decision support for ovarian stimulation , prediction for the day of triggering [33,34], and follicle-stimulating hormone dosage prediction for ovarian stimulation . All the mentioned references are depicted in Figure 1. Machine learning models are listed in Table 1 [6,7,10,17,18,27-29,31-37], while those corresponding to the deep learning subset can be found in Table 2 [8,9,11-16,19-24,26,38-43]. In these tables, the AI models are described with their sample size, results and limitations. The main limitation of all studies was their retrospective nature. A limited sample size, imbalanced dataset, and lack of multi-center evaluation were also common limitations found in the literature review.Commercial platforms or in-house algorithmsThe AI systems used in in vitro fertilization (IVF) clinics can be categorised into two types: commercial products and self-developed in-house solutions. While cloud-based systems can offer advantages for IVF clinics with lower workloads, such as leveraging data from other clinics, they may face challenges in maintaining predictive accuracy due to interference from individual clinic protocols or conditions. Notable examples of cloud-based products include Embryo Ranking Intelligent Classification Algorithm (ERICA) , intelligent Data Analysis Score (iDAScore) , and Life Whisperer .In contrast, adopting an in-house approach could offer certain advantages, such as greater control and customisation over the AI system and its workflow as well as the possibility to test own ideas without having to wait for commercial releases. Single-center studies such as Zeadna et al. or De Gheselle et al. represent this approach to AI in IVF.Requirements for implementing new AI systems in the laboratoryPrior to introducing a new AI system—or any other technique—it is essential to ensure that it satisfies certain criteria in a laboratory setting. At least one of the following criteria should be met for the new technique to be considered suitable: the candidate AI system should have the ability to improve results, such as the live birth (LB) rate, time to pregnancy, or any other key performance indicator. If the results are not worsened, other criteria to be met could include making work easier and more efficient, saving time and resources, offering greater safety through an improved error detection, or providing better explainability.Factors to consider when introducing AI in the laboratoryThere are several factors that cannot be overlooked when considering the integration of a new system into the laboratory. These factors must be carefully evaluated before making a decision. When introducing an AI system, the following factors must be taken into account:1. User interfaceThe user interface (the visual display on the screen) should be easy to understand and navigate.2. ScalabilityThe system should be capable of adapting to the laboratory's needs, including the volume of data and users, as well as being integrated into the laboratory's workflow and protocols. If the AI platform cannot be adapted to the laboratory’s existing workflow, it is necessary to evaluate the impact of adapting the lab workflow and the potential benefit of using that AI platform.3. TrainingThe manufacturer should offer information regarding the required training for users and how it will be delivered.4. SupportThe manufacturer should specify the type of technical support offered, who will be responsible in case of failure, and what the response time will be.5. Follow-upAs AI systems continuously learn, it is crucial to ensure that the algorithms are updated to accommodate new data. The manufacturer should provide information about the maintenance and monitoring plan to ensure that the system continues to provide accurate and unbiased results.6. CostThe cost of a system should be considered in relation to the center’s budget and investment capacity.7. EthicsTo ensure that an AI system is ethically sound, it is important to evaluate its impact on patient care and outcomes. The system should not only improve patient outcomes but also avoid any harm or negative impact on the patient. Moreover, the manufacturer should have measures in place to ensure the confidentiality and security of patient data, such as the ISO 27002-2021 and IEC 62304 standards. The most important ethical issue is the lack of randomised controlled trials. It is premature to implement a technology in the clinical setting before the trial results are made available . The nature of the mathematical algorithms performed during the AI process leads to a spectrum of transparency, ranging from the most interpretable models, such as linear regression-based algorithms, to the most cryptic models, also called black-box, such as neural networks. It is important to know the risks, side effects, benefits and the confidence of each clinical decision before delegating the decision-making process to machines. While transparent models enhance clinical decision-making, black-box systems replace human decisions, leading to uncertainty about the responsibility for treatment success. Black-box algorithms could build predictive models biased by cofounders, and the error-checking processes of each prediction could go unnoticed by human operators . Moreover, opaque models could increase the risk of imbalanced outcomes. For instance, if there exists a correlation between embryo quality assessed by AI and gender, there could be an intrinsic imbalance that could take more time to detect than in interpretable models.8. Data qualityThe quality of data refers to the data’s accuracy, completeness, timeliness, relevance, consistency, and reliability. It is crucial for an AI system to have access to high-quality data to provide accurate and reliable results. If the data used for building the model are not reliable and generalisable, then the AI model will fail when applied to new data in the near future. Some models are based on a concrete and certain population, and if data across populations are not as homogeneous, then the model will not be accurate enough. Furthermore, in embryology, confounding factors such as age should not be used as predictors in embryo quality models if it is desired to develop an embryo quality model instead of an age-based predictive model , as the AI algorithm could base its prediction mostly on data included in the age variable with no importance for embryonic features.9. PerformancePerformance refers to the effectiveness and efficiency of an AI system in achieving its intended objectives, such as accuracy, speed, and reliability. The system's performance should be evaluated based on relevant metrics and benchmarks to ensure that it meets the desired standards.Data annotationThe source of data is crucial in data annotation. The origin of data can vary (tabular, images, videos, audio, the outcome of a previous AI algorithm, etc.), and the annotation of data is expected to be more effective when automated, since automation removes the subjectivity of human-annotated parameters. However, the effectiveness of automated versus manual annotation depends on the degree of intra-individual and inter-individual variability for the target variable when annotated by humans and the reliability of the automatic annotation methods [45,46]. Features with higher variability or lower reliability can lead to lower performance of predictive models, since AI may use different values for data that are actually equivalent. Including such features in the models can introduce noise or inconsistencies, affecting the accuracy of predictions and the model’s overall performance. Determining whether manual or automated annotation is more suitable depends on each specific case. Factors such as data complexity, available resources, and the desired level of accuracy need to be considered. Manual annotation can provide more accurate and reliable results, but can be time-consuming and introduce human biases. Automated annotation methods can be more efficient and scalable, but may be less accurate or reliable, especially in cases with noisy data or lack of proper validation.It is not always possible for all values in a database to be filled. Not available (NA) values represent a problem when building AI algorithms and require proper handling. Several options exist for managing missing values. Some common approaches include discarding observations with NA values, imputing missing values using methods such as mean or median imputation, or utilising other AI algorithms such as k-nearest neighbour for imputation, as well as directly excluding the feature with NA values.Machine learning techniques are also sensitive to data points that deviate significantly from the majority of the data (outliers). Managing outliers involves deciding whether to integrate them into the analysis or discard them.Therefore, careful consideration is required when dealing with NA values and outliers. The choice of appropriate strategies for managing them depends on the specific context, the nature of the data, and the objectives of the analysis.Risk factors affecting data quality in model designEach predictive model has its unique characteristics and objectives, and is based on a specific experimental design that includes certain factors as inclusion and exclusion criteria. It is crucial to carefully review the experimental design, as there could be potential risks that may affect the quality of data used in the model. One such risk would be the possibility of data bias due to the inclusion criteria, which could compromise the generalisability of the results, particularly if there were confounding factors affecting predictive variables [47,48]. Three additional pitfalls to consider, as described by Curchoe et al. , are small sample sizes, imbalanced datasets, and limited performance metrics.Furthermore, in classification cases, there could exist a risk of mislabelling in the output variable. Mislabelling occurs when the categorical variable has incorrect labels for some of the data points. It is important to be aware of this risk, as the inclusion of mislabelled data decreases accuracy [50,51]. A potential example of mislabelling in embryology is evident in two embryo selection models with different labels for classification. One model compares implanted or LB embryos versus non-implanted or non-live birth (NLB) embryos , while the other compares euploid versus aneuploid embryos . In the LB versus NLB comparison, it is important to carefully consider the potential for mislabelling, as high-potential embryos with a negative outcome due to reasons unrelated to the embryo could be incorrectly labelled as NLB, which may negatively impact the performance of machine learning and deep learning algorithms [36,40,52]. Additionally, in ploidy models, undetected mosaicism can also lead to mislabelling. Moreover, the "Schrödinger embryo" paradox makes it impossible to assess the genetic status of the inner cell mass and trophectoderm until the whole embryo has been donated for research. Once an embryo has been donated, it becomes impossible for it to achieve LB, and its real potential for viability will remain unrealised. Besides, the algorithms’ performance may be distorted depending on the inclusion criteria in each experimental design. There is a risk of including embryos with low viability potential, those that have not yet been transferred, or even euploid embryos that were not cryopreserved due to low quality . Specifically, Tran et al. reported that the area under the curve (AUC) could be inflated by including many arrested embryos in the sample used to compute it. That predictive model could be considered proper for justifying automation for the quality assessment of arrested embryos, although random choice was supposed to be used for non-arrested embryos .Machine and deep learning modellingMachine and deep learning modelling refer to the process of creating and training mathematical models that can automatically identify patterns and make predictions or decisions based on data. Deep learning is included in the broader category of machine learning category. These models are built using algorithms and statistical techniques that allow computers to learn from large datasets and improve their performance over time . To emphasise the main differences, it is worth noting that machine learning typically requires fewer data points and provides greater interpretability than deep learning. As a rule of thumb, the sample size should be at least 10 times the number of parameters in an algorithm, and it is generally easier to determine this value for machine learning models than for deep learning models .There are two primary types of machine learning algorithms: supervised and unsupervised. On the one hand, supervised learning is an approach in which a model is trained using labelled data. After introducing input features (independent variables) along with corresponding target labels (dependent variable), supervised learning tries to learn a function or a relationship between the input features and the target labels. Once trained, the model can make predictions or classify new instances based on the input features. Supervised learning is commonly used in prediction and classification problems, where the objective is to predict a specific outcome or category, although numerical values can also be predicted through regression models. Decision trees, scoring systems, generalised additive models, and case-based reasoning are among the primary techniques used in various supervised learning algorithms . Each algorithm has its own specific characteristics and uses. Linear regression involves fitting a linear equation to the data, enabling the prediction of continuous target variables . Logistic regression is mainly used for binary classification tasks, although it could also be useful for multi-class problems, by modelling the probability of an event occurring based on input features . Recursive partitioning is a technique commonly used in decision trees, where the data are recursively split into subsets based on certain conditions of features . Random forest is an ensemble learning method that combines multiple decision trees to improve prediction accuracy and reduce overfitting [17,31]. The k-nearest neighbour method classifies or predicts the value of a data point based on the values of its k-nearest neighbours in the feature space [34,57]. Gradient boosting is an ensemble technique that builds a strong predictive model by iteratively combining multiple weak models, often decision trees, to correct errors made by the previous models . Support vector machines construct hyperplanes in a high-dimensional feature space to separate different classes or estimate continuous target variables [31,57]. Neural networks are complex and versatile machine learning algorithms capable of handling various tasks, including classification, regression and pattern recognition. They are inspired by the structure of the human brain. Image recognition models are based on this type of algorithms [13,16,19,20].On the other hand, unsupervised learning is employed in situations where the training data lack pre-existing labels or outcomes. Its objective is to discover patterns or structures inherent in the data without explicit guidance and to uncover similar groups or clusters of data. This type of learning is useful for exploring and comprehending the underlying structure in data and identifying hidden patterns. Clustering algorithms and dimensionality reduction methods are widely used in the field of unsupervised learning. K-means is a popular clustering algorithm aiming to divide a dataset into distinct groups or clusters based on similarity. The algorithm iteratively assigns data points to the nearest cluster centroid and updates the centroids until convergence . Principal component analysis (PCA) is a dimensionality reduction technique that transforms a high-dimensional dataset into a lower-dimensional space by identifying the principal components that capture the most significant variance in the data. These principal components are orthogonal and ordered in terms of their explanatory power. PCA is useful for simplifying complex datasets, visualising data in lower dimensions, and identifying the most important features driving variability in the data .Thus, the algorithms used in assisted reproduction to predict categories using labelled data are of the supervised learning type. When encountering AI-based predictive models, clinicians and embryologists should be familiar with the machine learning lifecycle (Figure 2): Collect and pre-process data: Collect relevant data and carry out pre-processing (cleaning, normalising, transforming, etc.) to prepare the data for machine learning algorithms. Train a machine learning model: Train a machine learning model on the pre-processed data using a suitable algorithm and hyperparameters. Test and evaluate the model: Test the trained model on a separate test dataset and evaluate its performance using suitable evaluation metrics. Deploy the model: Deploy the trained model to a production environment, such as a web application or a mobile app. Monitor the model: Continuously monitor the performance of the deployed model and collect feedback from users. Refine and update the model: Refine and update the deployed model periodically using new data and feedback to improve its performance and adapt to changing requirements.Performance evaluation and model validationWhen discussing performance, the first step is to define what is being evaluated. If one encounters studies that claim remarkable results on the training dataset, it is advisable to exercise caution. Predicting data that are already in the system makes it easier for the computer to find a previous pattern in the known model, leading to the overfitting effect. It is entirely normal, and almost necessary, for the training set results to be particularly good, as they do not represent the actual predictive potential of the model.As showed in Figure 3, the process of developing a predictive model involves an initial partition of the test set, which is kept separate from the algorithm's training. Cross-validation is performed on the training set by separating a certain percentage and creating the model with the training set, then predicting the validation set. This process can be repeated several times to obtain cross-validation metrics. This prediction can already be considered representative of the model's predictive potential. Cross-validation can be performed through k-fold cross-validation (e.g., 80% of the dataset for training and 20% for validation) [18,28]; as well as training the model with the full dataset except for one specimen, predicting it, and repeating the process for all specimens in the dataset (leave-one-out cross-validation) .Finally, the test dataset is used to validate how the method (training set+validation set) predicts data that are not in the database. Therefore, it can be considered representative of the model's predictive potential .Performance metrics for machine learningDepending on the type of algorithm, different metrics should be chosen to evaluate its performance . For regression models, common metrics include mean squared error, mean absolute error, root mean squared error, and r2 . For classification models, common metrics are obtained from a confusion matrix, which unfortunately is not always provided in studies. Common metrics include accuracy, AUC and AUC precision (positive predictive value), recall (sensitivity), negative predictive value and specificity . The F1-score and Matthews correlation coefficient are also metrics to be considered, especially in imbalanced datasets . It is important to ensure that the positive reference is correctly identified in order to avoid confusion when evaluating model performance. For example, in a comparison of euploidy, it may seem obvious that the aneuploid group should be considered as the negative reference. However, the computer may mistakenly assign the aneuploid group as the positive reference if not explicitly specified, such as in cases where alphabetical ordering is used. Therefore, it is crucial to carefully define the positive and negative references before assessing a model’s performance.Conclusions: time to implement?Different authors have expressed their thoughts on whether or not to implement predictive AI models into the daily practice [59-61]. From my point of view, it is worth considering implementing an algorithm if its result is robust enough to answer the initial question of the requirement. For instance, if the objective was to improve the implantation rate, it is not as crucial whether the embryo selection model is based on viability, genetics, or a combination of both [36,40], nor is the specific value of AUC achieved particularly relevant. While a better AUC is theoretically associated with a better implantation outcome, this cut-off value would not be relevant if the implantation rate with the AI score is superior to that without AI. Nevertheless, external validation should be carried out to verify that the response to the requirement for integrating an AI system in the laboratory is truly satisfactory when applying AI compared to not applying AI. From there, it will be necessary to consider verifying the data either prospectively or in a multi-center setting.

PMC

Journal of Clinical and Translational Science

PMC10129750

10.1017/cts.2023.226

145 A CTS Team Approach to Adapting an Evidence-Based Mindfulness Tool to Increase Trust of Reproductive Healthcare Providers

Nesbit Tyler S., Ronke Coker Karen Awura Adjoa, McKune Sarah, Forthun Larry

OBJECTIVES/GOALS: The goals of this study are to 1) adapt a mindfulness-based intervention that supports the development of trust-promoting behaviors of OBGYN providers with patients who identify as Black women based on the input of providers and patients, and 2) assess the feasibility of implementation for OBGYN healthcare providers. METHODS/STUDY POPULATION: Goal 1: Focus groups will be conducted with members of the populations of providers and Black women patients in Alachua County, Florida to identify essential intervention content to complement the central component of mindfulness and spiritually based practices. This complementary content will serve to address the institutional and cultural context of the intervention setting. Goal 2: Providers will be recruited to participate in interviews about their perceptions of intervention feasibility. These aspects include recruitment potential, acceptability of the intervention content and delivery, implementation practicality, identification of appropriate outcomes, and identifying strategies to recruit Black women patients to participate in program evaluation. RESULTS/ANTICIPATED RESULTS: Goal 1: We will elicit the perspectives of providers and Black women patients regarding the respective roles and relationship of mindfulness and spirituality to increase trust-promoting behaviors with patients who are Black women. We also anticipate identifying additional content to complement the core intervention components that participants perceive as necessary to develop the knowledge, skills, and behaviors which convey the trustworthiness of providers to patients. Goal 2: We expect to gain key insights into intervention design, implementation, and evaluation feasibility from the perspective of providers. Interview data will be aggregated and qualitatively analyzed for themes pertaining to feasibility. DISCUSSION/SIGNIFICANCE: An intervention that builds on mindfulness and spiritual practice is an innovative approach to improving interpersonal outcomes in provider-patient relationships. By investigating the feasibility of such an intervention, we will gain insight into how to design and deliver a program to increase the trust-promoting behaviors of OBGYN providers.

PMC

Jornal Brasileiro de Pneumologia

PMC10171262

10.36416/1806-3756/e20230100

Legal action in sleep medicine: new alternatives need to be sought!

Fagondes Simone Chaves, John Angela Beatriz

In this issue of the Jornal Brasileiro de Pneumologia, the article by Pachito et al. 1 raises the discussion of an increasingly common approach in Brazil, as well as in other countries, which is taking legal action for access to medical procedures and treatments. 2 The evolution of knowledge in health care has introduced more sophisticated diagnostic methods and therapeutic options, and, consequently, costs have increased. However, many of these methods and treatments are not covered by the Sistema Único de Saúde (SUS, Brazilian Unified Health System) or private health insurance plans, which, based on the premise that health is a universal right, makes legal action an alternative, with all the complexity that this approach imposes.From the perspective of sleep medicine, there is a great lack of public services that offer specialized care in this area. A recent study has identified the presence of 36 specialized centers in Brazil, with a great asymmetry in terms of geographic distribution, and 44% of those are concentrated in the southeastern region of the country (personal information). Regarding diagnosis, sleep laboratory beds accredited to perform tests by the SUS are a minority, totaling only 28 centers throughout Brazil (personal information). On the other hand, the use of portable polysomnograms, which are less expensive and dispense with sleep laboratories, still requires improvements in both logistics and operationalization.In addition to diagnostic limitations, we have an even greater challenge when we address issues related to treatment. The main treatment for moderate and severe obstructive sleep apnea is the use of a device that generates CPAP in the upper airways. It is a high-cost piece of equipment that is included neither in the SUS nor in most private health insurance plans. In our daily practice at a public tertiary university hospital, we have observed actions that aim to fulfill this need at the municipal level; however, these actions are generally restricted to patients with more severe disease and are concentrated in larger cities, closer to capitals.The magnitude of the problem, therefore, is directly related to the prevailing socioeconomic reality in our country and the limitations arising from an area of medicine that is still being consolidated, especially in the public sphere, as well as to a highly prevalent medical condition (approximately 30% of the adult population), 3 whose consequences have been widely documented in the literature. 4 , 5 One of the concerns pointed out in the article by Pachito et al. 1 is the high economic costs that the growing practice of legal action in sleep medicine imposes. The study presents an additional cost estimate of 588% for diagnostic tests and of 21.7% for treatment with CPAP. These values are substantially higher when we compare the public health care system with the private health insurance plans.The theme takes on an even more relevant and worrying role considering that the number of lawsuits identified in the manuscript seems to be underestimated. The authors performed an analysis based on information extracted from the judicial system database over a period of five years and identified only 1,462 lawsuits, that is, approximately 292 cases/year. Considering the already mentioned high prevalence of obstructive sleep apnea in a country with an estimated adult population of 159.2 million individuals, 6 the number of patients who would potentially seek public health care assistance should be much higher. Another aspect that deserves attention is the decrease in the number of lawsuits between 2017 and 2019 reported in that study. 1 This finding differs from our experience in a public hospital. In recent years, with the deepening of the socioeconomic crisis in Brazil and the consequent decrease in income, making it difficult to maintain a private health insurance plan and to acquire a CPAP device, we have observed a substantial growth in the number of patients referred to our sleep outpatient clinic.As it was already pointed out by the authors, 1 the need for public policies that include the training of physicians to care for those patients, the dissemination of diagnostic methods, a detailed review of health care staff wages, and the establishment of partnerships is vital in order to improve the offer of CPAP treatment. It is also necessary that these patients have access to follow-up by a qualified medical team in the various regions of the country.Definitely, legal actions regarding sleep medicine are far from being a solution. They should be an exception, and it is urgent that we seek new alternatives!

PMC

Preventive Medicine Reports

PMC10518787

7-27-2023

10.1016/j.pmedr.2023.102345

Correspondence to Corrigendum

We thank Dr. Li and his team for noting the error in the abstract of our paper: Rosal M.C., Lemon S.C., Borg A., Lopez-Cepero A., Sreedhara M., Silfee V., Pbert L., Kane K., Li W. . The Healthy Kids & Families study: Outcomes of a 24-month childhood obesity prevention intervention. Prev. Med. Rep. Dec 7;31:102086. PMID: 36820371; PMCID: PMC9938323. A corrigendum with the corrected abstract has been published in this journal (Corrigendum to “The Healthy Kids & Families study: Outcomes of a 24-month childhood obesity prevention intervention” [Prev. Med. Rep. 31 102086] – ScienceDirect). We regret any inconvenience this may have caused.Funding/SupportThis publication is a product of a Health Promotion and Disease Prevention Research Center supported by Cooperative Agreement Number U48DP005031 from the Centers for Disease Control and Prevention.Declaration of Competing InterestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Cancer Control : Journal of the Moffitt Cancer Center

37724508

PMC10510351

9-19-2023

10.1177/10732748231202470

Comparative Analysis of First-Line FOLFOX Treatment With and Without Anti-VEGF Therapy in Metastatic Colorectal Carcinoma: A Real-World Data Study

Brenner Ronen, Amar-Farkash Shlomit, Klein-Brill Avital, Rosenberg-Katz Keren, Aran Dvir

BackgroundFOLFOX (leucovorin calcium [folinic acid], fluorouracil, and oxaliplatin) combined with or without anti-VEGF therapy represents one of the primary first-line treatment options for metastatic colorectal carcinoma (mCRC). However, there is limited comparative data on the impact of anti-VEGF therapy on treatment effectiveness, survival outcomes, and tumor location.MethodsThis retrospective, comparative study utilized data from the AIM Cancer Care Quality Program and commercially insured patients treated at medical oncology clinics in the US. We analyzed 1652 mCRC patients who received FOLFOX, of which 1015 (61.4%) were also treated with anti-VEGF therapy (VEGF cohort).ResultsPatients in the VEGF cohort exhibited a higher frequency of lung (33% vs 23%; P < .001) and liver metastases (74% vs 62%; P < .001), underwent fewer liver surgeries prior to treatment (1.2% vs 3.6%; P = .002), and had a higher proportion of right-sided tumors (27% vs 18%; P = .001). Adjusted analysis revealed no significant difference in overall survival (OS) between patients treated with and without anti-VEGF (median survival: 25.4 vs 26.0 months; P = .4). FOLFOX-only treated patients experienced higher rates of post-treatment hospitalizations (22% vs 15%; P < .001). Notably, left-sided tumors treated with anti-VEGF showed a trend toward decreased OS (median survival: 26.8 vs 33 months; P = .09).ConclusionOur real-world data analysis suggests that the addition of anti-VEGF to FOLFOX offers limited and short-lived benefits in the context of mCRC and may provide differential survival benefit based on tumor sidedness.

IntroductionColorectal cancer, CRC, is the third most commonly diagnosed cancer in males and the second in females, worldwide. 1 Every year, approximately 150 000 new patients with CRC are diagnosed in the United States, and approximately 50 000 die of CRC. 2 Of new colorectal cancer diagnoses, 20% of patients have metastatic disease at presentation and another 25% who present with localized disease will later develop metastases. In early and locally advanced CRC, surgery and adjuvant chemotherapy are used with the aim to cure the disease, whereas treatment of metastatic disease is aimed at prolonging life and preserving quality of life.The treatment for metastatic colorectal cancer (mCRC) has advanced significantly over the past two decades. Two decades ago, 5-fluorouracil (5-FU) was the only active treatment with a median overall survival of approximately one year, 3 while nowadays with newer agents and treatment combinations median survival can reach 2–3 years. Treatment agents for colorectal cancer today include the anti-topoisomerase irinotecan, the platinum agent oxaliplatin, the anti-EGFR monoclonal antibodies cetuximab and panitumumab, and the anti-VEGF antibody bevacizumab or its biosimilars. 2 The optimal combination of these agents in the first- and later treatment lines is a paradigm that is continually evolving, and different biomarkers are used to determine which is the best treatment for an individual patient. Another recently evolving predictive factor identified from post hoc analyses of various phase 3 trials suggested that the location of the primary tumor is an important predictive factor for first-line (1L) usage of EGFR inhibitors; thus, these drugs are usually recommended as 1L just for patients with left-sided primary tumors.4,5 The evidence for an overall survival (OS) benefit of VEGF inhibitors in the context of oxaliplatin- or irinotecan-based chemotherapies is limited and inconsistent and is based on clinical trials conducted almost two decades ago, with some studies showing a significant benefit in OS to anti-VEGF treatment6–8 and others do not.9–11 It should be noted that VEGF inhibitors are associated with increased toxicities, including proteinuria, hypertension, bleeding, bowel perforation, impaired wound healing, and arterial thromboembolic events, 12 along with added costs, emphasizing the need to monitor their efficacy.Real-world studies are a very important tool to evaluate the actual usage and effectiveness of medical treatments which were already included into common practice by randomized control trials. Previous studies that compared real-world data to clinical trials and used proper adjustments have shown similar OS in NSCLC in the 2L setting. 13 Other studies have leveraged real-world data to provide evidence in the absence of a randomized phase 3 trial or to further characterize patient outcomes in the real world for metastatic breast cancer patients 14 or in metastatic pancreatic cancer,15,16 or to explore the prognostic and predictive role of tumor location in mCRC. 15 Although VEGF inhibitors together with FOLFOX are the most common treatment for mCRC in 1L setting, with strong evidence on positive impact on PFS, the evidence supporting the impact on survival is limited, as well as data supporting the role of tumor location for this treatment. Here, we utilized a large real-world clinical dataset from over 1600 mCRC patients across multiple regions and health care systems in the United States and explored survival outcomes and post-treatment hospitalizations associated with FOLFOX alone or in combination with VEGF inhibitors.Materials and MethodsData SourceThis retrospective analysis utilizes nationally representative clinical data provided to the AIM Cancer Care Quality Program, along with corresponding claims and lab data. 17 Submissions via the AIM portal encompass various details, including the planned treatment regimen, treatment date, TNM staging, therapy sequence, ECOG PS, ICD-10 diagnosis code, and KRAS status (available for 30% of patients). The claim data covers medical and pharmacy claims for nearly 40 million members under 14 U.S. regional health plans with commercial insurance, excluding government, Medicare, and Medicaid claims. All data were anonymized for patient privacy. Mortality information was gathered from discharge status, national death registries, and obituary data. The patients originate from multiple outpatient centers and medical oncology clinics in numerous U.S. states. The index date is established as the start of first-line therapy as per the request, confirmed via administrative claims. Patients without mortality events were assessed until their final medical claim date or the study’s conclusion. The study adheres to STROBE guidelines. 18 Covariates and OutcomesThe primary measure of effectiveness centered on overall survival (OS), which was defined as the period from the treatment index date to the time of death or the most recent follow-up in cases of censorship. A secondary outcome involved post-treatment hospitalizations, identified within 30, 60, and 90 days after the index date. Initial characteristics considered encompassed prognostic stage, ECOG PS, sites of metastasis, instances of liver surgery before or after treatment, existing medical conditions, sociodemographic particulars, tumor orientation, and KRAS status. The dataset employed for analysis was rendered anonymous following the principles of Safe Harbor privacy. It excluded personal identifiers like names, precise dates (only relative intervals were retained), postal codes (only rounded SDI), and any other information that could lead to identification.Covariate CalculationThe PS score is determined by the treating physician at the treatment’s outset. Comorbidities were evaluated using the Charlson Comorbidity Index (CCI) through patient claims within one year before and up to 10 days before the index date. The R comorbidity package 19 computed the CCI, consisting of 17 medical conditions; scores excluded metastasis and cancer diagnoses. Metastatic sites were identified using ICD-10 codes associated with metastasis in the year before and up to the index date. Sociodemographic data encompassed age, gender, and social deprivation index (SDI), determined from patient zip codes. ECOG PS, SDI score, and CCI, numerical variables, were normalized via min–max normalization. Prior liver surgeries were assessed using related CPT codes up to one year before the index date; post-treatment liver surgeries followed a similar evaluation with CPT codes within one year after the index date. KRAS status was available for only 30% of patients. Tumor sidedness was approximated using the ICD-10 diagnosis from the request and mapping provided in a previous study. 20 Rectal tumors were considered left-sided tumors after an analysis revealing comparable KRAS and MSI status in rectum patients and left-sided patients.AdjustmentMatchingEmploying the MatchIt R package (Version 4.3.1, R), 21 a 1:1 matching process was conducted, utilizing the Mahalanobis distance metric and incorporating all the mentioned covariates. Following matching, P-values were computed for the source variables to confirm the absence of significant disparities between the matched groups (see Table 1). Subsequently, the Kaplan–Meier estimator was applied to the matched cohort.IPTWInverse probability of treatment weighting (IPTW) was employed through a propensity score analysis. The propensity score was derived using a logistic regression model with treatment type as the dependent variable, factored against potentially confounding variables. To compute and assess inverse propensity weighting, the Python causalib package (Version .1.3, IBM Python) was utilized. Evaluation of IPTW adjustment quality included ROC curve analysis with and without weights, assessing the propensity distribution, Absolute Standard mean difference before and after adjustment, and utilizing a calibration curve (refer to Supplementary Figure 2). For survival assessment, the Kaplan–Meier estimator was adjusted by the inverse propensity score.Statistical AnalysisTable 1 displays a comparison of baseline characteristics between groups. Categorical variables underwent Chi-square or Fisher exact tests, while continuous variables were subjected to Mann–Whitney tests or Student’s t-tests. A significance level of P < .05 indicates statistical significance. Median survival time was computed via Kaplan–Meier estimation. For unadjusted survival, Kaplan–Meier survival curves were utilized, and distinctions among survival curves were evaluated through the log-rank test. 22 Unless specified differently, all presented analyses rely on either propensity score analysis with inverse probability of treatment weighting (IPTW) or 1:1 matching.Table 1.Patient Characteristics of the FOLFOX-Treated Cohorts.FOLFOX Only (N = 637)FOLFOX + VEGF (N = 1015)P-ValueAge, median [Q1,Q3]56.1 [50.1, 61.4]55.7 [48.9, 61.3].55Sex, n (%)Female261 (41.0)441 (43.4).35Social Deprivation Index,a median [Q1,Q3]46.0 [23.0, 70.0]46.6 [22.0, 69.0].89ECOG performance score,b n (%)0306 (48.0)479 (47.2).311293 (46.0)491 (48.4)238 (6.0)45 (4.4)Charlson Comorbidity Index Score,c mean (SD).6 (.8).7 (.8).34Metastasis site, n (%)Liver396 (62.2)756 (74.5)<.001Lung148 (23.2)338 (33.3)<.001Peritoneum136 (21.4)227 (22.4).67Brain18 (2.8)21 (2.1).41Bone61 (9.6)118 (11.6).22Liver surgery, n (%)Before treatment23 (3.6)12 (1.2).002Post-treatment60 (9.4)79 (7.8).28KRAS, n (%)Mutant48 (57.1)123 (54.2).74WT36 (42.9)104 (45.8)Colon sidedness, n (%)Left338 (81.6)476 (72.7).001Right76 (18.4)179 (27.3)aScores range from 1 to 100 with higher scores indicating greater deprivation (SDI).bScores range from 0 to 2, with higher scores indicating worse status.cCharlson index score was adjusted to exclude oncology diagnosis in the score.Ethical DeclarationThis study was designed as an analysis based on medical claims data, and there was no active enrollment or active follow-up of study subjects, and no data were collected directly from individuals. The study was not required to obtain additional IRB approval, as the HIPAA Privacy Rule permits protected health information (PHI) in a limited data set to be used or disclosed for research, without individual authorization, if certain criteria are met.ResultsStudy CohortThe study cohort included patients with a diagnosis of colorectal cancer stage IV between January 2016 and June 2021 at the start of their first-line treatment and ECOG performance score between 0 and 2. Based on this inclusion criteria, we identified 9003 patients. Only patients intended for treatment with FOLFOX-based regimens were included in analyses. Based on this inclusion criteria, we identified 6561 patients. Patients were followed until December 2021 or death (Figure 1). The treatment plan was defined using the clinical data available in the AIM Cancer Care Quality Program. Patients were excluded if the treatment plan could not be corroborated by the claims data, or if the line of treatment was higher than 1L, resulting in the “Treated cohort” with 2521 patients. Finally, only patients treated with FOLFOX only or FOLFOX and anti-VEGF were included in the final “FOLFOX cohort” resulting in 1652 patients.Figure 1.Flow chart of the FOLFOX mCRC 1L-treated patient cohorts.Trends in TreatmentsUsing clinical data and administrative claims, we identified 10 352 treatment requests (request for every treatment modification) for patients diagnosed with colorectal cancer stage IV and started between January 2016 and June 2021 their 1L treatment. The top regimens were FOLFOX- or FOLFIRI-based regimens (43% and 21%, respectively); however, many different combinations exist, including the FOLFIRINOX, Capecitabine, and CapeOX as other leading chemotherapies, and immune checkpoint blockade therapies as well (Figure 2A). We decided to focus our study on FOLFOX-based regimens, and for those requests we corroborated the clinical data with claims data to validate that the requests were executed, and patients were treated (Figure 1). In the validated “treated cohort.” FOLFOX combined with anti-VEGF (bevacizumab or MVASI) was the most common regimen accounting for 40.2% (1015/2521 patients), following FOLFOX-only (25.3%; 637/2521 patients). Interestingly, we observed a decline in the FOLFOX plus anti-VEGF combination in recent years with the FOLFOX-only regimen prevalence increasing (Figure 2B), suggesting clinicians started to reconsider the value of anti-VEGF treatment in the context of FOLFOX.Figure 2.Trends in treatments in 1L mCRC during 2016–2021. (A) Number of requests of different regimens for 1L mCRC in the intent to treat cohort. (B) Percentage of patients with first-line treatment of FOLFOX- and FOLFIRI-based regimens, normalized by year, between the years 2016 and 2021 in the treated cohort. (C) Length of cycles in days for patients treated with FOLFOX. (D–E) Stack density plot of the FOLFOX (in D) and FOLFOX plus VEGF patients (E) showing the percentage of patients who received any of these treatments: FOLFOX, FOLFOX + MVASI or VEGF, 5FU, 5FU + LV, 5FU + LV + Bev, or Bev only in each cycle (cycle length = 14 days) as well as patients that died, treatment was not reported, or don’t have a follow-up. (F–G) Similar plots, with only the treatment events in each cycle, in which case the 100% is different in each cycle and includes only patients who received any treatment from the list in this cycle.We observed that even in the limited set of treatment decisions we focused on in this study, there are still many differences among patients in the treatment plan. First, cycle length differs among patients, as most patients receive treatment every 14 days, but cycle length can be 21 or 28 days (Figure 2C). Additionally, the treatment protocol is dynamic as treatment progresses, with the addition and removal of chemotherapies. In the FOLFOX-only cohort, we observed that after 10 cycles (with a cycle length of 14 days) of FOLFOX, there was an increase in the percentage of 5FU + LV treatment, suggesting oxaliplatin was removed due to toxicity or low tolerance (Figure 2D and 2F). After 20 cycles, there are less than 10% of patients that are still treated with FOLFOX, while the 5FU + LV is most dominant. We also observed that anti-VEGF may be added to the FOLFOX treatment after a few cycles for about 10% of the patients and there too oxaliplatin will be removed after around 10 cycles for most patients (Figure 2D and 2F). In the FOLFOX plus anti-VEGF cohort, we observed similar patterns, where oxaliplatin was removed even earlier, at around 7 cycles, as we observe an increase in 5FU + LV + Bev, and after around 15 cycles, this becomes the dominant treatment (Figure 2E and 2G).Comparative Effectiveness of Addition of Anti-VEGF to FOLFOXTo study treatment effectiveness, we created a limited study cohort of 1652 patients treated with FOLFOX, with or without anti-VEGF treatment. Due to the observational nature of the study design, patients receiving FOLFOX differed in few demographic and clinical characteristics from those treated with FOLFOX plus anti-VEGF (Table 1). Some patients undergo liver resection (10.5% in our cohort), pre- or post-treatment, and treatment decision may be influenced by the planned surgery. Patients undergoing post-treatment liver resection may represent a better prognostic group and thus will be treated with FOLFOX only. Indeed, in our cohort there was a slight, but non-significant, enrichment of FOLFOX only compared to FOLFOX + anti-VEGF in patients undergoing post-treatment liver surgery (9.4% vs 7.8%; P-value = .28). Nevertheless, liver surgery was used as a confounder and was adjusted throughout our analyses. Of note, significant differences between the two groups are lung (33% vs 23% for FOLFOX plus anti-VEGF and FOLFOX alone, respectively; P-value <.001) and liver metastases (74% vs 62%; P-value <.001), liver surgeries before the treatment (1.2% vs 3.6%; P-value = .002), and right-sided tumors (27% vs 18% P-value = .001). There were no apparent socioeconomic differences in the decision to treat with anti-VEGF or not. Furthermore, we did not observe any significant difference between the treatment groups in terms of ECOG performance score, or biomarker status (KRAS), suggesting that both ECOG and KRAS status is not impacting the decision to treat with respect to anti-VEGF.We compared the real-world effectiveness of FOLFOX with and without anti-VEGF with an end point of all-cause mortality. To adjust for the covariates, we performed IPTW (see method validation in Supplementary Figure 2a and in methods). Interestingly, the adjusted Kaplan–Meier fit showed no survival difference between the treatments (median survival: 25.4 vs 26 months for FOLFOX and FOLFOX and anti-VEGF, respectively, P-value = .7; Figure 3A). Similarly, an adjusted Cox proportional model using the IPTW as weights resulted in an HR of 1.08 (P-value = .36), again, suggesting no survival benefit for anti-VEGF treatment in the context of FOLFOX. We also used 1:1 matching to account for the confounders which resulted in sub-populations of 613 patients for each treatment, with similar results (Supplementary Figure 1b).Figure 3.Overall survival and hospitalization analysis of VEGF benefit. (A) Adjusted Kaplan–Meier fit for FOLFOX- and FOLFOX and anti-VEGF-treated patients; dashed lines and numbers show the median survival. (B) Percentage of hospitalization post-treatment, 30, 60, or 90 days after treatment start for patients treated with FOLFOX only or FOLFOX and anti-VEGF.To study post-treatment effects, we compared hospitalization rates after the initiation of the treatment. Using both the matched cohort and IPTW adjustment, we found that anti-VEGF treatment leads to ∼29% less hospitalization events 90 days following the first treatment (P-value <.001; Figure 3B). A similar trend was observed for 30- and 60-day window. This analysis suggests some short-term benefits for anti-VEGF, supposedly reducing severe adverse reactions to the chemotherapies.Tumor SidednessWe next wanted to explore the impact of tumor sidedness on OS. About 65% of the patients could be annotated to right or left tumors based on their ICD-10 diagnosis code (Table 1). Right-sided CRC tumors are considered to have worse prognosis and different molecular landscapes. Similar to previous reports, 2 we found right-sided tumors were about twice as likely to be MSI-H (18% vs 9%, P-value = .37) and more commonly have KRAS sequence variations (59% vs 31%; P-value <.001). Tumor sidedness was also a strong prognostic feature in our data, where right-sided tumors are associated with shorter median OS (22.5 vs 28.3 months, P-value <.001; Figure 4A). KRAS mutational status was also shown to be associated with OS, 23 and indeed we observed worse survival for patients with KRAS mutation, although not significantly (24 vs 27 months, P-value = .19; Figure 4B). In terms of the benefit of anti-VEGF, in the left-sided tumors, after adjustments, anti-VEGF showed a trend toward decreased survival (26.8 vs 33.1 months, P-value = .09; Figure 4C), while in right-sided tumors, there was no difference in survival with or without anti-VEGF (22.3 vs 23.7 months, P-value = .9; Figure 4D). Interestingly, while 70% of the right-sided patients received anti-VEGF, only 58% of the left-sided patients received anti-VEGF (Figure 4E), suggesting clinicians might take tumor sidedness into their treatment decisions already. We also see within the tumor side a difference in anti-VEGF prevalence between KRAS status although less significant (Figure 4E).Figure 4.Overall survival analysis with sidedness of the tumor and KRAS status. (A–D) Adjusted Kaplan–Meier fits for left- and right-sided tumor patients treated with FOLFOX or FOLFOX + VEGF. Dashed lines and numbers show the median survival. (E) Pie charts representing how many patients were treated with FOLFOX (light blue) or FOLFOX + VEGF (orange) in different segments that include tumor sidedness (pink and gray chart) and KRAS status (green and red chart).DiscussionIn this study, we investigated the real-world effectiveness of the FOLFOX plus anti-VEGF combination in the first-line treatment of mCRC. Given the low probability of further randomized studies, real-world studies like ours play an important role in accumulating evidence on the efficacy of this treatment combination. Our results showed no statistically significant survival differences with the addition of anti-VEGF to the FOLFOX backbone. This finding is consistent with the phase 3 NO16966 trial, which also found no survival benefit when adding anti-VEGF to FOLFOX/XELOX regimens, 11 although there is conflicting evidence from other randomized controlled trials. 8 Although limited data support the OS benefit of anti-VEGF, it is still recommended for first-line treatment of mCRC by the NCCN guidelines. In our cohort, the FOLFOX plus anti-VEGF combination was the most prevalent treatment regimen. However, we observed a decline in the usage of this combination and a reciprocal rise in using FOLFOX alone. This might be due to the uncertainties regarding the efficacy of anti-VEGF, as well as concerns about toxicity and cost.Tumor sidedness is recognized as a prognostic factor in colorectal cancer. 5 In our study, we confirmed the well-known worse prognosis of right-sided tumors and their tendency to harbor RAS mutation and MSI high tumors. We also found that anti-VEGF has no survival benefit for right-sided tumor patients and shows a trend toward decreased OS for patients with left-sided tumors (P-value = .09). This finding is in line with recent evidence suggesting that tumor sidedness is an important predictive factor for treatment in first-line therapies involving anti-EGFR agents, 4 although the evidence for anti-VEGF agents is less consistent.24-26Treatment toxicity is an important consideration when evaluating the effectiveness of a treatment regimen. In our study, we measured treatment toxicity in terms of hospitalization during the treatment period. We found no evidence of increased toxicity when anti-VEGF was used in combination with FOLFOX, and in fact, we observed a significant reduction in the number of hospitalizations in the first months after treatment, suggesting a clinical and quality-of-life benefit from adding anti-VEGF to chemotherapy, consistent with previous reports of better PFS with the anti-VEGF. 11 Lastly, this study has several limitations. First, the use of claims data might result in coding differences by different practices and physicians. 16 However, our analysis is based on a combination of claims and clinical data from the AIM Cancer Care Quality Program. The clinical data was used to augment the administrative claims data, providing information such as the stage, line of treatment, ECOG performance status, and biomarkers. However, since the clinical data is only collected for pre-authorization decisions, information not relevant for the treatment decision is not collected, and therefore our data includes biomarker status data only for a subset of patients (in this case, KRAS status was only available for 30% of patients).Second, our main outcomes were overall survival and post-treatment hospitalizations, and we may have ignored other clinically relevant outcomes such as progression-free survival (PFS) and quality of life metrics. Third, some patients could have left the insurance plan or used private services that are not well-documented. Finally, the observational nature of the study means that there may be inherent limitations from time-related biases and residual confounding from unmeasured factors.Despite these limitations, our study provides valuable insights into the real-world effectiveness of the FOLFOX plus anti-VEGF combination in the first-line treatment of mCRC. Our findings support the use of this treatment combination in clinical practice, although the decision to use anti-VEGF should be made on a case-by-case basis, taking into account factors such as tumor sidedness and patient characteristics. Importantly, addition of anti-VEGF may not provide OS benefit but rather it seems to have more effect on toxic reactions. Further studies are needed to confirm our results and investigate the potential benefits and limitations of different treatment regimens in the first-line treatment of mCRC.ConclusionsAlthough anti-VEGF therapy is very common in the treatment of mCRC 1L, there are inconsistent reports as to the efficacy and toxicity of it. Our study based on large cohort real-world data reveals that anti-VEGF is not associated with improved survival, although in the short term, it is associated with fewer post-treatment hospitalizations. Lastly, our study suggests that sidedness should be considered when treating with anti-VEGF in 1L of mCRC.Supplemental MaterialSupplemental Material - Comparative Analysis of First-Line FOLFOX Treatment With and Without Anti-VEGF Therapy in Metastatic Colorectal Carcinoma: A Real-World Data StudyClick here for additional data file.Supplemental Material for Comparative Analysis of First-Line FOLFOX Treatment With and Without Anti-VEGF Therapy in Metastatic Colorectal Carcinoma: A Real-World Data Study by Ronen Brenner, Shlomit Amar-Farkash, Avital Klein-Brill, Keren Rosenberg-Katz, and Dvir Aran in Cancer Control.

PMC

Clinical and Experimental Pediatrics

37321570

PMC10839191

6-14-2023

10.3345/cep.2022.00346

Moderate to severe atopic dermatitis in children: focus on systemic Th2 cytokine receptor antagonists and Janus kinase inhibitors

Kim Jeong Hee, Samra Mona Salem

Atopic dermatitis (AD) is a lifelong disease that markedly impairs quality of life. AD is considered a starting point of the “atopic march,” which begins at a young age and may progress to systemic allergic diseases. Moreover, it is strongly associated with comorbid allergic and inflammatory diseases including arthritis and inflammatory bowel disease. Understanding the pathogenesis of AD is essential for the development of targeted therapies. Epidermal barrier dysfunction, immune deviation toward a T helper 2 proinflammatory profile, and microbiome dysbiosis play important roles via complex interactions. The systemic involvement of type 2 inflammation, wheather acute or chronic, and whether extrinsic or intrinsic, is evident in any type of AD. Studies on AD endotypes with unique biological mechanisms have been conducted according to clinical phenotypes, such as race or age, but the endotype for each phenotype, or endophenotype, has not yet been clearly identified. Therefore, AD is still being treated according to severity rather than endotype. Infancy-onset and severe AD are known risk factors leading to atopic march. In addition, up to 40% of adult AD are cases of infancy-onset AD that persist into adulthood, and these are often accompanied by other allergic diseases. Therefore, early intervention strategies to identify high-risk infants and young children, repair an impaired skin barrier, and control systemic inflamation may improve long-term outcomes in AD patients. However, to the best of our knowledge, no study has evaluated the effectiveness of early intervention on atopic march using systemic therapy in high-risk infants. This narrative review addresses the latest knowledge of systemic treatment, including Th2 cytokine receptor antagonists and Janus kinase inhibitors, for children with moderate to severe AD that is refractory to topical treatment.

Graphical abstract. Subtypes of adults with atopic dermatitis. Approximately 40%–60% of adults with atopic dermatitis develop them in infancy and persist into adulthood. At birth, reduced IFN-γ- and enhanced IL-4-producing CD4+ cord blood T cells are subsequently associated with infancy-onset atopic dermatitis. Early-onset atopic dermatitis and allergic sensitization at early age increase the risk of the early-onset persistent phenotype. Among infants with early-onset atopic dermatitis, some develop systemic allergic disease such as food allergy, allergic rhinitis, and asthma. The figure is based on the findings described by references 8–13, 15, and 16. IL, interleukin; IFN, interferon; Th1, T helper 1; Th2, T helper 2.IntroductionAtopic dermatitis (AD) was once considered an early-onset pediatric disease that usually resolves around the age of 2–3 years . Although approximately 40%–60% of infancy-onset AD cases achieve remission by 6–7 years of age, recent studies reported that AD is a lifelong disease with recurrent exacerbations [2-4]. AD is frequently accompanied by systemic allergic diseases, other inflammatory diseases, and/or psychosocial disorders such as depression, anxiety, sleep disorders, and attention deficit hyperactivity disorder [5,6]. Therefore, the burden of AD is quite high. Given the fact that 85% of all AD cases begin within the age of 5 years, this period may be critical not only for AD development but also for disease modification.Recent studies have provided new insights into the complex pathophysiology and phenotypes of AD. Moreover, based on an extended understanding of its pathogenesis, new agents are being tested in patients with moderate to severe AD. This review describes the latest knowledge about pediatric AD, the pathogenesis and phenotypes of it, and the currently available systemic therapeutics for children with moderate to severe AD that is refractory to topical treatment.Atopic march begins with atopic dermatitis“Atopic march” is the progression of allergic diseases from AD to other immunoglobulin E (IgE)-mediated diseases such as food allergy (FA), allergic rhinitis, and asthma. Over the past 20 years, the term atopic march has been widely used to describe changes in the temporal prevalence of allergic diseases reported in epidemiological studies ranging from AD and FA in infancy to allergic rhinitis and asthma in childhood . These results led to the hypothesis that AD is the first manifestation of an atopic phenotype starting in infancy and early childhood . There is epidemiological and experimental evidence supporting AD as the initiation of allergic diseases [8,9]. Several prospective birth cohorts have shown an association between early-onset AD and the development of asthma and allergic rhinitis at school age [8-10]. The risks of respiratory allergic diseases are greater in children with the early-onset persistent AD phenotype . AD children with specific IgE antibodies (extrinsic AD) by 2–4 years of age are at higher risk of the progression of atopic march to allergic rhinitis and asthma than those without (intrinsic AD) . A Canadian birth cohort study reported that a significantly increased risk of FA, asthma and allergic rhinitis was observed in 1-year-old children with AD and allergic sensitization versus those with neither condition . A defective skin barrier, an AD hallmark, has been suggested as a mechanism of atopic march [8,9].A recent systematic review and meta-analysis of 7 birth cohort studies evaluated AD prevalence across 3 to 6 time points among patients aged 3 months to 26 years and found no significant difference in AD prevalence before versus after childhood . The presence of AD symptoms varied among individuals. Multiple studies found that individuals with early-onset AD were more likely to have symptoms at older ages . The reason for the similar estimated prevalence across ages can be explained by the combination of 3 categories: active disease in both childhood and early adulthood, intermittent disease clearance periods, and later-onset disease.Two population-based birth cohort studies reported that only a small proportion (~7%) of children with AD experience the complete manifestations of atopic march . However, other studies reported that individuals with early-onset AD are more likely to be symptomatic until later in life, with approximately 17%–31% of patients who developed AD by 2 years of age had AD at all time points up to 18 years of age [15,16]. Approximately 40% of adults with AD have infancy-onset disease, 30% have chronic symptoms into adulthood (early-onset persistent phenotype), and 10% have intermittent symptoms (early-onset intermittent phenotype) . Therefore, it is important to identify high-risk infants with AD that will persist into childhood and adolescence. In addition, early intervention is required to modify atopic march (see Graphical abstract). However, to date, there have been no intervention studies aimed at modifying the atopic march in infants with moderate to severe AD.PathogenesisThe pathophysiology of AD is complex and multifactorial, caused by interactions between various factors, including epidermal barrier dysfunction, immune dysregulation, microbiome dysbiosis, and pruritus, with strong genetic susceptibility (Fig. 1) . A considerable body of evidence suggests that both epidermal barrier dysfunction and immune deviation to T helper 2 (Th2) inflammation play key roles in AD.Two hypotheses have been proposed: inside to outside and outside to inside. The first hypothesis is that abnormalities in the innate immune system cause skin inflammation, leading to barrier impairment upon antigen or irritant stimulation. Various mutations and polymorphisms of inflammatory genes have been associated with AD, such as interleukin (IL)-4 receptor α and the cluster of differentiation (CD)-14 genes, the serine protease inhibitor Kazal type 5, regulated on activation, normal T cell expressed and secreted (RANTES), IL-4, and IL-13 . Th2 lymphocyte-dominant immune dysregulation produces IL-4 and IL-13, which inhibit filaggrin (FLG) expression.The outside to inside hypothesis suggests that an impaired skin barrier is the first step in AD pathogenesis and causes immune dysregulation. FLG is an important structural protein in the stratum corneum (SC) that ensures proper epidermal differentiation and skin barrier function . FLG breakdown products produced in the cornified layer contribute to skin moisture retension, pH regulation, barrier permeability regulation, and microbial protection [19,20]. The FLG loss-of-function mutation and its effects on epidermal integrity provide strong evidence supporting outside to inside hypothesis. While there is ongoing debate regarding the sequence of events, it is evident that both epidermal barrier dysfunction and immune dysregulation significantly contribute to the pathogenesis of AD, as they intricately interact with each other.Recently, it has been suggested that barrier-initiated AD pathogenesis may induce immune dysregulation, further compromising permeability barrier function and forming a potential vicious outside-inside-outside circle in AD.Although AD is well known to be characterized by a strong Th2 immune response, it is recently recognized as a more heterogeneous disease with additional involvement of the Th22, Th17/IL-23, and Th1 cytokine pathways depending on disease subtype .The epidermal barrier consists of SC and tight junctions, and SC are composed of corneocytes and the extracellular matrix, called the brick and mortar structure. Intact skin is an important physical and immunological barrier to allergens, microbes, and chemicals. Skin barrier impairment, caused by inherited defects or acquired insults, is characterized by downregulated epidermal barrier structural proteins (including FLG, keratins, loricrin, involucrin, and cell adhesion molecules), decreased intercellular lipids and enzymes, decreased antimicrobial peptides (AMPs), increased skin pH, and reduced skin microbiome diversity with a greater abundance of Staphylococcus aureus . Most patients with AD have reduced epidermal terminal differentiation and SC ceramide levels, either primarily or secondarily by immunemediated mechanisms. A disrupted epithelium exposed to stimuli such as proteolytic allergens, bacteria, parasites, and chemicals promotes antigen penetration and triggers a variety of proteinase-activated receptors and pattern recognition receptors on barrier epithelial cells, inducing the release of epithelial-derived cytokines (alarmins) such as thymic stromal lymphopoietin (TSLP), IL-33, and IL-25. In the epithelial regulation of allergic-type 2 responses, 3 epithelial-derived cytokines are critical mediators of type 2 inflammation through the activation of dendritic cells (DCs) and type 2 innate lymphoid cells (ILC2s) [19-23]. DCs at barrier surfaces present processed allergens to naive T cells in the draining lymph nodes through major histocompatibility complex class II molecules. In the presence of IL-4, naive T cells differentiate into Th2 cells, the major cell type that skews the immune reaction to allergens by producing the cytokines IL-4, IL-5, IL-9, IL-13, and IL-31. Activated Th2 cells and ILC2s release IL-4 and IL-13, which promote IgE class switching [19-23]. In addition, IL-4 and IL-13 stimulate keratinocytes to produce TSLP. TSLP overexpression has been identified in the keratinocytes in both acute and chronic lesions of AD . TSLP activates OX40 ligand-expressing dermal DCs to induce differentiation of naive T cells to inflammatory Th2 cells. The epidermal production of TSLP is correlated with clinically observed lesions and AD severity and persistence .Cytokines and chemokines, such as IL-4, IL-5, IL-13, eotaxins, CC chemokine ligand (CCL)17, CCL18, and CCL22, are produced by Th2 cells and DCs and stimulate the infiltration of DCs, mast cells, and eosinophils into the skin. ILC2s are potent sources of IL-5 and IL-13.IL-22, an α-helical cytokine belonging to the IL-20 subfamily that is strongly upregulated in AD, is produced by the Th22 cell subset. IL-22 signals via a heterodimer of IL-22 receptor 1 and IL-10 receptor 2, which are expressed on epithelial cells in the skin (keratinocytes), lung, and gut . Increased IL-22 levels act as proinflammatory cytokines, leading to upregulation of AMPs in synergy with IL-17. IL-22 has also been suggested to induce epidermal hyperplasia by promoting keratinocyte proliferation and barrier defects by inhibiting terminal differentiation . IL-22 plays important pathogenic roles in AD initiation and development and is correlated with AD severity.Th1 and Th17 cells are suggested to play a role, especially in certain subtypes such as intrinsic, pediatric, and Asian phenotypes . However, Th2 and Th22 cells play predominant roles in all AD subtypes . Therefore, dupilumab, which blocks IL-4/IL-13 receptors, is equally effective for extrinsic and intrinsic AD as well as in pediatric and adult AD. Similar or higher Th2 and Th1 activity but much greater Th22 and Th17 immune responses are seen in the lesional skin of patients with intrinsic versus extrinsic AD .Pruritus is the most burdensome symptom in AD, leading to unremitting scratching and further damage to the epithelial barrier, and impairing quality of life of patients and their family. It is primarily a sensory perception of the skin mediated by unmyelinated C-fibers and thinly myelinated Aδ fibers originating from cell bodies in the dorsal root ganglion . It has been suggested that endogenous and exogenous pruritogens such as histamine, 5-hydroxytryptamine, proteases, substance P, various cytokines including IL-31 and TSLP, and environmental allergens can signal through specific itch pathways on nerve fiber endings [24,31]. IL-31 is a potent pruritogenic cytokine in AD. Physical damage due to chronic scratching significantly increases cutaneous TSLP levels. TSLP directly causes pruritus as a pruritogen and indirectly by inducing Th2-related cytokines that activate sensory neurons. Moreover, IL-4 enhances neuronal responsiveness to multiple pruritogens. Therefore, pruritogens, including TSLP and Th2 cytokines, are implicated in AD development and aggravation by inducing itching, scratching, and skin barrier dysfunction .Evidence is growing for an important role of the microbiome in AD pathogenesis: specifically, the abundance of S. aureus and relative reduction of commensal organisms that may play a role in regulating growth of S. aureus .SubtypesAD is a heterogeneous disease with several phenotypes and endotypes characterized by the activation of diverse cytokine signaling pathways, including Th1, Th2, Th22, and Th17 cells, depending on disease subtype. Phenotypes can be classified according to clinical features, such as age, severity, race, and therapeutic response. An endotype is a subtype of a health condition defined by distinct functional or pathobiological mechanisms such as extrinsic/intrinsic AD based on atopic status. The term endophenotype is used to connect the clinical phenotype and mechanical endotype. Defining a distinct endophenotype is a key to determining personalized therapy. Personalized targeted therapy is possible if there is a unique cytokine signature that characterizes an individual’s endotype. Studies on endophenotype based on race/ethnicity and age have been conducted. However, additional studies with a greater number of subjects are required to elucidate the characteristics of these subtypes. Here, we describe the subtypes according to age at onset, atopic status, and disease chronicity, which have shown several distinct characteristics.1. Subtypes based on age at onsetThe clinical AD phenotypes according to age at onset can be clearly defined. Generally, 4 types are classified: infantile (<2 years), early childhood (2–6 years), late childhood (6–12 years), and adolescence (12–18 years) [33,34].A European birth cohort study revealed that the prevalence of asthma and FA by 6 years of age strongly increased among children with early phenotypes (aged <2 years), especially those with persistent symptoms . Similarly, a recent Korean study of school-aged children and adolescents with AD found that comorbid FA, allergic rhinitis, and asthma as well as inhalant allergen sensitization were more prevalent in infancy-onset (<2 years of age) than childhood-onset AD (≥2 years of age) . While a significant proportion of patients with the early-onset phenotype can reach complete remission before 2 years of age, another proportion, estimated at up to 40%, continues to suffer from the disease over a long period of time , and this category of patients may be at high risk for atopic march .As the immune system changes with age, AD in different age groups may present diverse phenotypes and endotypes. Unique cytokine signatures characterizing individual pediatric endotypes may enable age-specific tailored treatment.The shape and distribution of lesions in AD vary among age groups: cheeks, scalp, and trunk in infants; extensors of limbs in younger children; flexural distribution of limbs in older children; and additional lichenified lesions on the forehead and neck in adolescents. These changes may be derived from background endotype skewing over time. Therefore, it is crucial to elucidate them to ensure proper treatment.In addition, even among children of a similar age, the underlying immunological profiles may differ according to atopic status (intrinsic vs. extrinsic), disease duration (acute vs. chronic), severity (mild vs. moderate to severe), and race. However, unlike in adults, making the distinction between extrinsic and intrinsic AD may not be clear in infants and young children because some intrinsic AD cases evolve to the extrinsic type through sensitization.Studies with peripheral blood samples suggested that infants present overexpression of regulatory T cells and a greater Th17 lineage capacity than adults [37,38]. At birth, immune responses are Th2 polarized, with low Th1/ interferon (IFN)-γ levels in healthy newborns and those with AD. The number of cutaneous lymphocyte antigen (CLA)+ Th1 cells was lower in infants and increased with age. Children (<5 years old) with moderate to severe AD showed suppressed and delayed development of skin-homing (CLA+) Th1 cells in the peripheral blood. CLA+ Th1 cell counts in AD infants were lower than those of age-matched controls and older children with AD . However, frequencies of CLA+ Th2 cells were similarly expanded across all age groups of infants, children, adolescents, and adults with AD and significantly higher than in age-matched controls . After infancy, systemic Th2 cells (CLA-Th2 cells) increased in AD patients of all ages, suggesting systemic immune activation with disease chronicity . In addition, IL-22 frequencies also increased from normal levels in infants to significantly higher levels in adolescents and adults versus their respective control subjects. Principal component analysis of the flow cytometric marker frequencies (percentages) in patients with AD by age showed 3 meaningful age clusters: infants (0–5 years), children and adolescents (6–17 years), and adults (≥18 years), suggesting unique molecular profiles of AD by age .Epidermal hyperplasia is more common in the lesional skin of children younger than 5 years of age who developed AD within 6 months of age than in adults. In addition, the nonlesional skin of infants and young children shows significant hyperplasia compared to that of adults . Epidermal TSLP expression as early as 2 months of age is associated with AD later in life . Taken together, true AD can be initiated before lesional skin appears in children with early-onset AD.A study of skin samples taken from moderate to severe AD patients of different ages (0–5, 6–11, 12–17, and ≥18 years) found common features of Th2 (Th2-related markers of IL-13, CCL17/thymus and activation-regulated chemokine, CCL18/pulmonary and activation-regulated chemokine, and IL-4R) and Th22 skewing (Th22-related markers of IL22 and S100As) . The differences in expression levels of cytokines between age groups of AD were as follows: infants showed the greatest Th17-related cytokines, whereas long-standing adults displayed Th1 skewing cytokines, including IFN-γ and C-X-C motif chemokine ligand (CXCL)9/CXCL10/ CXCL11, suggesting disease chronicity. The expression level of Th17-related genes was inversely related to the developmental age of children aged 0–11 years with or without AD, was generally higher in the skin of AD patients versus healthy controls, and presented 2 peaks, with the highest expression in infants followed by adults . Although the role of Th17 in AD has not yet been clearly elucidated, IL-17 is less important in AD than in psoriasis. Table 1 shows the characteristics of subtypes by age at onset.2. Subtypes by atopic statusAD has long been subdivided into extrinsic/atopic and intrinsic/nonatopic subtypes. The extrinsic phenotype (60%–80% of cases) is characterized by high serum total and specific IgE levels, eosinophilia, personal and familial atopic backgrounds, and a higher FLG mutation rate. In contrast, patients with intrinsic AD (20%–40%) have normal IgE levels, no other atopic background, a female predominance, a delayed disease onset, and preserved barrier function [44,45]. However, even in the same extrinsic subtype, there may be differences in the sensitized allergens as well as stage and site of AD lesion according to age. Sensitization to food allergens is common in infants and young children, whereas sensitization to inhaled allergens is more frequent in older children.Most studies in infants and young children have attempted to characterize disease phenotypes using peripheral blood analysis. The eosinophil count, eosinophil cationic protein level, and IL-5 detection rate were higher in infants with extrinsic versus intrinsic AD .Increased Th1 signals (IFN-γ, CXCL9, CXCL10, and MX-1) and more pronounced Th17/Th22 activation (IL-17A, CCL20, Elafin, and IL-22), which are linked to psoriasis, are noted in intrinsic AD. Levels of antimicrobial activity (S100A9 and S100A12), which are coregulated by IL-17/IL-22, are higher in intrinsic versus extrinsic lesions .In the skin, Th1/IFN-γ-related gene expression and levels of the Th17 chemokine CCL20 are correlated with disease severity in intrinsic AD. On the other hand, Th2 markers were positively correlated with disease severity but negatively correlated with the barrier products of loricrin, periplakin, and FLG in extrinsic AD . Intrinsic AD has inflammatory (IL-22, IL-36α/γ, IL-36RN, and CCL22) and lipid metabolism pathways that overlap with psoriasis, supporting Th17/IL-23 and IL-22 as common profiles of both conditions .Although each type has characteristic cytokine profiles, they share a similar clinical presentation in the lesional skin and a similar increase in Th2 markers; increased infiltration of T cells and DCs in the lesional and nonlesional skin of both AD (more cellular infiltrates of T cells, myeloid DCs, and Langerhans cells in intrinsic AD) and epidermal hyperplasia in the lesional skin versus nonlesional skin . Table 2 summarizes the characteristics of subtypes by atopic status.3. Subtypes by stageSkin lesions vary widely, but can be classified as acute or chronic. Acute lesions begin with erythematous papules and serous exudates with intense itching and include secondary lesions, such as excoriations and crusted erosions due to scratching. When acute lesions persist, subacute lesions such as erythematous scaly papules and plaques appear. Progressive itching and rashes result in chronic lichenified lesions characterized by prominent skin marks with hyper- or hypopigmentation.AD usually presents as multiple lesions of different stages at multiple sites. It is common to find overlapping acute and chronic lesions in the same patient. Acute lesions begin with a marked increase in AMP and a lesser increase in IL-17 levels as well as the upregulation of Th2 and Th22 cytokines. Intensification of the Th2 and Th22 cytokine axes with disease chronicity has been demonstrated along with significant increases in Th1 [48,49]. Taken together, acute inflammation in AD is driven by type 2 cytokines, while enhanced Th2 and Th22 as well as additional Th1 responses are involved in the chronic stage; changes from acute to chronic AD are quantitative rather than qualitative in terms of Th2, Th22, Th1, and Th17 responses, and additional features develop only in chronic inflammation (Table 3) [48,49].TreatmentMost patients with mild to moderate AD respond to standard topical anti-inflammatory therapies with optimized skincare. Nevertheless, it is often inadequately controlled by the avoidance of irritants or triggers (food and environment), application of emollients, and intensive topical treatments. The importance of treating children with AD comes from the fact that up to 80% of cases begin in infancy or early childhood and AD is an early presentation of the allergic march. Therefore, it is ideal to shift the treatment goal from symptom resolution to modulation of the immune response behind it. Therefore, early systemic treatment requires in young children with immune dysregulation. However, treatment options for this age group are limited due to the lack of the clinical trial data on the effectiveness and long-term safety of new agents. In addition, no clinical studies have determined whether early systemic treatment of immune dysregulation can modify the disease course in infants and young children. Here, we present a descriptive review of currently accepted new systemic therapies, Th2 cytokine receptor antagonists and Janus kinase inhibitors (JAKi), for children with moderate to severe AD. The results of new systemic agents other than those approved in pediatric AD are not discussed here.1. Th2 cytokine receptor antagonistsIL-4, IL-13, IL-5, and their respective receptors have been targets of drug development strategies to modulate the Th2 response, a core pathway in AD . IL-4 and IL-13 receptors are expressed in neurons and believed to play additional roles in the itch-scratch mechanism .1) IL-4/IL-13 antagonistsDupilumab is a human monoclonal antibody that inhibits downstream signaling of IL-4 and IL-13 by binding to IL4Rα . IL-4 and IL-13 share a heterodimeric receptor composed of IL-4Rα and IL-13Rα1, known as the type 2 receptor of IL-4 . It is approved (2022, U.S. Food and Drug Administration; 2022, Korean Ministry of Food and Drug Safety) for children aged 6 months and older with moderate to severe AD. Phase 3 studies evaluated the efficacy and safety of dupilumab for the treatment of moderate to severe AD in children and adolescents aged 6 months to 17 years and reported improvement in AD signs and symptoms, including itching, sleep loss, and quality of life (Table 4, Figs. 2 and 3) [53-55]. Adolescents were administered dupilumab (200/300 mg every 2 weeks or 300 mg every 4 weeks) for 16 weeks. The proportion of patients with 75% improvement in the eczema area and severity index score (EASI 75) from baseline was 41.5%, 38.1%, and 8.2% on 2-week and 4-week dupilumab and placebo regimens, respectively (Table 4, Fig. 2) . In a phase 2a open-label sequential cohort study with a phase 3 open-label extension, adolescents with moderate to severe AD were administered dupilumab 2 mg/kg or 4 mg/kg on a weekly basis. The percent change in EASI from baseline to week 52 for the 2 mg/kg and 4 mg/kg regimens was -85% and -84%, respectively . Almost all children reported at least one mild to moderate treatment-emergent adverse event (TEAE) with a dose-related trend; none led to interruption of treatment.In children aged 6–11 years, the administration of dupilumab with a weight-based regimen of 100/200 mg every 2 weeks (q2 wk) or 300 mg every 4 weeks (q4 wk) for 16 weeks combined with a medium-potency topical corticosteroid (TCS) improved AD; the proportions of patients who achieved an EASI 75 in q2w and q4w dupilumab regimens and placebo were 58.3%, 50.8%, and 12.3%, respectively (Table 4, Fig. 2) .Similar results were reported for younger age groups (6 months to <6 years) with moderate to severe AD. They were given 200/300 mg of dupilumab every 4 weeks for 16 weeks combined with TCS; more patients treated with dupilumab than placebo achieved an EASI 75 (53% vs. 11%) and Investigator’s Global Assessment (IGA) score 0/1 (clear/ almost clear, 28% vs. 4%) (Table 4, Fig. 2) . Itching, one of bothersome symptom, was also remarkably improved in the dupilumab group; the percentage change from baseline in worst itch Numerical Rating Scale score was -49.4% and -2.2% in the dupilumab and placebo groups, respectively. The incidence of conjunctivitis was higher in the dupilumab versus placebo group (5% vs. 0%). However, skin infections, excluding herpes virus infections, were less frequent in the dupilumab versus control group .An ongoing open-label extended phase 3 study (52 weeks) is evaluating the efficacy and long-term safety of dupilumab in patients with moderate to severe AD aged ≥6 months to <18 years participating in 3 parent studies [53,56-58]. Results of dupilumab in adolescents (≥12 to <18 years) among enrolled subjects were recently reported . Patients enrolled in the 3 parent studies and newly enrolled patients received 300 mg of dupilumab every 4 weeks regardless of body weight during the extended study period under the new protocol. At week 52, 42.7% of patients achieved an IGA score of 0/1, while 81% achieved an EASI 75 .Adverse reactions of special interest were mild to moderate and resolved with continued treatment. Clinical trials have reported an increased incidence of conjunctivitis with dupilumab versus placebo. Interestingly, it occurs more frequently in patients with AD versus other diseases such as asthma, chronic rhinosinusitis with nasal polyps, or eosinophilic esophagitis [53,59]. Dupilumab-associated conjunctivitis is less common in children versus adults . However, the exact pathophysiology of conjunctivitis remains unclear.In summary, dupilumab has a favorable safety profile, even in 6-month-old children and can be administered as a long term therapy in pediatric AD.2) IL-13 selective antagonistSelective IL-13 antagonists such as lebrikizumab and tralokinumab can manage AD and improve patients’ quality of life . The 2 agents differ in their binding epitopes and ability to block one or both IL-13 receptors; lebrikizumab does not block 13α2 receptor chains, whereas tralokinumab blocks binding of IL-13 to the IL-13Rα1 and IL-13α2 receptor chains, the decoy receptor, which may be involved in endogenous IL-13 regulation .Tralokinumab is a fully humanized antibody targeting IL-13 that blocks its binding to both IL-13Rα1 and IL-13α2 receptor chains [62,63]. It has been approved for the treatment of moderate to severe AD in adults after being studied for up to 52 weeks in phase 3 studies [64,65]. Significant improvements in AD assessment scores, pruritus, sleep interference, and quality of life were noted and maintained over time without the requirement for TCS [64,65]. The results of a phase 3 trial for tralokinumab monotherapy in adolescents (aged 12–17 years) were released . Tralokinumab (150 or 300 mg) was administered every other week; EASI 75 was above 25% by the end of week 16 and reached 44%–64% by the end of maintenance treatment at week 52 with a favorable safety profile. The proportion of patients who achieved an IGA score of 0/1 in the 150 mg and 300 mg tralokinumab and placebo groups was 21.4%, 17.5%, and 4.3%, respectively. The proportion of patients who achieved an EASI 75 in the 150 mg and 300 mg tralokinumab and placebo groups was 28.6%, 27.8%, and 6.4%, respectively (Table 4, Fig. 3). Upper respiratory tract infection was the most common TEAE during the maintenance and safety follow-up period (Table 4) .Lebrikizumab is a fully humanized anti-IL-13 antibody that specifically binds to soluble IL-13 and does not block cytokine binding to the receptor but impairs the heterodimerization of IL-4Rα and IL-13Rα1, thereby inhibiting signal transduction [62,67]. Multicenter double-blind placebo-controlled phase 3 randomized clinical trials evaluated the efficacy of lebrikizumab as monotherapy (ADvocate 1 and 2) and a combination therapy with TCS (Adhere) in the treatment of adolescents (aged ≥12 to <18 years; weight, ≥40 kg) and adults with moderate to severe AD [67,68]. At week 16, EASI 75 in the treatment group vs. placebo was 58.8% vs. 16.2%, 52.1% vs. 18.1% and 69.5% vs. 42.2% in ADvocate1, ADvocate2 and Adhere trials, respectively [67,68]. The TEAEs were mild or moderate in severity and did not lead to discontinuation of the study. The most frequently reposted TEAEs were conjunctivitis (7.4%, 7.5%, 4.9%) and headache (3.2%, 5%, 4.8%) herpes infection (3.2%, 2.8%, 3.4%) in ADvocate1, ADvocate2 and Adhere trials, respectively (Table 4, Fig. 3) [67,68]. A 52-week open label study evaluated the efficacy and safety of lebrikizumab exclusively in adolescent patients of ADvocate1, ADvocate2 and Adhere trials . Serious events leading to treatment discontinuation were infrequent, and 81.9% achieved an EASI 75 by Week 52 .In adults, the improvement in EASI scores after adjusting for placebo was comparable between dupilumab and lebrikizumab (32%–36% and 37%, respectively) and slightly lower in tralokinumab (12%–22%), which may be attributed to differences in the study designs, but because tralokinumab also blocks receptors involved in the endogenous regulation of IL-13 [61,70].3) IL-31 antagonistNemolizumab is a humanized monoclonal antibody against receptor A of IL-31, a prominent pruritogenic cytokine produced by infiltrating Th2 cells in AD, which correlates with disease severity and has been found to be excessively expressed in skin lesions in AD [31,71]. Therefore, IL-31 and its receptor are the focus of strategies to better control the itch-scratch cycle [72,73]. It has been approved for moderate to severe AD over the age of 13 years in adolescents and adults in Japan. In a double-blind, phase 3 trial, 60 mg nemolizumab was subcutaneously administered every 4 weeks with concomitant topical agents to subjects aged 13 years or older with moderate to severe pruritus unresponsive to topical agents, and adolescents aged 13–17 years accounted for approximately 5 percent of subjects . After week 16, the mean percent change in the EASI score was -45.9% with nemolizumab and -33.2% with placebo (Table 4, Fig. 3). Adverse events were generally mild to moderate; however, 1 in the nemolizumab groups discontinued treatment due to AD exacerbation . Although some patients reported exacerbation of AD as an adverse event, these patients also experienced less itching as measured by visual analog scale score .In an open-label phase 2 study of patients aged 12–17 years with moderate to severe AD, nemolizumab was administered subcutaneously as a 60-mg loading dose, followed by 30 mg every 4 weeks until 12 weeks and followed up for 8 more weeks . AD-related proinflammatory biomarkers changed more significantly in EASI responders than in EASI nonresponders .As nemolizumab appears to be a promising agent, large-scale studies are required to evaluate its long-term efficacy and safety. A phase 2 study is ongoing to evaluate the pharmacokinetics, safety, and efficacy of nemolizumab for moderate to severe AD in children aged 2–11 years (NCT04921345).2. JAK inhibitorsJanus kinase (JAK)s are a group of molecules comprising JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). Binding of different cytokines to their specific receptor subunits on different cell populations leads to the activation of a specific JAK-signal transducers and activators of transcription (STATs) pathway, and the different isoforms of JAKs are coupled to specific receptor/cytokine pairs. When a cytokine binds to its intracellular domains of type I or type II cytokine receptors, a conformational change is induced, which activates JAK-tyrosine kinases, resulting in phosphorylation of tyrosine residues in the receptor's intracellular domain [40,76]. The phosphorylation of receptor subunits allows for the recruitment of signal molecules, including latent cytoplasmic transcription factors such as STATs, phosphorylated STATs are activated, dimerized, and translocated to the nucleus to regulate target gene expression (Fig. 4). In general, all type I and II receptors rely on JAK1/JAK2 for signal transduction. Depending on the particular receptor, one or more members of the JAK family work together to mediate signal transduction. Therefore, each JAK is often involved in the downstream signaling of multiple cytokine receptors in association with other JAK family members. TYK2 can partner with both JAK1 and JAK2, whereas is a much less widely expressed JAK protein and restricted to receptors containing the common γ chain-containing receptors (Fig. 4).JAK inhibitors (JAKi) are small molecules that can be administered orally or topically and are recently introduced to treat AD. Currently, more than 90 JAKi are patented, many of which are in clinical development for various indications, such as inflammatory bowel diseases and rheumatoid arthritis [43,76]. As the binding of Th2 and Th22 cytokines to their receptors involves downstream JAK-STAT signaling, JAKi are emerging as attractive compounds for AD treatment.JAKi approved or under clinical development for AD can be classified into 3 main categories: nonselective (delgocitinib, cerdulatinib, jaktinib, CEE321), dual (baricitinib, ruxolitinib, brepocitinib, ATI-1777), and selective (upadacitinib, abrocitinib, SHR0302) .JAK inhibition exerts a broad immunopharmacological effect by blocking the signal transduction pathways of multiple cytokines. JAKi blocks numerous cytokines that are involved in many aspects of host defense, hematopoiesis, metabolism, cell growth, and cell differentiation; therefore, they can have multiple systemic effects . Serious adverse effects include infections, anemia, pulmonary embolism, malignancy risk, thromboembolic risk, and elevated serum cholesterol . Nonetheless, the hope is a second-generation JAKi with increased selectivity to reduce adverse effects and preserve efficacy. Here, we discuss only orally available JAKi that have been studied in pediatric patients (Table 4, Fig. 3).Baricitinib is a selective JAK1/JAK2 inhibitor that has been approved for the treatment of patients with moderate to severe AD aged 12 years and older. In 2 independent 16-week phase 3 trials, the participants who achieved EASI 75 reached 24.8% with baricitinib monotherapy and 36.6% with baricitinib plus TCS . The most common adverse events were nasopharyngitis and headache . No cardiovascular disease, venous thromboembolism, or serious hematological changes were detected during the 16-week treatment period. Unlike selective JAK1 inhibitors, no increase in acne incidence was noted . In a pooled safety analysis of cumulative data from 8 adult studies (n= 2,531), simple viral infection and headache were the most frequently reported TEAE, with 2 major cardiovascular events, 2 venous thrombosis events, and 1 death . One death reported after taking baricitinib for more than 12 months was due to gastrointestinal bleeding. The patient was randomized to 1 mg in the first study and then to 4 mg, which was reduced to a renally adjusted dose of 2 mg because of reduced glomerular filtration rate in the long-term extension study. With the satisfactory effect of improvement and onset of action as well as an acceptable safety profile, baricitinib has been studied in children aged 2–17 years with an inadequate response to topical treatment (Table 4) . The baricitinib 4-mg equivalent for 16 weeks achieved a significant improvement in EASI and itching versus placebo.As hematopoietic signaling of receptors depends crucially on JAK2 homodimers, JAK1 selective inhibitors are suggested as a safer option to avoid major JAKi adverse effects. Upadacitinib and abrocitinib are second-generation JAK1 inhibitors that have been studied in children. Upadacitinib is a selective JAK1 inhibitor approved for the treatment of moderate to severe AD in adults and children aged 12 years. Three phase 3 trials (Measure Up 1, Measure Up 2, and AD Up) evaluated the efficacy of upadacitinib for 16 weeks (15 or 30 mg, once daily) in the treatment of patients aged 12 years or older with moderate to severe AD [82,83]. Measure Up 1 and 2 evaluated upadacitinib as monotherapy, while AD Up examined it with TCS for all participants. The percentage of participants who achieved EASI 75 in Measure Up 1, Measure Up 2, and AD Up was satisfactory with the 15 mg dose (69.6%, 60.1%, and 65 %, respectively), and 30 mg dose (79.7%, 72.9%, and 77%, respectively); both regimens showed statistically significant improvements by week 2. Adverse reactions were mild to moderate in severity and included acne, upper respiratory tract infection, headache, oral herpes, and asymptomatic elevation of plasma creatine phosphokinase. Acne was the most common side effect in all 3 studies. Although acne was not serious and did not lead to treatment discontinuation, it was higher than that observed in previous studies of rheumatoid arthritis, which may be due to the relatively younger age of patients with AD (Table 4) [84,85]. These results demonstrate the potential of upadacitinib as a monotherapy to reduce the burden of TCS use [82,83]. In addition, the incidence of oral herpes infection was lower in upadacitinib monotherapy (3%) than in combination therapy with upadacitinib and TCS [82,83].Upadacitinib efficacy at week 16 was maintained through the 52-week follow-up studies [86,87]. EASI 75 was achieved in 82.0%, 79.1% and 50.8% of patients maintained on the 15 mg dose and in 84.9%, 84.3% and 69.0% of patients maintained on the 30 mg dose in the Measure Up 1, Measure Up 2 and AD UP studies, respectively. Both doses of upadacitinib were well tolerated, with no new critical safety issues, and a very low rate of treatment discontinuation due to adverse events [86,87]. An open-label multiple dose study in the younger age group (2 to <12 years of age) is currently in progress (NCT 03646604).Abrocitinib is a JAK1 selective inhibitor that has been approved for the treatment of moderate to severe AD in adolescents and adults. Three phase 3 trials evaluated the efficacy and safety of once-daily 100 mg or 200 mg of abrocitinib for 12 weeks; JADE Mono 1 and 2 studied abrocitinib monotherapy in adolescents and adults (adolescents were 22% and 10% of the study subjects, respectively), whereas JADE TEEN examined abrocitinib plus TCS in adolescents (Table 4, Fig. 3) [88-90].With the 100-mg dose, the percentage of participants achieved EASI 75 in JADE Mono 1 and 2 as well as JADE TEEN was 40%, 44.5%, and 68.5%, respectively and with 200 mg dose, it was 63%, 61%, and 72%, respectively. Patient-reported signs and symptoms, including sleep loss and quality of life, were substantially improved with abrocitinib monotherapy or combination therapy compared to placebo in adolescents enrolled in JADE TEEN as well as JADE Mono 1 and 2 . Commonly reported adverse events were nausea, vomiting, abdominal pain, headache, increased blood creatine phosphokinase, and acne [88-90]. Thrombocytopenia was noticed in 3 studies and resolved with continued treatment, except for 1 patient who had to withhold treatment for 8 days [88-91]. Regarding the serious side effects of JAKi, no thromboembolism or major cardiovascular events were reported. In upadacitinib studies, the elevation of liver enzymes in the Measure Up 1 and 2 groups and placebo group was 1.7% and 1.1%, respectively, which did not lead to discontinuation of treatment. An integrated safety analysis of a phase 2b study, 4 phase 3 studies and 1 long-term extension study was conducted to evaluate the long-term safety of abrocitinib in adolescents, who represented only 12.7% of all patients in the abrocitinib group . Four events of herpes zoster (0.2%; all in the abrocitinib 200 mg group) resulted in permanent discontinuation of study treatment; abrocitinib 200 mg, age ≥65 years, and severe disease at baseline were associated with higher risk of herpes zoster . Incidence rates presented as numbers of patients with events per 100 patient-years (PYs) were 2.33/100 PY and 2.65/100 PY for serious infection, 4.34/100 PY and 2.04/100 PY for herpes zoster, and 11.83/100 PY and 8.73/100 PY for herpes simplex in the 200-mg and 100-mg groups, respectively. Five venous thromboembolism events occurred (IR 0.30/100 PY) in the 200-mg group .Comparative studies were performed between selective JAKi and dupilumab in adults [93,94]. Upadacitinib 30 mg and abrocitinib 200 mg were superior to dupilumab 300 mg in terms of onset of itch improvement and EASI 75 at 16 weeks. The adverse effects of the 3 drugs were consistent with those reported in previous studies and were mild to moderate in severity. The risk of adverse events was numerically higher in the upadacitinib 30 mg and abrocitinib 200 mg groups than in the dupilumab 300 mg group; however, serious adverse events during treatment were similar across study groups [93,94]. Each drug has its own characteristics including pharmacokinetics. Therefore, this finding needs to be carefully interpreted. JAKi are administered orally every day, and their efficacy is maintained constantly, whereas dupilumab is injected subcutaneously at 4-week intervals; therefore, the concentration before administration is relatively low, and the main outcomes were measured at 4-week intervals, except for weeks 1 and 2.ConclusionAD is a heterogeneous systemic inflammatory skin disease associated with immune dysregulation, epithelial barrier dysfunction, and pruritus. Approximately 60%–80% of adults with AD develop the disease during the first 2 years, and 85% develop the disease before 5 years of age, although the rates vary between studies. Up to 40% of patients with infancy-onset AD suffer from the disease into adulthood, and some progress to the atopic march. In addition to concerns about AD chronicity, the systemic Th2 inflammatory response in moderate to severe AD, even at a young age, indicates the need for early appropriate systemic treatment. However, for this very important period in young children, we have limited options for disease intervention. Fortunately, dupilumab, an IL-4 and IL-13 antagonist, has recently been approved for use in children aged 6 months and older. Currently, it is time to focus on whether early treatment for high-risk infants and young children can modify the disease course. For this purpose, background immunologic profiles and clinical features including skin characteristics in young children with AD should be further elucidated.

PMC

ACS Omega

PMC10809706

01-08-2024

10.1021/acsomega.3c07983

Simulation Study of N

Meng Bingbing, Shi Bin, Cao Yunxing, Wang Li, Liu Shimin

N2–hydraulic compound fracturing (NHCF) is an innovative technology aimed at addressing coalbed methane development challenges in low-permeability, low-pressure coal reservoirs in China. However, limited research has been focused on the evolution of damage zones, pore pressure fields, and fluid pressure characteristics in this context. In this paper, we establish a finite element seepage equation based on the volumetric opening model and construct a finite element model for horizontal well stage fracturing. We used the physical and mechanical parameters specific to coal reservoirs in the Xinjing coal mine. Subsequently, we conducted numerical simulations of N2 fracturing (NF), hydraulic fracturing (HF), and NHCF using ANSYS. The results indicate that the initiation-fracturing pressure of NHCF is lower than that of HF but higher than NF, but the steady-fracturing pressure is higher than HF and NF. Moreover, numerical simulation shows that under the same water injection volume, the total volumetric opening formed by NHCF is about 2 times that of HF, NF is the smallest, and the damage zone and pore pressure field caused by NHCF are the largest. Finally, when comparing the casing pressure curve of NHCF by field test with the fluid pressure curve of wellbore obtained from numerical simulation, we observe a strong correlation; the steady fracturing pressure of NF is about 13 MPa, which is basically consistent with the numerical simulation, and the steady- fracturing pressure of HF after NF is about 27 MPa, which is slightly lower than the 30 MPa in numerical simulation. This is because in the numerical simulation, the reservoir parameters after NF can be inherited to the subsequent HF, which cannot be done in the field test. This study presents a novel method for numerical fluid fracturing simulation, offering a fresh perspective on the subject.

1IntroductionCoalbed methane is a clean unconventional energy source with great development prospects and significant reserves in China.1−3 Its efficient development can alleviate the country’s energy crisis and is also of great significance for achieving the dual carbon goals. However, due to the complex geological conditions of coalbeds and the prevalence of low-pressure, low-permeability reservoirs, commercializing coalbed methane is a significant challenge in China.4−6Hydraulic fracturing (HF) is a widely employed technique for enhancing reservoir permeability.7−9 N2 fracturing (NF) can elevate reservoir pressure and facilitate the rapid desorption of methane.10 there is an observed increase in fracture complexity,11,12 a reduction in rock initiation-fracturing pressure,13,14 and an expansion in the influence radius for subsequent HF.15 Consequently, N2-hydraulic compound fracturing (NHCF) represents a promising frontier in the advancement of unconventional coalbed methane.16−18 However, owing to the multitude of factors involved in NHCF and the high cost associated with field testing, the propagation behavior of fracture and seepage fields in NHCF remains unclear at present.Numerical simulation is a crucial method for illustrating the evolution of fracture and seepage fields, playing a vital role in enhancing our comprehension of the physicomechanical processes involved in fluid fracturing. It aids in predicting fracturing expansion and provides valuable guidance for the fracturing engineering design. The damage finite element method is a commonly utilized technique in fluid fracturing simulations. It relies on the coupling principle of continuous medium seepage, stress, and damage to depict the expansion and evolution of the fracturing zones. In this method, the fracturing zone is characterized by unit damage, and the direction of fracturing expansion is governed by the anisotropy of the unit material properties.19−22 Compared with the predefined fracture method23−27 and extended finite element,28−30 this method is particularly suitable for fracturing zone extent identification and prediction in large-scale HF projects in the field because no predefined extension path and no special cell technology are required. However, a current limitation of this method is that the fracture damage unit lacks the concept of fracturing opening width and does not adequately capture the influence of the fracture propagation mechanism on the width of the fracturing opening.Wang31,32 established a volumetric opening model (VOM) to couple the pore opening and fracturing propagation mechanisms, thereby compensating for the deficiencies of the damage finite element method. In this paper, we introduced the process of establishing a VOM and the finite element control equation of the VOM has been studied. On this basis, we investigate the evolution of the damage zone, pore pressure field, and injection pressure in horizontal well staged fracturing using NF, HF, and NHCF based on Xinjing coal mine, numerically characterize the impact of these three fracturing techniques on reservoir reconstruction, and validate the simulation results through field tests involving NHCF. This study introduces a novel method to numerical simulation research on NHCF.2Volume Opening Model2.1Fluid Fracturing Volumetric OpeningTake the rock representative volume element (RVE) in the fluid fracturing expansion path. It is assumed that its initial state before fracturing is homogeneous and isotropic, contains various forms of fractures, pores, and microfractures internally, which are collectively referred to as pores, and is closed under in situ stress. With fluid injection, the rock matrix skeleton undergoes elastic opening, plastic damage, and macro fracture stages, as shown in Figure 1. Accordingly, the fluid inflow of the RVE evolves gradually from the initial (a–d) four evolutionary states.Figure 1RVE evolution of fluid fracturing. (a) Initial compression state, (b) stress balanced state, (c) critical state, and (d) fracture opening state.Let σh be the in situ stress in the fracturing opening direction, Pp be the fluid pore pressure, and σI′ be the effective stress in the matrix skeleton in the same direction as σh. The first stage (Stage I) is the compressive stress relief stage, the second stage (Stage II) is the elastic opening stage, and the third stage (Stage III) is the fracture opening stage. The evolution of pore pressure and effective stress throughout the process is shown in Figure 2, where the red curve represents the evolution of effective stress σI′ and the blue curve represents the evolution of pore pressure Pp.Figure 2Evolution of pore pressure and effective stress with state (a–d) of fluid fracturing.From the red curve in Figure 2, it can be seen that the RVE is in compression in state (a) and the effective stress of the skeleton σI′ is −σh. As the fluid is injected, the pores are slowly filled to state (b) and the effective stress σI′ rises to zero. From state (b–c) process, the effective stress continues to increase, reaches a peak, and then decreases slightly, and the matrix skeleton begins to undergo cohesive fracturing. By the state (d), the matrix macroscopically fractures and the effective stress drops to zero. Since the pore pressure, effective stress, and in situ stress are in equilibrium, σI′ + σh = αPp, it is known that the evolution of pore pressure is consistent with the effective stress.A total of three volumetric openings occur as the effective stress evolution. Stage I and Stage II involve only pore opening, so only the pore volumetric opening is produced. Stage III produces macroscopic fracturing of the matrix, but also accompanied by pore opening, so both the fracture volumetric opening and the pores volumetric opening are produced, and the total volumetric opening is recorded as1where Vpe is defined as pore volumetric opening per unit bulk volume, Vpd is defined as trunk fracture volumetric opening per unit bulk volume, and Vp is defined as the total volumetric opening per unit bulk volume.The incremental volumetric openings are obtained as2where Cm is compressibility of rock matrix; = (1 – α0)f(D), α0 is initial Biot coefficient, where D, representing the damage due to cohesive breakdown of rock skeleton,33 which is defined as3in which εs is a finial strain for a brittle fracture, εb is a transition strain from microcracks evolution to macro fractures propagation, m is the brittle index, kb is the ratio of stress drop at εb, representing the intensity of stress drop, and Δσbr is the stress drop from the start of a macro crack propagation to the residual stress.f(D) is the evolution function of the damage in the fluid fracturing process, f(D) = 1 – D)1/n, where n is the evolution index, which represents the coupling of the fluid flow mechanism with the VOM, the specific coupling steps are described in the literature and will not be reviewed in this paper.312.2Finite Element Governing EquationWithout considering the fluid generation rate and the compressibility of injection fluid, incremental rate of volumetric opening dVp/dt equals to that of fluid content; thus, the mass transfer equation is4where K is the permeability matrix, μ is fluid viscosity, ρw is mass density of injection fluid, and dζ is volume increment of rock fluid content per unit volume, consistsof volumetric opening and fluid compressibility. dζ = dVp + ϕCfPp, where Cf is fluid compressibility coefficient and ϕ is permeability. Combined with eq 2 expressed in terms of volumetric opening as5Equation 5 is written into the conventional form as6where C = Cm (1 – ϕ0 – ), the first term on the right side of eq 6 represents the amount of fluid change caused by the rate of fluid pressure change, the second term represents the rate of fluid content change due to matrix skeleton stress change and fracture tension, which is a coupling term from the stress field.The conventional FEM equation for transient analysis of fluid pressure is7Combining with the FEM equation in solid domain, the weakly coupling equations are written as8in which the first equation is established in the solid domain, a is unknown vectors of nodal displacements, Ks is global stiffness matrix, Fs is the load vector of body forces and surface forces, and Ff is the body load vector from fluid pressure, K̅ is the equivalent permeability vector, and Q̅ is the equivalent load vector.2.3Iterative AlgorithmThe fluid fracturing finite element flow-solid weak coupling process is implemented by using the load transfer method. The fluid pressure is output from the flow field calculation, which is transferred to the stress field calculation as a volumetric force load, and the stress field output, which is used to calculate skeleton fracturing, volumetric opening, and permeability. The updated permeability is input as a parameter, and the volumetric opening is input to a new round of flow field calculations as a transient fluid volume increment in the fluid field. In this cycle, the fracture propagates forward by extension driven by a constant flow rate continuous loading. The whole flow-solid coupling process is display in Figure 3.Figure 3Flowchart of fluid–street interaction analysis of VOM.3Numerical Simulation of NF, HF, and NHCF3.1Geological Background of Xinjing Coal MineA horizontal well staged fracturing model was developed for the no. 3 coal seam at the Xinjing coal mine in the Yangquan mining area. The Xinjing coal mine covers an area of 64.75 km2. The Taiyuan Formation and Shanxi Formation are the most significant coal-bearing strata within the mining area, comprising a total of 16 coal seams. The primary coal-bearing layers are the third, eighth, ninth, and 15th seams. The third coal seam has a depth ranging from 300 to 700 m and a thickness ranging from 0.755 to 4.31 m, with an average thickness of 2.33 m. It is characterized as having poor quality coal to sub-bituminous coal.Due to structural influences, the coal seams in this mining area have a rock strength coefficient of approximately 0.3. The well testing results indicate that the reservoir pressure in the exploration well ranges from 1.19 to 1.6 MPa, with an average of 1.4 MPa, and the reservoir pressure gradient is approximately 0.23 MPa per hectometer, averaging at 0.25 MPa per hectometer. The permeability of the reservoir varies from 0.36 to 0.43 mD, classifying it as a low-pressure, low-permeability reservoir. Therefore, it is planned to employ a NHCF for production enhancement and reservoir improvement, and in order to improve the fracturing effect and range, horizontal well staged fracturing is adopted.3.2FEM Model EstablishedThe finite element model comprises a total length of 402 m, a width of 200 m, and a height of 10 m, as depicted in Figure 4a. The roof has a thickness of 3 m, the coal seam measures 2.5 m in thickness, and the floor has a thickness of 4.5 m, as shown in Figure 4b. The model includes three perforation intervals labeled as sections A, B, and C; the interval between each section is 40 m. Within each section, there are four perforation clusters, and the interval between these perforation clusters is 30 m. The horizontal well has a diameter of 0.1246 m, and the perforation clusters are distributed along the axial direction of the horizontal wellbore in a 60-degree spiral pattern, the perforation depth is 0.8 m, and each cluster has a length of 1 m, the horizontal well and perforation clusters in section B, as shown in Figure 4c. The model is divided into 52,900 units; the SOLID70 elements are used for the seepage field units, and SOLID185 elements are used for the stress field units. The values for each property parameter of the seams are presented in Table 1.Figure 4Geometric model of horizontal well fracturing in a coal reservoir. (a) overall diagram of the FEM model, (b) thickness of coal, roof, and floor, (c) partial enlarged view of horizontal well and perforation clusters.Table 1Basic Parameters of Coal, Roof, and Floorrock stratumrock propertythickness/mE0/GPaΝσt/MPak0/mDϕ0/%α0roofsandy mudstone3.05.610.253.510.00011.00.6coalcoal2.51.520.350.270.434.80.6floorargillaceous sandstone4.520.250.204.440.00011.00.6The in situ stress state imposed by this finite element model is as follows: the maximum principal stress σH is 17.3 MPa, minimum principal stress σh is 12.5 MPa, and vertical stress is 15.7 MPa. Stress field boundary conditions are as follows: the outer boundary of the model and the inner wall of the horizontal well had zero displacement constraints. The INISTATE command is applied to initialize the model for static calculations, allowing for the determination of the in situ stress field. Seepage field boundary conditions are defined as follows: the outer boundaries of the model are configured with infinite flow field pressure conditions. The inner wall of the entire length of the horizontal well, except for the location of the perforation cluster, is set to an isolated flow boundary condition with zero flow velocity. In the region where the perforation cluster is located, the fluid is subjected to a flow velocity of q on the inner surface. The flow rate q is9where Qm is the fracturing fluid flow rate in the horizontal well, d is the diameter of the inner wall of the horizontal well, 0.1246 m, and h is the distribution width of a single perforation clusters, 1 m.3.3Numerical SchemeTo simplify the calculation process, NHCF simulations were conducted only in the middle perforation segment of the aforementioned finite element model, which is Section B. NHCF were performed on the same finite element model based on the ANSYS, with the fracturing area being section B. The fracturing sequence involves NF first, retaining the model parameters after the NF, and then proceeding with HF. This approach is used to achieve NHCF in numerical simulations. The fracturing programs are shown in Table.2. In order to compare the effectiveness of single-fluid fracturing, numerical simulations of both NF and HF were conducted separately using parameters consistent with NHCF.Table 2Fracturing Parameters for the Three Fracturing Methodsfracturing methodsfluidviscosity/pa·scompressibility coefficientinjection flowinjection time/minnoteNHCFN22.3 × 10–50.9973300 N m3/min100NF is performed first, followed by HF water0.001012 m3/min50 3.4Determination of Parameters in the VOMThe VOM is a constitutive relationship that describes the evolution of the volume opening in rock reservoirs represented by the RVE under the influence of fluid forces. Determining its parameters involves considering the mechanical characteristics of fluid fracturing, specifically how the fluid flow rate and viscosity affect the cohesion fracture of the rock. Detournay34−36 identified four limiting fracture propagation regimes by examining fluid properties, solid permeability characteristics, and the toughness of the fracture, and also provided corresponding analytical solutions. The four limiting propagation regime are storage-viscosity regime (M regime), leak-off-viscosity regime (M̃ regime), storage-toughness regime (K regime) and leak-off-toughness (K̃ regime). These four regimes describe the primary controlling factors for fracture propagation under different conditions and are essential for understanding and modeling fracture behavior in various engineering applications.The essence of the VOM is to take the width of a fracture under a specific fracture propagation regime as a known condition, fit the evolution pattern of VPd(t) and W(0,t), and thereby obtain a reasonable parameters group {εb,εs,m,n} for the evolution of VOM.Based on the geological parameters, fluid flow rates, viscosity, and injection time of the Xinjing coal mine, it has been determined that both NF and HF conform to the M regime.The parameter group evolution of volumetric opening for HF and NF has been determined through curve fitting; as shown in Table 3, εt0 stands for ultimate uniaxial tensile strain. The curve fittings of W(0,t) and VPd(t) by NF and HF can be observed in Figure 5.Table 3Parameter Group of NF and HF for VOMfluid fracturingεbεsmnNF0.565εt00.11εt01.951.092HF0.56εt00.11εt01.61.18Figure 5Curve fitting of W(0,t) and VPd(t) by NF and HF.4Results and DiscussionNumerical simulations were executed following the aforementioned fracturing procedures, and various parameters such as the pore pressure field, damage zone, horizontal well pressure, and total volumetric opening induced by the three fracturing methods were monitored. Data and graphs were extracted for analysis and comparison.4.1Evolution of Laws of Damage Zone and Pore Pressure FieldFigure 6a–c represent the evolving cloud images of the damage zone and the pore pressure field generated by NF, HF, and NHCF. From Figure 6, it can be observed that the evolution pattern of the pore pressure field and the damage zone created by the three fracturing methods is generally consistent, with the pore pressure field being larger than the damage zone. This occurs because the pressure at the leading edge of the pore pressure field is lower than the fracture pressure of the coal seam, which results in an increase in pore pressure without causing damage to the coal seam.Figure 6Evolution laws by simulation for NF, HF, and NHCF.Among these methods, it is worth noting that the pore pressure field and damage zone generated by NF have the most limited impact area. The pore pressure field is primarily confined to an elliptical shape around each perforation cluster, with relatively low intercluster communication. Additionally, evolution of the damage zone is predominantly localized around the perforation clusters. This phenomenon is closely linked to the physical properties of the coal reservoir and the rate of N2 injection. It is noteworthy that the extent of the damage zone tends to increase with a higher N2 injection rate. HF, in contrast to NF, has a significantly broader influence range, characterized by a higher degree of communication between perforation clusters. The overall evolution pattern appears elliptical, and the damage zone expands to reach the model boundary when the injection time reaches 50 min. NHCF stands out with the most extensive reservoir modification impact. It exhibits an overall walnut-shaped pore pressure field and damage zone. Interestingly, its evolution is more pronounced toward sections A and C of the model compared to HF.4.2Evolution of Horizontal Well PressureFigure 7 displays a curve depicting the monitored pressure in units around the wellbore. The fluid pressure curve can be categorized into two stages: the initial phase of rapid pressure increase referred to as the initiation-fracturing stage, followed by the subsequent stage marked by pressure fluctuations termed the steady-fracturing stage.Figure 7Fluid pressure on the borehole wall.As depicted in Figure 7, the initiation-fracturing stage of NF has the longest duration, approximately 40 min, with the fluid pressure reaching the steady-fracturing stage at a lower level, around 12.5 MPa. In contrast, HF has the shortest initiation-fracturing stage, lasting about 10 min, and the fluid pressure required to reach the steady-fracturing stage is higher than that of NF, approximately 27.2 MPa. For NHCF, the initiation-fracturing stage has a duration of about 16 min, but the fluid pressure needed to transition to the steady-fracturing stage is the highest among the methods, approximately 30 MPa.Compared to water, N2 has a lower viscosity and higher compressibility, making it highly penetrative. N2 can easily access micropore tips, generating a substantial stress intensity factor and reducing the initiation-fracturing pressure in coal. However, N2 struggles to form a fluid capable of driving stable fracture expansion at the micropore scale, resulting in less damage to the matrix structure. Consequently, it exhibits a lower steady-fracturing pressure. Water, as a noncompressible fluid, efficiently propels the primary fracture forward, leading to rapid fluid pressure growth. Therefore, the steady-state pressure in HF is greater than that in NF, and the evolution range of the pore pressure field and damage zone is much larger than that in NF.NHCF combines the advantages of both NF and HF. Initially, N2 injection enhances micropores, increasing volumetric opening and effectively damaging the coal seam matrix. As a result, NHCF boasts a lower initiation pressure than HF. However, due to water’s incompressibility, the pressure growth rate surpasses that of NF. The damage zone and pore pressure field evolution range are greater than in both HF and NF. Nonetheless, the frictional resistance faced by fluid flow in NHCF is higher than in HF, contributing to a greater steady-fracturing pressure.4.3Evolution of Total Volumetric OpeningThe evolution curve in Figure 8 displays the total volumetric openings of all units within the model. It is evident that the growth rate of volumetric opening induced by NF is relatively slow, whereas HF results in a significantly larger increase compared to NF. The growth rate of volumetric opening during the early stages of NHCF falls between NF and HF. However, during the later stages, it surpasses both NF and HF. This difference can be attributed to the initial NF phase, which increases the volume of pore-fracturing, in the early stages, the hydraulic force predominantly follows the fractures created by N2. Once HF extends beyond the region affected by N2-induced damage, hydraulic energy dissipates during fracture expansion. As a result, the growth rate of volumetric opening in the subsequent phases is much higher than that observed in HF and NF.Figure 8Total volumetric opening of NF, HF, and NHCF.Additionally, as depicted in Figure 8, it becomes evident that under the same volumetric opening (568 m3), HF necessitates 50 min of water injection, whereas NHCF only requires 10 min of water injection, with the simultaneous water injection rate being identical. The total volumetric opening caused by NHCF is about 2 times that of HF under the same water injection amount. This suggests that NHCF has the potential to conserve water resources, reduce the volume of HF operations, and consequently reduce the introduction of water-based fracturing fluids into the coal reservoir. This can contribute to a reduction in the pollution of water resources.4.4Verification of Numerical Simulation ResultsTo confirm the reliability of this numerical simulation, two staged NHCF casing pressure and flow curves from horizontal well staged fracturing by field test at the Xinjing coal mine were extracted, as illustrated in Figure 9.Figure 9Field curve of NHCF in the Xinjing coal mine.The N2 flow rate during field fracturing is approximately 300 N m3/min, while the water flow rate is 12 m3/min, which aligns with the fracturing flow rates used in the numerical simulation. The growth rate of the casing pressure curve for NF is notably lower than that of HF, with the stable casing pressure ranging from around 13–15 MPa, closely resembling the numerical simulation results. For HF conducted after NF, the stable casing pressure is approximately 23–27 MPa, which is more in line with the steady-state pressure of HF (28 MPa) from the numerical simulation and slightly lower than the steady-state pressure of 30 MPa observed in the NHCF numerical simulation. This observation highlights an important distinction between numerical simulation and field implementation. In numerical simulation, the model can accurately inherit the material properties after NF and apply them to the subsequent FEM model for HF. However, in practical NHCF field operations, there may be short gaps between the transition from NF to HF. During this transition, the elastic openings created by NF at the micropore scale slowly close and the inflow of water into these micropores may be reduced compared to what is represented in the numerical simulation. This reduction can limit the influence range of subsequent HF and decrease the frictional resistance, resulting in a lower steady-state pressure compared with the numerical simulation results. Nonetheless, the overall trend remains largely consistent. This underscores the reliability of the chosen flow loading method, VOM, and parameter selection, offering valuable insights into the numerical simulation of fluid fracturing.5ConclusionsIn this study, the finite element control equations were established based on the VOM. The geological overview of the Xinjing coal mine was analyzed, and numerical simulations for the horizontal well staged NHCF were conducted. To investigate the superiority of NHCF compared to that of NF and HF, simulations for both NF and HF were also carried out. Finally, the measured NHCF casing pressure curve was compared with that of numerical simulations. The following conclusions were drawn.As obtained from simulations, in comparison with NF and HF, NHCF exhibits a higher initiation-fracturing pressure than NF but lower than HF. Additionally, the steady-state fracturing pressure is also higher than that of HF.The total volumetric opening, pore pressure field, and damage zone of NHCF is much larger than HF and NF from simulation. At the same modified volume, NHCF can play a role in reducing water usage, thus preventing water damage to the coal matrix.Under the same fluid parameters, a comparison between the simulated wellbore pressure curve and the field-measured casing pressure reveals a strong alignment, demonstrating the practicality of the numerical simulation method.

PMC

Translational Oncology

37329828

PMC10366638

6-16-2023

10.1016/j.tranon.2023.101715

Exosomal miRNA-223-3p derived from tumor associated macrophages promotes pulmonary metastasis of breast cancer 4T1 cells

Wang Ziyuan, Zhang Chen, Guo Jian, Wang Wei, Si Qin, Chen Chong, Luo Yunping, Duan Zhaojun

Highlights•Exosomal miRNAs including miR-223-3p from tumor associated macrophages can be delivered into 4T1 cells.•miR-223-3p promotes pulmonary metastasis of 4T1 cells.•Cbx5 is a novel target of miR-223-3p. Research about the effect of exosomes derived from tumor associated macrophages (TAM-exos) in the distant organ metastasis of breast cancer is limited. In this study, we found that TAM-exos could promote the migration of 4T1 cells. Through comparing the expression of microRNAs in 4T1 cells, TAM-exos, and exosomes from bone marrow derived macrophages (BMDM-exos) by sequencing, miR-223-3p and miR-379-5p were screened out as two noteworthy differentially expressed microRNAs. Furthermore, miR-223-3p was confirmed to be the reason for the improved migration and metastasis of 4T1 cells. The expression of miR-223-3p was also increased in 4T1 cells isolated from the lung of tumor-bearing mice. Cbx5, which has been reported to be closely related with metastasis of breast cancer, was identified to be the target of miR-223-3p. Based on the information of breast cancer patients from online databases, miR-223-3p had a negative correlation with the overall survival rate of breast cancer patients within a three-year follow-up, while Cbx5 showed an opposite relationship. Taken together, miR-223-3p in TAM-exos can be delivered into 4T1 cells and exosomal miR-223-3p promotes pulmonary metastasis of 4T1 cells by targeting Cbx5.

IntroductionAccording to the Global Cancer Statistics 2020, breast cancer ranks first for its highest incidence and mortality in female malignancies . Estimated new cases of breast cancer in the United States in 2022 are projected to account for nearly one-third of all new cancer diagnoses . Distant organ metastases are responsible for the incurability with current therapies and have a significant impact on prognosis . The organ tropism of metastatic breast cancer includes bone, lung, liver and brain in sequence according to the occurrence rate in metastatic cases . Pulmonary metastasis has drawn much attention as it is associated with both a relative high morbidity and mortality rate. Investigation of the molecular mechanisms behind the metastasis can offer some new targets and strategies in the future therapies. The process of cancer spread is multi-step and inefficient. Only when the tumor cells have escaped from the primary lesion, succeeded in the intravasation, survived in the circulation, adapted to the new microenvironment after extravasation, can they colonize in the distant organs and form metastatic lesions. This complicated process can't be completed without supports from other cells, such as macrophages.Macrophages is the major component of tumor infiltrating immune cells, which accounts for about 30-50% of the total. It has been summarized that through expressing cytokines, chemokines, enzymes, exosomes et al, tumor associated macrophages (TAMs) could promote invasiveness and metastasis of tumor cells both in the primary lesions and in the pre-metastatic niches . Exosome is a type of membranous vesicle which could be produced by various kinds of cells. It communicates between cells and delivers messages by encapsulating proteins, lipids and nucleic acids. It is reported that integrins α6β4 and α6β1 on tumor cells exosomes determine lung metastasis while αvβ5 is related to liver metastasis . Exosomes from macrophages have also been found to help cancer cells to spread . Yue et al demonstrated that when the gene progranulin in macrophages is knocked out, the ability of macrophages derived exosomes to accelerate invasion and migration is impaired . However, Xu et al discovered that exosomes from THP-1 induced macrophages could significantly inhibit proliferation and metastasis of breast cancer cells . Treating tumor bearing mice with exosomes from M1-polarized macrophages obviously reduced the tumor burden . These seemingly contradictory results suggest that the contents in the exosomes is of great importance. microRNAs are common cargoes loaded by exosomes that can be transferred into another cell. As reported by Zhang et al, PTEN-targeting miRNAs derived from exosomes of astrocytes initiate brain metastasis outgrowth after being transferred into breast cancer cells . miR-16 and miR-148a contained in the cancer-associated fibroblasts exosomes function as inhibitors of tumor cells migration and proliferation . Our previous data also proved that tumor exosomal miR-183 induced IL-1β, IL-6 and TNF-α secretion from macrophages . Since most of the current research focuses on exosomes secreted by cancer cells, the relationship between exosomes produced by TAMs, especially miRNAs contained within them, and metastasis of breast cancers remains largely unclear.In this study, we aim to figure out the role of exosomal miRNAs derived from TAMs in the lung metastasis of breast cancer.Materials and MethodsStimulation of TAMs and cell cultureFor the induction of bone marrow derived macrophages (BMDMs), primary cells were isolated from tibias and femurs of Balb/c mice and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 30 ng/ml recombinant murine macrophage colony-stimulating factor (M-CSF). Replacing with fresh medium containing M-CSF of same concentration every third day, BDMDs can be obtained at day 6. If the BMDMs were further cultured with 4T1-conditioned medium for 2 days, TAMs were generated in vitro (Fig. 1A). The murine breast cancer cell line 4T1 was a kindly gift from Prof. R. A. Reisfeld (The Scripps Institute, La Jolla, CA). 4T1 cells and its derivatives, whose construction will be introduced in the following parts, were cultured in RPMI-1640 medium with 10% FBS. Human-derived breast cancer cell line MDA-MB-231 and human acute monocytic leukemia cell THP-1 were purchased from American Type Culture Collection (ATCC) and cultured as guidelines. THP-1 cells were stimulated into macrophages with a final concentration of PMA at 320nM 6h. Then the culture supernatant of MDA-MB-231 was added to induce tumor associated macrophages.Fig. 1TAMs derived exosomes promote tumor cells migration. (A) Preparation of TAMs derived exosomes. (B) TEM images of exosomes. (C) exosomes detected by NTA. (D) exosomes identified by western blot. (E) transwell assays of 4T1 cells co-cultured with exosomes from BMDM (BMDM-exos) or TAMs (TAM-exos). (F) statistics of migrated cells in transwell assays. (G) Wound healing assays of 4T1 cells co-cultured with BMDM-exos or TAM-exos. (H) statistics of wound closure in wound healing assays.Fig 1Isolation and characterization of exosomesSupernatants of around 1 × 108 BMDMs or TAMs cultured for three days were collected. Centrifuging at 1000 g for 10 mins to remove cells and cell debris. Then the supernatants were filtered through 0.22 µm filter (SLGLP033RB, Millipore) to remove larger vesicles, following by ultracentrifuging at 100,000 g for 4 h at 4°C (optima L-100XP, 70Ti, BECKMAN). The pellets were supposed to be exosomes and dissolved in PBS for identification.Samples at a concentration of 3 × 107 particles/ml were used for nanoparticle tracking analysis (NTA) (Particle Metrix, Meerbusch, Germany). The data was analyzed by ZetaView 8.04.02 SP2.For transmission electron microscopy (TEM) detection, drop 50 µl exosomes suspension onto a 200-mesh copper grid and incubate at room temperature for 5mins. 1% phosphotungstic acid was used for negative staining. Wash the copper grids with distilled water droplets for twice. When the copper grid dries, it can be examined under a TEM-1400plus transmission electron microscope at 80 KV.Alix and TSG101 are typical biomarkers often used to identify exosomes [14,15] Their levels in our extracts were detected by western blot. GAPDH was used as a negative control. Antibody against Alix (ab117600, abcam,1:1000 dilution), TSG101 (ab30871, abcam, 1:1000 dilution), GAPDH (ab36233, Multi Sciences, 1:2000 dilution) were used. The final images were recorded by chemiluminescence image analysis system (5200, Tanon).Construction of 4T1miR-223-3p and 4T1miR-379-5pThe precursor sequence of miR-223 or miR-379 was respectively inserted into lentivirus backbone plasmid LV10N (U6/mcherry&Puro) provided by Shanghai GenePharma Co., Ltd. 4T1 cells were infected by packaged lentivirus with 8 µg/ml polybrene (107689, sigma). 48 hours after infection, 2 mg/ml puromycin was used to screen for the cells with a stable expression of corresponding microRNA.Animal experimentsFemale Balb/c mice were purchased from SiPeiFu (Beijing) Biotechnology Co., Ltd. 2 × 105 breast cancer cells (4T1, 4T1miR-223-3p or 4T1miR-379-5p) resuspended in 100 µl PBS were inoculated into the fourth mammary pad of each mouse. The weight of mouse and volume of tumor were recorded every three days since day6. All the mice were sacrificed at day26. Tumor and lung of each mouse were obtained for further analysis. These experiments were approved by the Animal Ethics Committee of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.miRNA sequencingRNAs in exosomes derived from TAMs or BMDMs were extracted by miRNeasy Mini Kit (217004, Qiagen). For each group, three repetitive samples were prepared and sent to Guangzhou Ribobio Co., Ltd. The miRNA sequencing was conducted by Hiseq 2500 system, and the data was analyzed by Agilent 2200. The miRNA sequencing data has been submitted and can be achieved from BioProject (ID:PRJNA807949. Website: real-time PCRTaqMan MicroRNA Reverse Transcription Kit (4366596, ThermoFisher) was used for reverse transcription of microRNA. TaqMan Fast Advanced Mix (4444556, ThermoFisher) was used for quantitative real-time PCR (qPCR). TaqMan MicroRNA Assays for miR-223-3p/miR-379-5p/U6 (ID: 002295; 001138; 001973) (4427975, ThermoFisher) included the primers and probes needed for reverse transcription and qPCR.For the detection of miRNA target genes, TransScript® Uni All-in-One First-Strand cDNA Sysnthesis SuperMix (AU341-02, TransGen) and PerfectStart Green qPCR SuperMix (AQ601-04, TransGen) were used. qPCR primers for each target gene in mice were listed as follows: Rasa1-F: TGTGGTGATTACTACATTGGTGG; Rasa1-R: CGCCTTCTATCTTCTACTGGCTC. Fat1-F: CTACGGAGGAACGTGCATGG; Fat1-R: ATCTTTGCAGTACGGACTAAGC. Mef2c-F: ATCCCGATGCAGACGATTCAG; Mef2c-R: AACAGCACACAATCTTTGCCT. Ankrd17-F: TTGGACCAGGATGATTTGGAGA; Ankrd17-R: GCCGTCGATAACTTGCCTATTC. Rbpj-F: AGTTGCACAGAAGTCTTACGG; Rbpj-R: CCTATTCCAATAAACGCACAGGG. Kpna3-F: TCGGGAACTTCTGCACAGAC; Kpna3-R: ACACCGCTTGTTCACAAACATT. Pds5b-F: GGACTGCTCAAGCTATTGAACC; Pds5b-R: ACCTGGAGGCGTTCTTCATTAT. Mbnl1-F: CAAATGCAGTTAGCCAATGCC; Mbnl1-R: GTAAGGGTTAAAGGCTGCTGAT. Ptbp2-F: ATGGACGGAATTGTCACTGAGG; Ptbp2-R: TGCCACTCATATTAGAGTTGGGG. Cbx5-F: GACAGGCGCATGGTTAAGG; Cbx5-R: CCTGGGCTTATTGTTTTCACCC. Cbfb-F: CCGCGAGTGCGAGATTAAGTA; Cbfb-R: GTTCTGGAAGCGTGTCTGG.Transwell and wound healing assaysThe concentration of 4T1 cells was adjusted to 2 × 106 cells/ml and 100 µl cell suspension was seeded into transwell inserts (3422, Corning). The outer well was filled with complete medium with or without exosomes. After incubation for 12 h, the permeable supports were fixed and stained with 0.1% crystal violet (G1063, Solarbio). Take pictures under a microscope and the analysis was performed with Image J software.For wound healing assays, 2 × 105 4T1 cells were seeded into 24-well plate. When the confluence of cells was close to 90%, a straight scratch wound was created with a sterile pipette tip. Then the width of the scratch was measured at 0 h, 12 h and 24 h.Hematoxylin and eosin stainingParaffin-embedded sections were deparaffinized by immersing the slides into ethanol with gradient concentration. After rehydration, the slides were stained with Harris hematoxylin (BSBA-4097, ZSGB-BIO), differentiated with acid alcohol, blued with ammonia water, and counterstained with eosin (ZLI-9613, ZSGB-BIO) in sequence. Apply a drop of neutral balsam (10004160, Sinopharm) to each slide and cover it with a coverslip. The slides can be observed using a microscope.Dual-luciferase reporter-system assaysThe sequence predicted to be bound by miR-223-3p in the 3’UTR of Cbx5 was cloned into psiCHECK-2 vector (C8021, Promega), locating at the 3’UTR of synthetic renilla luciferase gene. A mutant vector of which the miR-223-3p binding base pairs were mutated was constructed at the same time. After co-transfection of the Cbx5-WT-psiCHECK-2 vector or the Cbx5-mut-psiCHECK-2 vector with miR-223-3p, the firefly luciferase and renilla luciferase activities were detected using a dual-luciferase reporter assay system (E1910, Promega).Statistical analysisData for survival analysis were obtained from the KMplotter database ( Statistical analysis and plots were drawn with Hiplot ( Correlation analysis of miRNA and target genes was performed using the ENCORI Pan-Cancer Analysis Platform. The rest of statistical analyses were performed using GraphPad Prism 8 software. Quantitative data were analyzed using a two-tailed, unpaired Student's t-test (two groups). The growth curves of tumor between two groups were compared using two-way ANOVA. Data are shown as mean ± sem. p < 0.05 was used as a criterion for statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001)Resultsexosomes derived from TAMs (TAM-exos) promote tumor cells migrationAccording to our previous data, BMDMs stimulated with 4T1 cells conditional medium shared similar characteristics with TAMs isolated from 4T1 tumor-bearing mice . Exosomes were collected from supernatant of conditional medium induced TAMs in vitro as specified in Fig. 1A. And the extracts were identified by TEM (Fig. 1B), NTA (Fig. 1C) and western blot (Fig. 1D). Then we added the exosomes into 4T1 culture medium. The exosomes derived from BMDMs (BMDM-exos) were used as a control. Transwell assay proved that TAM-exos significantly enhanced the migration ability of breast cancer cells (Fig. 1E-F). The wound healing assay showed the same trend (Fig. 1G-H). Similar tests were repeated in human-derived breast cancer cell line MDA-MB-231. Human acute monocytic leukemia cell line THP-1 was induced into tumor associated macrophage and its exosomes (TAM-exos) were collected. Exosomes from original THP-1 (THP-1-exos) were used as a control. After stimulation with the TAM-exos, the migration and invasion abilities of MDA-MB-231 were also significantly elevated (Fig. S1B and S1C).miR-223-3p and miR-379-5p are differentially expressed microRNAs in TAM-exosIn order to figure out the key microRNAs in the TAM-exos which contributed to the migration of cancer cells, next-generation sequencing was performed. The expression levels of key microRNAs that delivered from TAM-exos to cancer cells were supposed to be higher in TAM-exos than in BMDM-exos. In addition, their constitutive expression in the 4T1 cells should be low, so that the transmitted microRNAs could cause obvious phenotypic changes in recipient cells. Thus, the eligible microRNAs were clustered and listed in Fig. 2A. Then we checked the levels of these microRNAs in 4T1 cells before or after co-culturing with TAMs by qPCR. As shown in Fig. 2B, six microRNAs (miR-210-3p, miR-223-3p, miR-412-5p, miR-652-3p, miR-16-5p, miR-379-5p) increased significantly after the co-culture for 72h and the fold change is greater than two. In the meanwhile, the qPCR results confirmed that the expression levels of the six microRNAs were hundreds of times higher in TAMs derived exosomes than that of 4T1 cells (Fig. 2C). Further comparison between TAM-exos and BMDM-exos narrowed the candidates down to three microRNAs, that is miR-223-3p, miR-412-5p, miR-379-5p (Fig. 2D). Analysis based on the Oncomir database ( showed that the expression level of miR-412-5 was very low, while miR-223-3p and miR-379-5p were highly expressed in breast cancer (Fig. S1E). In addition, an overall survival analysis of miR-412-5p demonstrated that people with high and low expression of miR-412-5p in breast cancer did not have a bias in the overall survival time (Fig. S1F). Since sequences of mature miR-223-3p and miR-379-5p in mice were highly homologous to those in human (Fig. 2E), these two microRNAs were selected for further analysis. When TAM-exos were added into the cultural supernatant, the levels of both miR-223-3p and miR-379-5p (Fig. 3A and B) were significantly elevated in 4T1 cells, comparing to BMDM-exos. This result was confirmed in MDA-MB-231. After stimulating with the THP-1 induced TAM-derived exosomes (TAM-exos), the expression of miR-223-3p in MDA-MB- 231 was significantly higher than that with THP-1-derived exosomes (THP1-exos) (Fig. S1A).Fig. 2Screening for differentially expressed miRNA in TAMs derived exosomes. (A) cluster of differentially expressed miRNAs among 4T1, TAM-exos, BMDM-exos. (B) comparing the expression levels of 28 candidate miRNAs in 4T1 cells before and after co-culture with TAMs for 72h by qPCR. (C) comparing the expression of 6 further screened candidate miRNAs in 4T1 cells with those in TAM-exos. (D) comparing the expression of 6 candidate miRNAs in BMDM-exos with those in TAM-exos. (E) sequence of mature miRNAs in human and mice.Fig 2Fig. 3The influence of miR-223-3p and miR-379-5p on the migration of 4T1 cells in vitro. (A-B) confirming the expression of miR-223-3p or miR-379-5p in 4T1 cells co-cultured with BMDM-exos or TAM-exos. (C-D) confirming the expression of miR-223-3p or miR-379-5p in 4T1 cells stably overexpressed with miR-223-3p or miR-379-5p. (E) transwell assays of 4T1NC, 4T1miR-223-3p and 4T1miR-379-5p cells. (F) statistics of migrated cells in transwell assays. (G) Wound healing assays of 4T1NC, 4T1miR-223-3p and 4T1miR-379-5p cells. (H) statistics of wound closure in wound healing assays.Fig 3miR-223-3p enhance the migration and metastasis of 4T1 cellsThen 4T1 cells stably over-expressed with miR-223-3p or miR-379-5p (marked as 4T1miR-223-3p or 4T1miR-379-5p) were constructed (Fig. 3C and D). It is proved that both miR-223-3p and miR-379-5p could increase the migration of 4T1 cells (Fig. 3E and F). So were their effects on the motility of 4T1 cells (Fig. 3G and H). When inoculating these breast cancer cells into the mammary fat pad of Balb/c mice, there is a tendency for the 4T1miR-223-3p group or the 4T1miR379-5p group to gain weight at the point of last observation, comparing with the 4T1NC group (Fig. 4A). Tumor volume was measured every three days and the data showed that tumors induced by 4T1miR-223-3p but not 4T1miR-379-5p grew more quickly (Fig. 4B). After the mice were sacrificed, tumors and lungs were removed and photographed (Fig. 4C). Statistical results demonstrated that both the tumors and lungs were heavier in the 4T1miR-223-3p group (Fig. 4D and E). However, no statistically differences exist between the 4T1miR-379-5p and 4T1NC group. Hematoxylin-eosin staining of lung tissues suggested that miR-223-3p could promote pulmonary metastasis of breast cancer 4T1 cells. Representative images near pulmonary alveoli, bronchus and blood vessels were displayed in Fig. 4F. It is worth mentioning that no increase but a decrease in proliferation can be observed in 4T1 cells stably over-expressed with miR-223-3p comparing to 4T1NC cells, indicating that the differences in migration between these two groups were not caused by proliferation (Fig. S1D). There were no more pulmonary metastatic nodules in 4T1miR-379-5p than 4T1NC group (data not shown).Fig. 4miR-223-3p enhance the metastasis of 4T1 cells. (A) weight of mice inoculated with 4T1NC, 4T1miR-223-3p or 4T1miR-379-5p cells. (B) tumor volume. (C) pictures of tumors and lungs from 4T1NC and 4T1miR-223-3p cells inoculated mice. (D) tumor weight. (E) lung weight. (F) hematoxylin and eosin staining of representative images near pulmonary alveoli, bronchus and blood vessel of lung.Fig 4miR-223-3p is up-regulated in lung-metastatic 4T1 cellsSince it is not easy to obtain the tumor cells at the metastatic nodules of breast cancer patients, metastatic cancer cells were isolated from lungs, livers, brains and kidneys of tumor-bearing mice which were inoculated with 4T1 cells in the mammary fat pad 28 days after the tumor in situ were removed (Fig. 5A). The isolated cancer cells from lung were inoculated in mammary fat pad and isolated from lung again. The screening was repeated for three times. The similar strategy was used in cancer cells isolated from livers, brains and kidneys. It is worth mentioning that in this experiment, a luciferase gene had been transferred into 4T1 cells by lentivirus before inoculation. So when the 4T1lung, 4T1liver, 4T1brain or 4T1kidney was respectively injected into the mammary fat pad of mice, their metastasis can be observed by Xenogen IVIS spectrum imaging system. And the image confirmed that 4T1lung had a high propensity for pulmonary metastasis (Fig. 5B). But the 4T1liver, 4T1brain and 4T1kidney cells failed to metastasize to corresponding organ specifically. Then the expression level of miR-223-3p and miR-379-5p were detected in 4T1 and 4T1lung. As expected, the expression level of both miR-223-3p and miR-379-5p were elevated in 4T1lung (Fig. 5C).Fig. 5Verification of miR-223-3p and miR-379-5p in 4T1lung. (A) strategy of isolating metastatic 4T1lung, 4T1liver, 4T1brain and 4T1kidney from mice inoculated with 4T1 in mammary fat pad. (B) Imaging of mice inoculated with 4T1lung, 4T1liver, 4T1brain and 4T1kidney (from left to right: 4T1lung, 4T1liver, 4T1brain and 4T1kidney). (C) expression of miR-223-3p and miR-379-5p in 4T1 and 4T1lung.Fig 5Cbx5 is a target of miR-223-3pTo explain how miR-223-3p functioned as a pulmonary metastasis-promoting factor, its target genes were screened by following rules: 1) the gene was predicted to be a target by no less than two databases among Targetscan, miRDB, PicTar and miRWalk; 2) the gene was down-regulated in lung metastases of breast cancer according to two or more downloaded GEO datasets (GSE54773, GSE63627, GSE110101, GSE56493); 3) it has not been reported as a potential oncogene in breast cancer. Thus, eleven candidate genes (Rasa1, Fat1, Mef2c, Ankrd17, Rbpj, Kpna3, Pds5b, Mbnl1, Ptbp2, Cbx5, Cbfb) were selected. When the mimic of miR-223-3p was transiently transfected into 4T1 cells, the mRNA level of Fat1, Ankrd17, Rbpj, Kpna3, Pds5b, Mbnl1, Ptbp2, Cbx5 were significantly decreased (Fig. 6A). In addition, all the candidate target genes except Mef2c had a lower expression in 4T1lung. As Cbx5 had been widely reported as a down-regulated gene in invasive breast cancer, a suppressor of cell migration and invasion, a positive prognostic factor in breast cancer, and it had not been found to be a target gene of miR-223-3p, we chose it for further confirmation , , , . When co-culturing 4T1 cells with BMDM-exos or TAM-exos, the expression of Cbx5 were significantly reduced in the latter group (Fig. 6C). This reduction reappeared in 4T1 cells stably over-expressed with miR-223-3p (4T1miR-223-3p) (Fig. 6D). In parallel, we also verified the protein expression of Cbx5 in 4T1 cells transiently transfected and stably over-expressed with miR-223-3p (Fig. 6E). Additionally, dual-luciferase report assays demonstrated that miR-223-3p inhibited the luciferase activity, of which 3’UTR was inserted with a fragment of Cbx5 3’UTR sequence (Fig. 6F). However, this inhibition was relieved (Fig. 6F) after the sequence in Cbx5 3’UTR matching with the seed sequence of miR-223-3p was mutated (Fig. 6G).Fig. 6Identifying the target genes of miR-223-3p. (A) mRNA levels of candidate target genes in 4T1 and 4T1 cells transiently transfected with mimic of miR-223-3p. (B) mRNA levels of candidate target genes in 4T1 and 4T1lung cells. (C) expression of Cbx5 in 4T1 cells co-cultured with BMDM-exos or TAM-exos. (D) expression of Cbx5 in 4T1NC and 4T1miR-223-3p cells. (E) Expression of cbx5 by western blot in 4T1 and 4T1miR-223-3p. (F) dual-luciferase report assays. (G) predicted miR-223-3p binding sequence in 3’UTR of Cbx5.Fig 6The expression of miR-223-3p is associated with the prognosis of breast cancer and negatively correlated with that of Cbx5Furthermore, the association between miR-223-3p and overall survival rate of breast cancer patients were analyzed based on GSE40267. The patients with higher expression of miR-223-3p had a relative lower survival rate during 36 months of follow-up (p=0.042) (Fig. 7A). A significant negative correlation existed between the expression of miR-223-3p and Cbx5 based on 1085 breast cancer patients (r=-0.153, p=4.24e-07) (Fig. 7B). In line with previous findings, Cbx5 was strongly associated with good overall survival (p=0.009), recurrence-free survival (p=1.3e-04), distant metastasis-free survival (p=0.0021) of breast cancer patients, lung metastasis-free survival (p=0.035)(Fig. 7C-F).Fig. 7Association between miR-223-3p/Cbx5 and survival rate in breast cancer patients. (A) relationship between miR-223-3p and overall survival rate. (B) correlation of miR-223-3p and Cbx5. (C-F) relationship between Cbx5 and overall survival, recurrence-free survival, metastasis-free survival rate, lung metastasis-free survival rate.Fig 7DiscussionIt has been summarized that exosomal microRNAs had a close relationship with organotropism in breast cancer metastasis . Our results provided new evidence which came from TAM-exos. According to our data, TAM-exos encapsulating with plenty of differential microRNAs enhanced the capability of tumor cells to metastasize. In particular, exosomal miR-223-3p was confirmed to be delivered into 4T1 cells and function as a tumor-promoting factor. miR-223-3p had been reported to be an oncogene in various cancers including breast cancer by a lot of researches. It could promote proliferation, invasion and migration of breast cancer cells and activate Hippo/Yap signaling pathway . In addition, miR-223-3p promotes epithelial-mesenchymal transition and metastasis through the Wnt/β-catenin signaling pathway or different targets [22,23]. Our study confirmed that the way of mediating metastasis is not through cell proliferation. It was also found to be elevated in serum exosomes of breast cancer patients and FOXO1 was its target gene . Simultaneous inhibition of miR-223-3p in breast cancer patients treated with celastrol significantly enhanced the anti-tumor effects . Gap junction-mediated transportation of miR-223 from bone marrow stromal cells reduced the expression of CXCL12, contributing to the quiescence of breast cancer cells . There was also evidence indicated that miR-223-3p was shuttled into breast cancers by microvesicles produced by IL-4-activated macrophages . Since IL-4 activated macrophages didn't fully represent TAMs, our rounds of selection demonstrated that TAM-exos was an important source of elevated miR-223-3p in breast cancer cells and this miRNA played a notable role in the pulmonary metastasis of breast cancer cells.In addition, this is the first time to prove that Cbx5 is a target of miR-223-3p. Cbx5 encodes heterochromatin protein 1α (HP1α), which is one of the chromobox proteins that recognizes histone 3 lysine tri-methylation at residues K9 (H3K9me3). H3K9me3 preferentially appears in heterochromatin and represents a common marker of transcriptional silencing . The revolutionary conserved protein HP1α contains a chromo domain in the N-terminus, a chromo shadow domain in the C-terminus and a hinge domain linking them. It combines with methylated histones on the chromatin through chromo domain while interacting with other proteins through chromo shadow domain . Structural analysis showed that a symmetric HP1 dimer could provide a bridge between two H3K9me3 nucleosomes . Several mechanisms about heterochromatin proteins especially HP1α in modulating gene transcription have been discovered. For example, the interaction between HP1α and heterochromatin brings two distant regions on the genomic DNA together, leading to a compact state of the chromatin which represses transcription . Besides the interaction between chromo domain and H3K9me3, the flexible hinge domain is essential for the establishment and maintenance of heterochromatin through recognizing parallel RNA and DNA G-quadruplexes . In addition, phosphorylated or DNA-bound HP1α has the ability to form phase-separated droplets, which could induce DNA strands to compact into puncta . Since only 2% of HP1α is located in gene promoter, transcription repression could just explain a small part of its function mechanisms . It is hypothesized that, HP1α may be involved in the process of epithelial-to-mesenchymal transition, which usually occurs at the initial stages of metastasis, through regulating alternative splicing directly or indirectly . More mechanisms are still under exploration. Other genes predicted to be the targets of miR-223-3p from database have also been found to have some anti-tumor effects, such as FAT1, whose significantly decrease has been reported to be associated with poor prognosis and invasiveness in invasive ductal carcinoma ; MBNL1 is demonstrated to be significantly downregulated in breast and gastric cancers in several studies, and MBNL1 can interact directly with ZFP36 to downregulate CENPA, thereby inhibiting the proliferation and stemness of breast cancer cells [36,37]. CBFB, together with its binding partner RUNX1, regulates multiple signaling pathways, and breast cancer cells may evade both translational and transcriptional surveillance simultaneously caused by CBFB downregulation . These targets above also corroborate the role of miR-223-3p in promoting metastasis of cancer cells.In summary, our data screened out that miR-223-3p was enriched in the exosomes of TAMs. It can be delivered into recipient cells along with the uptake of these exosomes by 4T1 cells, leading to a marked elevation of miR-223-3p. Through targeting Cbx5, miR-223-3p played an important role in promoting pulmonary metastasis of 4T1 cells. These findings suggested that exosomal miR-223-3p could possibly be an indicator of breast cancer metastasis and targeting TAMs exosomal miR-223-3p-Cbx5 axis might be a potential treatment strategy to inhibit breast cancer metastasis.FundingThis project was supported by the Fundamental Research Funds for the Central Universities (3332022181, 3332020033, to Zhaojun Duan), the National Natural Science Foundation of China (NSFC; 81672914, to Yunping Luo) and the Bilateral Inter-Governmental S&T Cooperation Project from Ministry of Science and Technology of China (2018YFE0114300, to Yunping Luo).CRediT authorship contribution statementZiyuan Wang: Data curation, Formal analysis, Validation. Chen Zhang: Supervision. Jian Guo: Methodology. Wei Wang: Methodology. Qin Si: Investigation. Chong Chen: Investigation. Yunping Luo: Conceptualization, Visualization, Writing – original draft, Writing – review & editing. Zhaojun Duan: Conceptualization, Visualization, Data curation, Formal analysis, Validation, Writing – original draft, Writing – review & editing.Declaration of Competing InterestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Nucleic Acids Research

36951106

PMC10123102

3-23-2023

10.1093/nar/gkad205

Design principles and functional basis of enantioselectivity of alanyl-tRNA synthetase and a chiral proofreader during protein biosynthesis

Sivakumar Koushick, Venkadasamy Vinitha Lakshmi, Amudhan Gurumoorthy, Ann Kezia J, Goud Gadela Karteek, Nayani Kiranmai, Gogoi Jotin, Kuncha Santosh Kumar, Mainkar Prathama S, Kruparani Shobha P, Sankaranarayanan Rajan

AbstractHomochirality of the cellular proteome is attributed to the L-chiral bias of the translation apparatus. The chiral specificity of enzymes was elegantly explained using the ‘four-location’ model by Koshland two decades ago. In accordance with the model, it was envisaged and noted that some aminoacyl-tRNA synthetases (aaRS) that charge larger amino acids are porous to D-amino acids. However, a recent study showed that alanyl-tRNA synthetase (AlaRS) can mischarge D-alanine and that its editing domain, but not the universally present D-aminoacyl-tRNA deacylase (DTD), is responsible for correcting the chirality-based error. Here, using in vitro and in vivo data coupled with structural analysis, we show that AlaRS catalytic site is a strict D-chiral rejection system and therefore does not activate D-alanine. It obviates the need for AlaRS editing domain to be active against D-Ala-tRNAAla and we show that it is indeed the case as it only corrects L-serine and glycine mischarging. We further provide direct biochemical evidence showing activity of DTD on smaller D-aa-tRNAs that corroborates with the L-chiral rejection mode of action proposed earlier. Overall, while removing anomalies in the fundamental recognition mechanisms, the current study further substantiates how chiral fidelity is perpetuated during protein biosynthesis.

INTRODUCTIONBiological macromolecules are homochiral, which is essentially due to the use of homochiral building blocks . The chiral specificity of enzymes towards their substrate was explained by using the ‘four-location’ model proposed by Koshland. According to this model, which is a modification of the ‘three-point attachment (TPA)’ model, three points of attachment and the direction of entry of the ligand are required for selecting chiral entities based on insights from high resolution isocitrate dehydrogenase structures . In the context of translation apparatus, it is well known that there is an absolute bias against the usage of D-amino acids to perpetuate homochirality during synthesis of proteins, universally across the tree of life . Aminoacyl-tRNA synthetases (aaRS) are a specialized class of enzymes that dictate the genetic code by preferentially pairing specific L-amino acids onto their corresponding tRNAs and thereby contributing significantly to proteome homochirality . However, aaRSs are known to commit errors occasionally by charging a wrong amino acid on a cognate tRNA (amino acid misselection), and rarely, charging a correct amino acid on the non-cognate tRNA (tRNA misselection) (4–10). Amino acid misselection is of two types: charging (i) a non-cognate L-amino acid or (ii) a D-amino acid . Ten out of the 20 aaRSs are equipped with proofreading domains to avoid the substitution of non-cognate L-amino acids (except AlaRS editing domain, which can act on achiral glycine also) . In case of non-cognate D-amino acids, in vivo studies on Escherichia coli and Saccharomyces cerevisiae have shown that chiral errors are committed by only a few aaRSs such as aspartyl-tRNA synthetase (AspRS), phenylalanyl-tRNA synthetase (PheRS), tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase (TrpRS) as envisaged by Koshland's ‘four-location’ model (2,12). Unlike the L-amino acid misselection, these erroneously formed D-aminoacyl-tRNAs are not proofread by their respective editing domains but by a specialized chiral proofreader called D-aminoacyl-tRNA deacylase (DTD) (13,14).Extensive genetic and biochemical studies have shown that DTD is selective towards mischarged D-amino acids but not L-amino acids (12,15,16). The structural basis of such absolute chiral specificity of DTD was deciphered by solving the crystal structure of DTD in complex with D-aminoacyl-tRNA analogue. DTD achieves such specificity by employing an invariant cross-subunit Gly-cisPro motif, which captures the chiral centre of the incoming D-amino acid by interacting with the amino group and the β-carbon . The side chain of the D-amino acid protrudes out from the active site pocket, which provides the basis of how a single enzyme, DTD, can act on multiple D-amino acids with varying side chains attached to tRNAs. Interestingly, this mode of operation allows DTD to act on achiral glycine, which is advantageous for avoiding the mistranslation of Ala codons . Such a discrimination profile can be explained only by the L-chiral rejection mechanism of DTD . However, the cognate Gly-tRNAGly escapes DTD action due to the presence of an anti-determinant in the acceptor arm of tRNAGly, whereas the non-cognate Gly-tRNAAla possesses a positive determinant at the same position (N73) that enable its proofreading. Thus, DTD employs a discriminator base based sorting of cognate and non-cognate species of Gly-tRNAs for proofreading . However, the activity of DTD was not tested on the tRNAs charged with smaller side-chain D-amino acids such as alanine, which is necessary to establish the L-chiral rejection mode of substrate discrimination across the entire spectrum of amino acid sizes.Alanyl-tRNA synthetase (AlaRS) charges alanine on tRNAAla and presents an interesting paradigm of mischarging smaller glycine and larger serine compared to its cognate amino acid . The mistranslation of alanine to glycine and serine is effectively prevented by employing both cis- and trans-editing domains including DTD that decouples erroneously attached Gly on tRNAAla (17,20). Very recently, it was shown that AlaRS also mischarges D-alanine on tRNAAla apart from glycine and L-serine, which is proofread by the AlaRS cis-editing domain and surprisingly not by DTD . These results deviate from previous works on multiple counts viz. the ‘four-location’ model of Koshland, in vivo studies in yeast and bacteria showing lack of growth defects in D-alanine enriched media, and the mechanistic model of DTD’s function proposed earlier (2,12,15,16). Considering the disagreement between the results of Rybak et al., NAR, 2019 with multiple earlier works, it is imperative that the results are thoroughly evaluated to aid in making any conclusion on the fundamental mechanisms involved in protein biosynthesis and chiral proofreading.Here, by using in vitro and in vivo experiments, our study unequivocally demonstrates that the L-chiral rejection mechanism of DTD holds true even on the smallest side chain proteogenic amino acid substrate, i.e. D/L-Ala-tRNAAla as well. Based on the analysis of the available AlaRS structures in the Protein Data Bank (PDB) , we show that D-alanine is sterically excluded from the active site and hence cannot be charged by AlaRS. Our studies further show that the cis-editing domain of AlaRS is inactive on D-Ala-tRNAAla thus disproving all the major claims made in the previous work and clears the serious anomalies ensued on the most fundamental reaction mechanisms that ensure chiral fidelity of the cellular aminoacyl-tRNA pool. Overall, the work experimentally substantiates the design principles governed by Koshland's ‘four-location’ model to explain the size-based disparity in activation of D-amino acids by aaRSs and the universality of L-chiral rejection based chiral proofreading by DTD.MATERIALS AND METHODSCloning, expression and purificationDTD genes with C-terminal 6x-His tag from E. coli (Ec), S. cerevisiae (Sc), Danio rerio (Dr) and Mus musculus (Mm) were cloned in pET28b (Novagen) expression vector. Similarly, N-terminal 6x- His tagged EcAlaRS (wildtype), EcAlaRS C666A mutant and TtAlaRS genes were also cloned in pET28b vector. C-terminal 6x His tag T. thermophilus (Tt) DTD is cloned in pTRC99 vector. The vectors carrying genes were first confirmed by Sanger sequencing (Eurofins Genomics, India) and then transformed in E. coli BL-21(DE3) expression cells for IPTG induction-based protein overexpression . TtDTD was expressed in Terrific Broth (TB) under IPTG induction. All the His-tagged proteins were purified from cell lysate of respective overexpression strain using immobilised metal affinity chromatography (Ni-NTA) in buffer containing 50 mM Tris (pH 8), 150 mM sodium chloride, 5% Glycerol and 5 mM β-mercaptoethanol. Proteins were eluted by gradient elution between 10 mM to 250 mM imidazole. Obtained fractions were further purified using size exclusion chromatography in buffer containing 150 mM NaCl, 50 mM Tris (pH 7.5). The purified and concentrated proteins were stored in -20°C in buffer containing 150 mM NaCl, 100 mM Tris (pH 7.5) and 50% glycerol for biochemical assays. All the steps after expression were carried out on ice or 4°C.Biochemical assays EctRNAAla was generated by in vitro-transcription using MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific, USA). Transcribed tRNA was 3′ end radiolabelled with [α-32P]-ATP (BRIT-Jonaki, India) using E. coli CCA adding enzyme. L-alanine was charged onto 3′ radiolabelled EctRNAAla (10 μM) in a reaction mix containing ATP (2 mM), β-mercaptoethanol (5 mM), L-alanine (100 mM) and EcAlaRS-WT (500 nM) . Alternatively, D-Ala-tRNAAla was generated by two strategies, 1) by EcAlaRS-WT with the same aminoacylation procedure used for L-Ala-tRNAAla and 2) Flexizyme-based charging method described in the following section . All the deacylation assays were carried out as reported earlier (15,16). In brief, all the aminoacylated substrates (2 μM) were incubated with 20 mM Tris pH 7.2, 5 mM MgCl2, 5 mM DTT and 0.2 mg/ml BSA along with 10 pM to 1μM DTD or 100 nM of EcAlaRS-WT/C666A mutant or 500 nM of TtAlaRS. Time course studies were performed by aliquoting 0.8 μl of the deacylation mix for each time point (2, 5, 10 and 15 minutes) and followed by S1 nuclease digestion. The incubated samples were then fractionated in Thin Layer Chromatography (TLC) and the proportion of aminoacylation in the samples were estimated after phosphor imaging the TLC exposed storage phosphor screen (Typhoon FLA 9000 biomolecular imager, GE Healthcare). All the aminoacylation and deacylation reactions were carried out in 37°C. EctRNAAla was used throughout the study.Aminoacylation titration assays with decreasing concentration of amino acid were performed using serially diluted amino acid substrates (L/D-alanine) from 100 mM to 100 nM in the reaction containing 500 nM of EcAlaRS WT or editing defective C666A enzyme.Flexizyme based aminoacylationFlexizymes were generated through in vitro-transcription as reported earlier . All the amino acid substrates were activated by either CME (Cyanomethyl ester) or DBE (Dinitro benzyl ester). These activated amino acid substrates were charged using enhanced Flexizyme (eFx) (for CME activated substrate) and di-nitro flexizyme (dFx) (for DBE activated substrate) based on the previous reports (25–28). Briefly, flexizyme is mixed step wise with 3′ radiolabelled tRNA, MgCl2 and respective amino acid substrate and then incubated for 12–14 h on ice. After incubation, all the aminoacylated-tRNAAlas were then subjected to ethanol precipitation and the resultant pellets were washed twice with 70% ethanol and 0.1% sodium acetate. Air-dried pellets were resuspended in 5 mM sodium acetate (pH 4.5) and used for further deacylations.Viability assaysViability assays were performed with both the MG1655 single mutant ΔalaS (contains editing defective AlaRS) and as well as double mutant ΔalaSΔdtd (dtd gene deleted strain containing editing defective AlaRS) in M9 minimal agar as per the protocol described in Pawar et al. . Briefly, ΔalaS, Δdtd, ΔalaSΔdtd mutant strains of E. coli MG1655 were inoculated in the minimal media containing 0.002% arabinose and 0.2% maltose, till the cultures reach 0.6 OD600. The cultures were serially diluted from 10−1 to 10−5 and 5 μl of each dilution was spotted on the agar plates containing respective amino acids (L-serine (3 mM), L-alanine (10, 50, 100 mM), D-alanine (10, 50, 100 mM), L-tyrosine (6 mM) and D-tyrosine (6 mM)) along with the above-mentioned concentration of arabinose and maltose.Growth curve experiments were done with the same strains which we utilised in the spot dilution assay, in M9 minimal liquid media containing 0.0002% of arabinose and 0.2% of maltose and supplemented with the amino acid of interests (L-serine (3 mM), L-alanine (100 mM), D-alanine (100 mM)) in the specified concentrations. Since D-tyrosine is known to get turbid at higher concentration (>5 mM) in the liquid culture , we used a mixture of D-amino acids containing 3mm of D-tyrosine, 10 mM of D-aspartate and 1.5 mM of D-tryptophan for the growth curve assay. The secondary cultures were initiated with 1% of overnight grown culture and allowed to grow till 0.6 OD600. Grown secondary cultures were utilised to initiate the tertiary cultures with the initial OD600 of 0.06Cell density was measured at an interval of 2 h from the time of inoculation. All the viability assays were done in triplicatesMass spectrometryBoth L-Ala-tRNAAla and D-Ala-tRNAAla were generated using EcAlaRS WT as above mentioned. The resultant products were ethanol precipitated and digested using aqueous ammonia solution (25% of v/v NH4OH) at 70°C for 12 h. These samples were then dried and resuspended in 10% methanol and 1% acetic acid. ESI-based mass spectrometry analysis of the selected precursor ion was carried out exactly as previously reported . Briefly, the samples were subjected to Heated Electrospray Ionization (HESI) and the spectra of the selected ions were analysed using Xcalibur™ 4.0 software (Thermo Fisher Scientific, USA).Structural analysisHighest resolution structures of both HsAlaRS (PDB id: 4XEM, resolution-1.28Å) and EcAlaRS (PDB id: 3HXU, resolution-2.1Å) bound with L-AlaSA (5'-O-(N-(L-alanyl)-sulfamoyl adenosine) were selected from Protein Data Bank (PDB) and used to model D-AlaSA in the active site. Pymol plugin ‘ProteinInteractionViewer’ was used with default parameters to visualize and represent the small and bad overlaps in both of the AlaRS structures with L-AlaSA and modelled D-AlaSA in the active site. MolProbity server ( was used to evaluate the small, bad, and worse clashes/overlaps based on van der Waals radii of the individual atom in the model . It was also employed to confirm and quantify clashes in AlaRS structures with D-AlaSA and L-AlaSA. From the resultant clash score tables, values that were common in both the structures were eliminated to obtain scores for unique clashes ensued by modelling D-AlaSA in the active site pocket.Detection of chiral compoundsTo probe the chiral impurity in the amino acid supply, we utilised Marfey's reagent (Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide) to derivatise the D-alanine and L-alanine reagents to separate and quantitate the D- and L-enantiomeric levels in each of them by following the method as previously mentioned . Briefly, amino acid dissolved in autoclaved milliQ water was added with acetone, 1% of Marfey's solution prepared in acetone and aqueous sodium bicarbonate. After incubating the mixture at 37°C for 90 min, the reaction was quenched with formic acid. The resultant mixture was further diluted 10-fold with 1% acetone and subjected to UPLC (Acuity H-Class UPLC connected to Xevo TQ-S micro-MS system) with Agilent ZORBRAX Eclipse Plus C18 (2.1 mm × 100 mm) reverse phase column. Gradient elution was done using a mobile phase consisting of solution A (ammonium acetate, pH 4.6) and solution B (Acetone). A linear gradient from 20% to 80% of solution B was developed in 10 min.RESULTSAlaRS editing defective strain of bacteria is not sensitive to D-alanineRecently it has been shown in vitro that AlaRS charges D-alanine on tRNAAla, and is proofread by the cis-editing domain of AlaRS . In order to test these observations physiologically, we hypothesized that bacterial stain with AlaRS editing defective (AlaRSED) gene would be sensitive to excess D-alanine supplementation in growth media, while the wild type bacterial strain would be insensitive to the same. To test this, we have used E. coli MG1655 strain with AlaRS gene deleted at the genomic level carrying a shelter plasmid bearing editing defective EcAlaRS (T567F, S587W, C666F-AlaRSED) gene under arabinose inducible promoter (MG1655 ΔalaS Para::AlaRSED) . As expected, the MG1655 ΔalaS Para::AlaRSED strain was sensitive to glycine and 6 mM L-serine supplementation, while the growth of E. coli strain containing wild type AlaRS was unperturbed (Figure 1A). Similarly, both the strains did not show any growth defect on L and D-tyrosine supplementation (Supplementation Figure S1A). Surprisingly, MG1655 ΔalaS Para::AlaRSED had no growth defect upon supplementation of excess D-alanine, ranging from of 10 to 100 mM, similar to that of the wild type strain (Figure 1B). The spot dilution assay results were further confirmed by growth curve assays with the same strains in the liquid media supplemented with respective concentration of amino acids (Supplementary figure S1B and C). These results were perplexing as to how AlaRS, which was earlier shown to misacylate and edit both L-serine and D-alanine under in vitro conditions , shows toxicity towards L-serine but not to D-alanine in viability assays. These observations raise the possibility of two scenarios: that there might be a redundant proofreader for D-Ala-tRNAAla or D-alanine charging by AlaRS in vitro may not have any physiological relevance.Figure 1.D-alanine supplementation is not toxic to AlaRS editing defective bacterial strain. Spot dilution assay of E. coli wild type MG1655 strain and E. coli MG1655 ΔalaS/para :: alaS (TM)-T567F, S587W, C666F supplemented with (A) No amino acid, 20 mM glycine and 6 mM L-serine, (B) 10, 50 and 100 mM of L-alanine and D-alanine. Increasing concentration of D-alanine even to 100 mM in the growth media does not cause any toxicity in both MG1655 wild type and MG1655 ΔalaS/para :: alaS TM strains.DTD’s L-chiral rejection-based mechanism is amino acid size independentLack of toxicity in MG1655 ΔalaS Para::AlaRSED strain to D-alanine in our viability assays raised a possibility for presence of a redundant proofreader for D-Ala-tRNAAla in the cells other than AlaRS editing domain. Based on earlier studies on D-aminoacyl-tRNAs proofreading modules, the only possible redundant factor for editing D-Ala-tRNAAla could be D-aminoacyl-tRNA deacylase (DTD) (13,15). Insights from multiple ligand-bound crystal structures of DTD and its biochemical activity, led to the proposition that DTD is a strict L-chiral rejection module (15–17). Previous NMR and modelling studies clearly showed that D-Ala-tRNAAla could be accommodated and acted upon by DTD (16,33). D-alanine fits snugly in the active site pocket of DTD when modelled based on the D-Tyr-3AA that was captured in the co-crystal structure (PDB id: 4NBI). Like any other D-amino acid, R group (methyl group)/ Cβ of D-alanine makes a C-H…O hydrogen bond with the carbonyl oxygen of Pro150 from the cross-subunit Gly-cisPro motif. This further reinforces the fact that D-aa-tRNAs are positioned in such a way that the side chain beyond Cβ atom is protruding outside the active site of DTD (Figure 2A). However, the biochemical activity of DTD was shown only on tRNAs charged with larger D-amino acids like tyrosine, phenylalanine, aspartate and tryptophan (11,15). Therefore, the activity of DTD on tRNAs charged with smaller D-amino acids was only a logical extrapolation. We checked whether DTD’s L-chiral rejection based enantioselectivity is effective on smaller amino acid charged tRNAs or not, experimentally, by performing in vitro deacylation assays. We generated L-Ala-tRNAAla, L-Phe-tRNAAla, D-Ala-tRNAAla, D-Phe-tRNAAla, Gly-tRNAAla and D-Ser-tRNAAla using flexizyme-based aminoacylation system. The substrates were incubated with E. coli DTD (EcDTD) and analysed for its ability to deacylate the aminoacyl-tRNAs. As expected, DTD was inactive on both L-Ala-tRNAAla (Figure 2B) and L-Phe-tRNAAla (Figure 2C) but efficiently deacylated all tRNAs charged with D-amino acids irrespective of the side chain size (Figure 2D, E and G) and glycine (Figure 2F). To show the conservation of this activity across species, we also tested the D-Ala-tRNAAla deacylation activity of DTD from various organisms such as P. aeruginosa (PaDTD), D. melanogaster (DmDTD), M. musculus (MmDTD) (Supplementary figure S2). In line with the EcDTD, all DTDs tested were able to deacylate D-Ala-tRNAAla at 100 pM concentrations. Thus, the above biochemical results experimentally substantiate the side chain independent L-chiral amino acid rejection mechanism of DTD. This observation contradicts the previous report that DTD cannot act on D-Ala-tRNAAla , thereby implying its possible role as a redundant proofreader for D-Ala-tRNAAla in the cells other than AlaRS editing domain.Figure 2.DTD is a strict L-Chiral rejection module. (A) D-alanine modelled in the active site of DTD, showing D-alanine can fit in the active site like any other larger side chain D-amino acids (D-tyrosine). Deacylation assay at 10 pM and 100 pM concentrations of EcDTD on (B) L-Ala-tRNAAla, (C) L-Phe-tRNAAla, (D) D-Ala-tRNAAla, (E) D-Phe-tRNAAla, (F) Gly-tRNAAla and (G) D-Ser-tRNAAla (all the substrates from B to F were generated using flexizyme) (*:modelled). E. coli lacking both DTD and AlaRS editing activity is not sensitive to D-alanineMany trans-editing factors act in redundancy with the aaRS borne cis-editing domains towards correction of mischarged aa-tRNAs, which can obscure the physiological impact of abrogation of the aaRS cis-editing domain activity (6,17,34,35). Our viability assay with MG1655 ΔalaS Para::EcAlaRSED upon supplementation of excess D-alanine in growth medium did not lead to any growth defect (Figure 1). Moreover, our biochemical data confirms that DTD can effectively deacylate D-Ala-tRNAAla generated with the help of flexizyme (Figure 2D; Supplementary figure S2). To probe the possibility of DTD being a redundant proofreader of D-alanine charged by AlaRS in vivo, we employed MG1655 bacterial strains lacking dtd gene (MG1655 Δdtd), AlaRS editing defective (MG1655 ΔalaS Δdtd Para::EcAlaRSED – Double knockout editing defective (DKO-ED)) and AlaRS-wild type complemented (MG1655 ΔalaS Δdtd Para::EcAlaRSWT – DKO-WT) background. As expected, all the three strains were sensitive to D-tyrosine and the DKO-ED showed sensitivity towards glycine and L-serine supplementation (Figure 3A). In addition, the growth of the all the strain remains unperturbed even after supplementing with 100 mM D-alanine in the media. However, high concentration of L-alanine (50 mM and 100 mM) showed severe growth perturbation as it is known to result in metabolic imbalance or growth defect (Figure 3B) . Similarly, growth curve assays with DKO-ED strain in the presence of 100 mM D-alanine, did not exhibit any cellular toxicity or reduced growth (Supplementary figure S3A) in comparison with ΔDTD and DKO complemented with wildtype EcAlaRS (Supplementary figure S3B and C). The absence of any toxicity towards D-alanine in ΔDTD and DKO-ED strain raise the question as to whether D-alanine charging by AlaRS is at all physiologically relevant or is it just an in vitro artefact .Figure 3.DTD knockout and AlaRS editing defective strain is insensitive to D-alanine. Toxicity assay of E. coli MG1655 Δdtd compared with that of E. coli MG1655 Δdtd::ΔalaS/para:: alaS-(TM)T567F, S587W, C666F and E. coli MG1655 Δdtd::ΔalaS/para::alaS in the presence of (A) no amino acid, 3 mM of L- and D-tyrosine, 20 mM glycine 3 mM and 6 mM L-serine, (B) Supplementation of 10, 50 and 100 mM of L-alanine and D-alanine, wherein even at 100 mM concentration of D-alanine all the three strains does not show any toxicity.Aminoacylation domain of AlaRS operates by strict D-chiral rejection modeThe Protein Data Bank has multiple entries of high-resolution structures of AlaRS aminoacylation domain with substrate analogue bound to the catalytic pocket. These substrate analogue bound structures helped glean mechanistic understanding at atomic level resolution of the alanylation reaction carried out by AlaRS . We sought to understand the structural basis of the in vitro D-alanine charging by AlaRS reported in previous study . In order to figure out how D-alanine fits in the aminoacylation site of AlaRS, we choose the highest resolution (1.28 Å) structure of AlaRS catalytic domain complexed with L-AlaSA (‘5’-O-(N-(L-alanyl)-sulfamoyl adenosine) (PDB ID: 4XEM- HsAlaRS) (Figure 4A) from PDB and modelled D-AlaSA in the active site. The model showed that D-alanine could not fit inside the active site pocket of AlaRS aminoacylation site as Cβ of D-alanine had a serious clash with Cγ2 atom of V218 and Cδ2 of Trp176 (Figure 4B). Furthermore, we confirmed the clashes using MolProbity server ( With the bad clash default overlap cut-off of 0.4Å, the modelled D-AlaSA exhibited severe clash with Val218 and Trp176 residues with the highest clash score (Figure 4C). To test the universality of the clashes, we repeated similar analysis with the high-resolution structure of EcAlaRS (2.1Å) with L-AlaSA in the active site (PDB: 3HXU). Expectedly, same clashes were observed when D-AlaSA was modelled in the active site but not for L-AlaSA (Supplementary figure S4A and B). MolProbity scores from D-AlaSA modelled EcAlaRS also gave similar scores as that of HsAlaRS (PDB ID: 4XEM) (Supplementary figure S4C). Moreover, the residues (valine and tryptophan) that are clashing with modelled D-alanine are conserved across the species (Figure 4D). Interestingly, Thermus thermophilus (Tt) AlaRS, which is shown to charge D-alanine by Rybak et al., NAR, 2019 is also possess the invariant valine and tryptophan in the active site pocket (Figure 4D). Structural modelling and alignment clearly suggest that AlaRS catalytic pocket has conserved features responsible for rejecting D-alanine that supports the ‘four-location’ model proposed by Koshland. In the case of AlaRS bound with L-AlaSA, the amino group of L-alanine is fixed by the side chain of residue Asp236, carbonyl group is held by guanidinium group of Arg70 (10,19). With this mode of selection of the amino acid, the residues Val218 and Trp176 sterically excludes Cβ of any D-amino acid, thus serving as the third attachment point and the fourth constraint is imposed by the orientation of amino acid to form amino acyl-AMP intermediate and aminoacyl-tRNA. Therefore, based on the structural insights from multiple substrate analogue (data not shown) bound crystal structures of AlaRS aminoacylation domain, D-alanine cannot bind to AlaRS aminoacylation domain and hence cannot be aminoacylated.Figure 4.AlaRS active site operates as a strict D-chiral rejection module. (A) L-AlaSA in the active site of the HsAlaRS catalytic domain (PDB id:4XEM). (B) D-AlaSA modelled in the active site of the HsAlaRS catalytic domain shows serious clashes with Val-218 and Trp-176. (C) Table showing clash distances calculated using MolProbity server between modelled D-AlaSA and surrounding residues in AlaRS aminoacylation domain. This suggests that D-alanine cannot fit into AlaRS catalytic pocket (with clash distance in red). (D) Structure based sequence alignment showing the invariant valine and tryptophan (marked by black arrows) in the catalytic site of AlaRS in all the domains of life.The species charged by AlaRS in D-alanylation reaction is a contaminantThere exists a contradiction between the proposed AlaRS aminoacylation of D-alanine in earlier work and the observed in vivo D-alanine toxicity data as well as structural insights from the substrate analogue bound AlaRS aminoacylation domain. To resolve this, we probed the reported aminoacylation of tRNAAla with D-alanine by editing defective C666A mutant of EcAlaRS. In our aminoacylation reactions, along with L-alanine, L-serine, and glycine, EcAlaRS C666A also charged D-alanine (albeit at a very high concentration of 100 mM of amino acid) on tRNAAla. We then tried deacylating the L-Ala/D-Ala aminoacylated tRNAs by DTD and AlaRS. Interestingly, both L-Ala-tRNAAla and D-Ala-tRNAAla were not deacylated by DTD and AlaRS (Figure 5A and B, respectively). Next, we used L-Ala-tRNAAla and D-Ala-tRNAAla generated by flexizyme as substrates for deacylation by AlaRS and DTD. Unlike aaRSs, flexizyme do not possess features for substrate specificity, therefore, this method allows aminoacylation irrespective of the side chain chemistry or Cα chirality . Surprisingly, AlaRS could not deacylate either L- or D-Ala-tRNAAla and D-Ser-tRNAAla substrates generated using flexizyme. However, flexizyme generated D-Ala-tRNAAla and D-Ser-tRNAAla were deacylated by DTD at 100 pM but not L-Ala-tRNAAla (Figure 5C and D; Supplementary figure S5A). The above results clearly indicate the possibility of some unknown contaminant in D-alanine reagent being charged in aminoacylation reaction with AlaRS. Yet another noteworthy observation is that AlaRS was not able to edit the presumed AlaRS generated D-Ala-tRNAAla substrate as effectively as L-Ser/Gly-tRNAAla (Figure 5B and supplementary figure S5B and C, respectively). It is important to note that the earlier paper authors have used Thermus thermophilus DTD (Tt) and TtAlaRS. Though the residues responsible for the D-alanine rejection is present in the active site of TtAlaRS, we wanted to check the ability of the same and TtDTD to deacylate the D-Ala-tRNAAla generated using flexizyme. Similar to EcDTD, TtDTD was inactive against L-Ala-tRNAAla but readily deacylated the flexizyme generated D-Ala-tRNAAla (Supplementary figure S6A and C, respectively). Tt AlaRS efficiently deacylated L-Ser-tRNAAla but was inert against D-Ala-tRNAAla (Supplementary figure S6B and C, respectively). Taking the structural, biochemical and in vivo experiments into account, it is evident that AlaRS cannot charge or deacylate D-alanine. Hence, it is most likely that the observed D-alanine aminoacylation might be a contaminant, which is present in trace amount in the D-alanine reagent.Figure 5.Chiral selectivity of AlaRS aminoacylation site. (A) Deacylation of L-Ala-tRNAAla by both EcDTD and EcAlaRS-WT. (B) Deacylation of D-Ala-tRNAAla (AlaRS generated aminoacylated substrate) by EcAlaRS-WT and EcDTD showing that D-Ala-tRNAAla is not cleaved by either of them. (C) Deacylation of L-Ala-tRNAAla charged by flexizyme by EcAlaRS-WT and EcDTD showing similar results as AlaRS charged substrate. (D) Deacylation of flexizyme charged D-Ala-tRNAAla by EcAlaRS-WT and EcDTD shows that EcDTD can effectively cleave D-Ala-tRNAAla but not by EcAlaRS-WT. (E) Aminoacylation of L-Ala and D-Ala on tRNAAla by EcAlaRS C666A with reducing concentration of the amino acid substrate in the reaction (100 mM, 10 mM, 1 mM, 100 μM, 10 μM, 1 μM and 100 nM) showing L-alanine getting charged at 10 μM amino acid concentration, which is 1000-fold less than the minimum concentration at which D-alanine charging is seen, i.e. 10 mM.To investigate the possibility that the aminoacylated specimen in D-alanine aminoacylation reaction could be a contaminant, we titrated a range of amino acid (D-alanine) concentrations lesser than the set concentration in the standard alanylation protocol (from 100 to 100 nM). Considering the contaminant in the D-alanine reagent is expected to be in trace amount, only a high amount of D-alanine in the reaction will have optimal amount of the contaminant for charging to happen. Indeed, upon reducing the concentration of D-alanine in the reaction with EcAlaRS C666A, there was a clear reduction in formation of aminoacyl-tRNAAla, however reducing L-alanine in aminoacylation reaction did not lead to reduction in formation of aminoacyl-tRNAAla as abruptly like in case of D-Ala (Figure 5E). Compared to L-Ala, D-Ala could be charged on tRNAAla only at 1000-fold excess amino acid concentration in the reaction. To probe the effect of AlaRS editing domain, we repeated the amino acid titration in aminoacylation reaction for both L-and D-alanine with EcAlaRS WT. Remarkably, EcAlaRS WT enzyme could aminoacylate D-alanine at around 1000-fold more concentration than L-Ala (Supplementary Figure S7). The ∼1000-fold excess concentration of D-Ala required to get comparable aminoacylation to that of L-Ala by AlaRS raises the possibility of enantiomeric contaminant L-alanine being the species charged on tRNAAla in D-alanine aminoacylation reactions.AlaRS charges enantiomeric contaminant L-alanine in D-alanylation reactionBased on the previous studies, AlaRS can charge L-serine, glycine and non-proteogenic amino acids such as azetidine-2-carboxylic acid (AZE) and βN-methylamino-L-alanine (BMAA) apart from L-alanine (31,33,34). Among these, it is known that AlaRS-editing domain can deacylate all the above-mentioned amino acids but not its cognate L-alanine and BMAA. To find out the nature of the contaminant present in the D-alanine reagent which can be charged by AlaRS catalytic domain, we performed mass spectrometry-based analysis of D-alanine and L-alanine for the presence of above-mentioned substrates. Interestingly, we could see a single prominent peak corresponding to alanine in both the samples whereas contaminant peaks were negligible (Supplementary Figure S8A and B). To probe the identity of the charged contaminant, we charged both L-alanine and D-alanine with EcAlaRS-WT on EctRNAAla and subjected the charged product to mass spectrometry. We observed similar mass spectrum for both the samples with a single predominant peak with m/z corresponding to alanine (Figure 6A). The mass spectra from both the sample suggests identical species alanine being charged by AlaRS, but the chirality of the charged alanine remain undetermined.Figure 6.AlaRS charges L-Ala contaminant in D-alanylation reaction. (A) ESI-MS profile of resultant product after aminoacylated by EcAlaRS using D- and L-alanine. (B) Chromatograms showing that area of the peak corresponding to L-alanine in D-alanine sample is increasing upon titration of increasing concentration of D-alanine in the reaction.In order to identify the enantiomeric status of the alanine charged onto tRNAAla, we utilised Marfey's reagent which helps in achieving enantio-separation and quantitation owing to the formation of diastereomers with different polarity with D- and L-enantiomers (32,37). When the derivatised D-alanine was subjected to reverse phase chromatography, the chromatogram showed a prominent D-alanine peak along with a comparatively smaller but observable peak. The retention time of the smaller peak correspond to that of L-alanine (Supplementary Figure S9A). To further validate this observation, we increased the concentration of D-alanine by 1:1, 2:1, 5:1, 10:1 and 20:1 with Marfey's reagent in the reaction. Concomitantly, the smaller peak gradually increased with the increasing D-alanine concentration in the reaction (Figure 6B). Notably, when the same set of reactions were repeated using L-alanine, conversely a smaller peak corresponding the retention time of D-alanine was observed (Supplementary Figure 9B). These observations clearly indicate that the D-alanine supply contains trace amount of enantiomeric impurity and vice versa. Relative quantification of the peaks suggests that the enantiomeric impurity contributes to around 0.1% of total derived amino acids. This result is also in line with the reported enantiomeric excess (ee) of D-alanine supply of 99% (Sigma-Aldrich). Ee of 99% denotes that ≤0.5% of L-alanine contamination is always possible in the commercially available D-alanine, which is in agreement with the aminoacylation titrations, where ∼1000-fold excess of D-alanine was required to achieve comparable aminoacylation with L-alanine (Figure 5E). This also highlights the strict chiral selective nature of the AlaRS aminoacylation site that even a 0.1% L-alanine in the enantiomeric mixture can be selected and charged onto tRNAAla leading to inadvertent synthesis of cognate L-Ala-tRNAAla that does not get deacylated by both AlaRS editing domain and DTD (Figure 5B). Moreover, using chirality non-discriminatory aminoacylation system Flexizyme, we generated D-ala-tRNAAla that is effectively deacylated by DTDs but not AlaRS (Figure 5D).Taken together, it is evident that AlaRS cannot charge or deacylate D-alanine and DTD can proofread small side chain D-aa-tRNAs although some of them may not be physiologically generated (Figure 7). Combining mass spectroscopy analysis of the charged species and derivatization of the D-alanine followed by HPLC analysis concludes that the observed aminoacylation is due to a trace amount of enantiomeric L-alanine contaminant in D-alanine reagent. Hence, DTD and AlaRS editing domain were not able to deacylate it. Our results conclusively show that it is indeed the aminoacylation site and not the editing site of AlaRS that enforces enantiomeric fidelity.Figure 7.Overall model showing the modus operandi of AlaRS and DTD. AlaRS sterically excludes D-alanine from the aminoacylation site but can charge L-serine and glycine along with L-alanine and its editing domain can deacylate mischarged L-Ser-tRNAAla and Gly-tRNAAla. DTD acts on D-aminoacyl-tRNAs independent of side chain size and deacylates the misacylated product into free amino acids and tRNAs.DISCUSSIONProofreading by aaRSs have been studied majorly from the point of view of non-cognate L-amino acids charging by aminoacyl-tRNA synthetases. Several cis- as well as trans-editing domains have been characterized for their mechanistic and functional basis of operation (34,38,39). It is now well known that nearly ten aaRSs charge non-cognate L-amino acids and proofreading functions are invoked to clear the errors generated using both double- and triple-sieve models (39–43). However, in the context of opposite chiral D-amino acids, only the larger side chains have been shown to be activated biochemically by tRNA synthetases and this phenomenon could be explained by Koshland's ‘four-location’ model (2,12,14). Therefore, it was puzzling to note from a recent work that AlaRS mis-aminoacylates D-alanine . Our modelling and biochemical work identifies the residues responsible for the L-chiral selection mechanisms and shows that AlaRS does not charge D-alanine as previously reported (Supplementary Figure S10A). AlaRS being one of the early synthetases evolved an efficient mechanism which allows it to discriminate and exclude D-alanine from the active site. The high enantio-specificity towards L-alanine permits it to effectively select and activate even ∼0.1% of L-alanine from the D-alanine mixture.The casualty of misinterpreting observation arising due to the interference by trace amount of contaminant present in reagent used in reaction is not new in the field. Classic examples of the presence of L-enantiomeric contamination in the D-amino acids and its effect on experimental outcome have been well documented in the case of D-aspartate and D-lysine . Similar instances of contaminants were reported and rectified in other studies (44–46). In this regard, the present study emphasises that even a ∼0.1% contamination in the starting material, would lead to misinterpretation and affects well defined mechanistic principles.While showing D-alanine charging, the Rybak et al. study also presented the first evidence of a cis-editing domain of an aaRS to clear such a chiral error . It would imply that the editing domain of AlaRS could remove opposite chiral D-alanine in addition to non-cognate L-serine and achiral glycine. Our experimental work unambiguously demonstrated that the AlaRS editing module, while retaining the long-established proofreading activity against L-serine and glycine (10,47–50), does not possess any detectable deacylase activity against D-alanine. The fact that AlaRS editing domain being inactive against flexizyme generated D-Ala-tRNAAla is in line with the literature that an aminoacyl-tRNA synthetase that charges D-amino acid cannot deacylate it using its cis-editing domain (Supplementary Figure S10B). As observed in the case of PheRS, which mischarges D-phenylalanine and L-tyrosine, the editing domain can only deacylate L-tyrosine but not D-phenylalanine . This is in accordance with the proposition that editing domains would have evolved to effectively deacylate the mischarged, chemically similar, non-cognate amino acids by aaRSs. It would be startling to identify a cis-editing domain with an ability to clear both mischarged non-cognate amino acid and D-form of amino acid, if at all it exists, as this would have important implications for the evolution of chiral fidelity in protein biosynthesis.Earlier, we proposed based on the mechanistic mode of action, DTD can carry out chiral proofreading of smaller amino acids (16,33). Now with the direct experimental proof of it acting on both smaller and larger D-amino acids, it is highly likely that DTD will act on the entire range of D-amino acids charged on tRNAs. The early translation machinery, which includes aaRSs, EF-Tu and the ribosome, in a pre-LUCA era may not have been as specific for L-chiral forms as the evolved ones that are present today. It is therefore tempting to propose that a DTD with its activity on the entire spectrum of D-aa-tRNA would have been beneficial in clearing the chiral mistakes thus enforcing homochirality during evolution of the protein biosynthetic machinery. Also, as we begin to understand how the metabolite fluxes are changing under different environmental conditions in organisms, retaining activity towards the total range of D-amino acids mischarged on tRNA may be beneficial and this aspect of DTDs physiological role, particularly in eukaryotes, needs to be explored further.DATA AVAILABILITYAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.Supplementary Materialgkad205_Supplemental_FileClick here for additional data file.

PMC

Heliyon

37251877

PMC10162472

5-05-2023

10.1016/j.heliyon.2023.e16024

Corrigendum to “The development of coronaphobia scale and psychometric effect among UAE people” [Heliyon 8(10) (October 2022) e10875]

Abdelrahman Rasha, Aldawash Fouad Mohammed

In the original published version of this article, the second author's middle name was misspelt, and their affiliation was incorrectly written. Their name and affiliation have now been corrected. The authors apologize for the errors. Both the HTML and PDF versions of the article have been updated to correct the errors.

PMC

Indian Journal of Ophthalmology

37322670

PMC10418014

6-01-2023

10.4103/IJO.IJO_25_23

Netarsudil monotherapy as the initial treatment for open-angle glaucoma and ocular hypertension in Indian patients: A real-world evaluation of efficacy and safety

Mathur Manoj Chandra, Ratnam P. Venkat, Saikumar S.J., John Manuel, Ravishankar Siddharth, Dinesh M.B, Chandil Priya, Pahuja Kishore, Cherlikar Vidya, Wadhwani Sunny, Bendale Pankaj, Hazari Ajit, Mishra Rajesh, Deshmukh Susheel, Achlerkar Rahul Raja, Shah Devang Tilak, Hingorani Chanda, Shah Kaivan, Topiwala Pratik, Jani Sheetal, Rana Viral G., Majumdar Nilay Kumar, Chakrabarti Debasis, Dey Rituparna, Halder Debabrata, Choudhury Sumit, Kumar Ajeet, Das Sasmita, Nanda Ashok Kumar, Kumar Vidya Bhushan, Dubey Rama, Kamdar Gulam Ali, Pandey Alka, Kishanpuria Sheetal, Srivastava Rajat Mohan, Singh Parul, Verma Sunil Kumar, Sharma Neha, Gupta Rajeev

Purpose:Glaucoma is the second leading cause of blindness worldwide, affecting more than 64 million people aged 40–80. The best way to manage primary open-angle glaucoma (POAG) is by lowering the intraocular pressure (IOP). Netarsudil is a Rho kinase inhibitor, the only class of antiglaucoma medications that reorganizes the extracellular matrix to improve the aqueous outflow through the trabecular pathway.Methods:An open-label, real-world, multicentric, observation-based 3-month study was performed for assessing the safety and ocular hypotensive efficacy of netarsudil ophthalmic solution (0.02% w/v) in patients with elevated IOP. Patients were given netarsudil ophthalmic solution (0.02% w/v) as a first-line therapy. Diurnal IOP measurements, best-corrected visual acuity, and adverse event assessments were recorded at each of the five visits (Day-1: screening day and first dosing day; subsequent observations were taken at 2 weeks, 4 weeks, 6 weeks, and 3 months).Results:Four hundred and sixty-nine patients from 39 centers throughout India completed the study. The mean IOP at baseline of the affected eyes was 24.84 ± 6.39 mmHg (mean ± standard deviation). After the first dose, the IOP was measured after 2, 4, and 6 weeks, with the final measurement taken at 3 months. The percentage reduction in IOP in glaucoma patients after 3 months of once-daily netarsudil 0.02% w/v solution use was 33.34%. The adverse effects experienced by patients were not severe in the majority of cases. Some adverse effects observed were redness, irritation, itching, and others, but only a small number of patients experienced severe reactions, as reported in a decreasing order: redness > irritation > watering > itching > stinging > blurring.Conclusion:We found that netarsudil 0.02% w/v solution monotherapy when used as the first-line treatment in primary open-angle glaucoma and ocular hypertension was both safe and effective.

Glaucoma is a chronic, progressive optic neuropathy characterized by loss of retinal ganglion cells, resulting in characteristic optic nerve head cupping and visual field defects. By 2040, it is anticipated that the global glaucoma burden will increase by more than 110 million. It is the second leading cause of blindness worldwide, affecting more than 64 million people aged 40–80. Furthermore, it is the leading cause of irreversible blindness in India, with at least 12 million people affected and approximately 1.2 million blind. According to the Directorate General of Health Services, the Ministry of Health and Family Welfare of India reports that glaucoma leads to blindness in 5.80% of cases. More than 90% of glaucoma cases in the community remain undiagnosed. In addition, the prevalence of glaucoma increases with age. According to the World Health Organization (WHO), there are several types of glaucoma; however, the two most common are primary open-angle glaucoma (POAG), having a slow and insidious onset, and angle-closure glaucoma (ACG), which is less common and tends to be more acute.Intraocular pressure (IOP) elevation is a major risk factor for POAG, and in most cases, the only way to slow or stop disease progression is to use pharmaceuticals to reduce IOP. Lowering IOP is the best way to manage POAG. The main contributors to IOP are episcleral venous pressure (EVP), aqueous humor flow rate, and resistance to outflow. IOP is regulated by a fine balance between aqueous humor production and the rate of aqueous outflow through the trabecular meshwork (TM) outflow and the uveoscleral outflow pathway. The majority of the aqueous humor is filtered through the TM pathway. Instead of directly targeting the TM pathway, current therapeutic options primarily increase uveoscleral outflow or decrease aqueous humor production to lower IOP. The TM is abnormal in POAG and ocular hypertension (OHT), but few medications specifically address this issue. Inadequate IOP treatments exist, and older drugs that targeted the TM either had a limited effect or were poorly tolerated.Netarsudil is a Rho kinase inhibitor, the only class of antiglaucoma medications that reorganizes the extracellular matrix to improve the aqueous outflow through the trabecular pathway. Its multimodal mechanism also includes a decrease in aqueous secretion and EVP. Furthermore, netarsudil is also believed to possess inhibitory action against the norepinephrine transporter (NET). Inhibition of NET prevents the reuptake of norepinephrine at the noradrenergic synapses, which increases the strength and duration of endogenous norepinephrine signaling. As a consequence of this enhanced signaling, norepinephrine-induced vasoconstriction that can reduce blood flow to the ciliary body may subsequently be responsible for a mechanism in which the formation of aqueous humor may be delayed, prolonged, or reduced as well.Major trials (like ROCKET-1, ROCKET-2) have evaluated the efficacy and safety of netarsudil in patients with open-angle glaucoma (OAG) or OHT. ROCKET-4 compared the efficacy and safety profile of netarsudil once daily versus timilol twice daily.Using a post-marketing surveillance trial, we evaluated the real-world efficacy of netarsudil ophthalmic solution (0.02% w/v) monotherapy as the first treatment in Indian patients to determine its safety and ocular hypotensive efficacy in patients with elevated IOP.MethodsStudy designAn open-label, real-world, multicentric, observation-based 3-month study for assessing the safety and ocular hypotensive efficacy of netarsudil ophthalmic solution (0.02% w/v) in patients with elevated IOP was performed using Goldmann applanation tonometry (GAT). The 6-month study involved 39 clinical sites in India (1 month enrollment, 3 months study period, and 2 months of data collection as well as analysis). Netarsudil ophthalmic solution (0.02% w/v) was given to patients as a first-line therapy.Eligibility criteriaAdults (18 years of age or greater) with a diagnosis of POAG or OHT, deemed to be appropriate for starting netarsudil at the treating ophthalmologist’s discretion, were included in the study. At the qualification visit, unmedicated IOP had to be >17 mmHg and 21 mmHg), but also in those with optic disk changes and normal IOP (i.e., patients with normal-tension glaucoma). Reducing the IOP reduces aqueous humor’s mechanical strain on the eye’s posterior structures, halting glaucomatous optic disk changes. β-adrenergic blockers, α-adrenergic agonists, and carbonic anhydrase inhibitors can reduce the rate of aqueous humor production. Cholinergic drugs (primarily trabecular outflow), α-adrenergic agonists, and prostaglandin (PG) analogs can improve aqueous humor drainage (largely uveoscleral outflow).Netarsudil reduces fibrotic material deposition in the TM and relaxes the overall tone of contractile cells, enhancing aqueous humor outflow and reducing the IOP. Netarsudil reduces aqueous humor production and improves drainage (trabecular pathway) by inhibiting NET, which lowers the IOP. Netarsudil topical has good corneal penetration. Eye esterases metabolize it to netarsudil-M1, a five-fold more potent active metabolite.[7,8] The IOP-lowering effect of netarsudil and its metabolite peaks 8 h after dosing and lasts 24 h. Once-daily netarsudil lowered the IOP across all baseline pressures. Netarsudil and its active metabolite are highly protein bound (>60%) to plasma proteins. Netarsudil has a 3-h half-life (approximately 175 min).Consequently, the active duration of IOP reduction justifies a single daily dose of netarsudil 0.02% w/v solution. Similar to previous studies in the American population by Zaman et al., our findings demonstrate the effect of netarsudil on IOP reduction in OAG and OHT in the Indian population. The safety and efficacy of netarsudil 0.02% w/v solution over 3 months were excellent, and minimal treatment-related adverse effects were observed. Some adverse effects observed were redness, irritation, itching, and others, but only a small number of patients experienced severe reactions, as reported in a decreasing order: redness > irritation > watering > itching > stinging > blurring. In 60% of patients, the right eye was examined, while in 40% of patients, the left eye was examined [Fig. 3]. The pharmacology and dynamic IOP-lowering effect of netarsudil monotherapy as the First-line treatment in Indian patients suggest its utility in the treatment of OAG and OHT.Figure 3Eyes examined at 39 centers; right = right eye, left = left eyeAdverse effects experienced by patients were not severe in the majority of cases.Possible retinal side effects were not evaluated in this study because retinal examination before and after administering netarsudil was not included in this study protocols. This should be considered as a limitation of this study.ConclusionWe found that netarsudil 0.02% w/v solution when used as monotherapy as the first-line treatment in POAG and OHT was both safe and effective.Financial support and sponsorshipThis study was funded by Ajanta Pharmaceuticals Inc.Medical writing support for the development of this manuscript, under the direction of the authors, was provided by Medwiz Healthcare Communication Pvt. Ltd.Conflicts of interestThere are no conflicts of interest.

PMC

BMB Reports

36443003

PMC10068341

3-31-2023

10.5483/BMBRep.2022-0137

Preventive effects of nano-graphene oxide against Parkinson’s disease via reactive oxygen species scavenging and anti-inflammation

Kim Hee-Yeong, Yoon Hyung Ho, Seong Hanyu, Seo Dong Kwang, Choi Soon Won, Ryu Jaechul, Kang Kyung-Sun, Jeon Sang Ryong

We investigated the neuroprotective effects of deca nano-graphene oxide (daNGO) against reactive oxygen species (ROS) and inflammation in the human neuroblastoma cell line SH-SY5Y and in the 6-hydroxydopamine (6-OHDA) induced Parkinsonian rat model. An MTT assay was performed to measure cell viability in vitro in the presence of 6-OHDA and/or daNGO. The intracellular ROS level was quantified using 2’,7’-dichlorofluorescein diacetate. daNGO showed neuroprotective effects against 6-OHDA-induced toxicity and also displayed ROS scavenging properties. We then tested the protective effects of daNGO against 6-OHDA induced toxicity in a rat model. Stepping tests showed that the akinesia symptoms were improved in the daNGO group compared to the control group. Moreover, in an apomorphine-induced rotation test, the number of net contralateral rotations was decreased in the daNGO group compared to the control group. By immunofluorescent staining, the animals in the daNGO group had more tyrosine hydroxylase-positive cells than the controls. By anti-Iba1 staining, 6-OHDA induced microglial activation showed a significantly decrease in the daNGO group, indicating that the neuroprotective effects of graphene resulted from anti-inflammation. In conclusion, nano-graphene oxide has neuroprotective effects against the neurotoxin induced by 6-OHDA on dopaminergic neurons.

INTRODUCTIONParkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN). The cardinal symptoms of PD include resting tremor, rigidity, and bradykinesia . The most common treatment for this condition is L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine, which can improve the motor symptoms of affected patients. However, as the loss of dopaminergic neurons gradually progresses, motor complications such as L-DOPA-induced dyskinesias, wearing-off, and motor fluctuations may occur . Electrical deep brain stimulation (DBS) has been widely used to alleviate these symptoms in advanced PD patients .There is currently no approved treatment that blocks or modulates the progression of PD. It is still therefore necessary to develop new disease-modifying therapies based on the pathophysiology of PD as an alternative to the existing dopamine-dependent treatments . In the pathogenesis of PD, among the most important disease causes are neuroinflammation and oxidative stress (5, 6). Graphene oxide (GO) has thus emerged as a possible candidate treatment for PD as it can suppress both of these processes (7, 8). Graphene quantum dots (GQD), which are graphene fragments of less than 20 nm in diameter , have been demonstrated to have biocompatibility and anti-inflammatory effects in patients with colitis . Moreover, GQDs have been shown to dissociate α-synuclein fibril by decreasing β-sheet structure, thereby preventing dopaminergic neuronal loss by reducing α-synuclein toxicity . However, the effects of GO on mitochondria can be both beneficial and detrimental. GO protects neuroblastoma cells against the prion-mediated mitochondrial toxicity via an autophagic flux . By contrast however, GO-induced reactive oxygen species (ROS) can disrupt mitochondrial homeostasis . Since the effects of GO greatly depend on its size (13-16), we manufactured GO with a 10 nm lateral size on average to minimize the possible induction of oxidative stress. We termed these deca nano-graphene oxide (daNGO). We here describe our investigation of the neuroprotective effects of the daNGO against ROS and inflammation in the human neuroblastoma cell line SH-SY5Y and in vivo in the 6-hydroxydopamine (6-OHDA) induced Parkinsonian rat model.RESULTSCharacterization of the deca nano-graphene oxidedaNGOs were synthesized using a modified Taylor method to obtain a single- or few-layers of graphene oxide with high oxidation efficiency (Fig. 1A). The lateral size of the daNGO particles was determined using a particle size analyzer (PSA) and was found to vary from 5 to 25 nm. The vast majority (99%) of the particles were less than 18.2 nm in size, and 50% of the distributed particles showed a size of 10.1 ± 7.3 nm. These data indicated that the average lateral size of the daNGOs was 10 nm (Fig. 1B). Images and the height profile of the daNGOs were characterized using AFM analysis. AFM images of 4 μμ2 indicated a nanoparticle with the height of 1.44 ± 0.14 nm (Fig. 1C, D). These results suggested that the daNGOs were composed of a single- or few layer graphene oxides of a nanoscale diameter.daNGOs protect SH-SY5Y cells against 6-OHDA-induced neurotoxicityAn in vitro MTT assay was performed to determine the level of cytotoxicity generated by 6-OHDA and to verify the neuronal protective effects of daNGO. SH-SY5Y cells were treated with 6-OHDA (5-400 μM) and/or daNGO (300 ng/ml-60 μg/ml) for 24 hours. Treatment with 6-OHDA alone decreased the cell viability in concentration-dependent manner but there was no significant change in cell viability upon exposure to various daNGO doses (Fig. 2B, C). When the cells were treated with both 6-OHDA and daNGO, the cell viability significantly increased as the concentration of daNGO increased compared to the cells treated with 6-OHDA only (Fig. 2D; *P < 0.05; ***P < 0.001).daNGOs scavenge 6-OHDA-induced ROS in SH-SY5Y cellsIt has been reported previously that 6-OHDA increases the intracellular oxidative stress level through ROS production in SH-SY5Y cells . To investigate the protective effects of daNGOs against oxidative stress caused by scavenging ROS, we measured the intracellular ROS levels using a DCF-DA assay in 6-OHDA-treated SH-SY5Y cells. The cells were stained with DCF-DA and analyzed using FACS equipment. Exposure of the cells to 6-OHDA increased the ROS level as compared with the control group. However, co-treatment with both 6-OHDA and daNGO significantly decreased the ROS level compared with the control group and 6-OHDA only group (Fig. 2E, F; **P < 0.01; ***P < 0.001).daNGOs improve the symptoms of forelimb akinesiaWe conducted in vivo analyses of the effects of daNGOs in a rat model of PD. daNGOs (30 mg/kg, 5 days, i.p.) were injected into the animals immediately after 6-OHDA injection. All the rats in the PD control group showed considerable contralateral forelimb akinesia in stepping tests (Fig. 3B, D). In contrast, this symptom was significantly improved in the daNGO group (Fig. 3B, D; ***P < 0.001). These beneficial effects of the daNGOs persisted two weeks after 6-OHDA injection (Fig. 3B, D). No effect of a unilateral 6-OHDA injection was seen on the number of ipsilateral forelimb steps in any of the rats (Fig. 3C).daNGOs alleviate apomorphine-induced rotationApomorphine-induced rotation tests were performed in the PD model rats at 7 days after 6-OHDA injections. The daNGO group showed a significant decrease in net (contra-ipsi) rotations per minute compared with the PD control group (Fig. 3E; *P < 0.05). In the 5 min interval record, the number of net rotations decreased significantly in the daNGO group until 15 minutes after apomorphine injection compared with the PD control group (Fig. 3F; *P < 0.05; **P < 0.01).daNGOs protect dopaminergic neurons from 6-OHDA-induced neuroinflammationImmunofluorescent staining of sections of lesioned SNs from the PD rat model revealed a considerable loss of tyrosine hydroxylase (TH)-positive cells in the PD control group (−87.4 ± 1.8%; Fig. 4A) compared with the daNGO group (−67.9 ± 3.6%; Fig. 4B). There were significant differences found between these two groups (**P < 0.01, *P < 0.05; Fig. 4C, D). We next conducted anti-Iba-1 staining to explore whether the daNGOs modulated 6-OHDA-induced neuroinflammation. The results indicated a significant decrease in the large size of the Iba-1 positive cells of ipsilateral SN in the daNGO group compared with the PD control group, meaning that the number of activated microglia (which have an increased cell body) was decreased in the daNGO group (*P < 0.05; Fig. 4E).DISCUSSIONGO is a promising therapeutic agent that consists of oxidizing graphite carbon atoms . It has a large surface area, contains functional groups, and has a high biocompatibility, which can be used as a drug carrier in cancer therapy (19, 20). Graphene nanostructures can also cross the blood brain barrier and thereby provide a highly efficient delivery to targeted brain areas. Regarding the pathophysiology of PD, ROS- and glial cell-induced neuroinflammation are known to be principal factors in the generation of sporadic PD . In the current literature, ROS have been reported to also induce mitochondrial dysfunction and oxidative stress . In previous studies, GQD was demonstrated to have anti-inflammatory effects against colitis, and we expected that it would exert these same effects in the brain as it can cross the blood-brain barrier . In addition to this, GO quantum dots (lateral sizes, 20-40 nm) can reduce the ROS level and exert neuroprotective effects . However, some studies have also reported that GO can produce ROS and oxidative stress, causing cytotoxicity (25-27). Previous studies have shown that larger GOs (lateral sizes, 750-1300 nm) increased macrophages and induced stronger inflammation (14, 15). In contrast, smaller GOs showed a high potential for ROS scavenging . Moreover, nanoscale GO had great anti-oxidant and anti-inflammatory effects in vivo (13, 24). Hence, we chose to use nanoscale GO in this study. We consider therefore that the opposing functions of GO regarding neuroprotective and neurotoxic effects are dependent on size i.e. nanoscale GO particles have ROS scavenging and neuroprotection characteristics whereas a larger GO size has contrary effects. In addition, GO size could be a crucial factor to enhance GO permeability across the blood-brain barrier . In our current study, we manufactured nanosized GO, which we termed daNGOs due to the 10 nm lateral size of these particles on average. To evaluate the potential neuroprotective, anti-neuroinflammatory, and ROS reducing effects of the daNGOs, we used 6-OHDA and the neuroblastoma cell line SH-SY5Y. 6-OHDA is a neurotoxin that specifically destroys dopaminergic neurons by causing oxidative stress and inflammation . SH-SY5Y cells are widely used and thus well established in PD studies . In these initial in vitro experiments, we found that daNGO treatments had neuroprotective effects against 6-OHDA-induced toxicity and acted as a scavenger of ROS, thus leading to a suppression of inflammation for which the ROS are signal molecules .We further tested the protective effects of daNGOs against 6-OHDA induced toxicity in vivo using the well-known 6-OHDA-induced rat model of PD. The use of 6-OHDA in these animals induces oxidative stress at mitochondrial complexes, resulting in more than 90% of dopaminergic neuronal death in a few days and also clear motor symptoms . In our current experiments, we found that the akinesia symptoms were improved in the daNGO group compared with the control group when we conducted a stepping test. This result suggests that daNGOs will prevent dopaminergic cell loss from 6-OHDA-induced toxicity because the stepping test is well-established method for evaluating forelimb akinesia and for predicting the extent of dopaminergic depletion . Stepping test scores have also been reported to be well correlated with striatal dopamine depletion . Furthermore, our present results from an apomorphine-induced rotation test of our PD model animals indicated a lower loss of dopaminergic neurons in the daNGO group.By immunofluorescent staining of our experimental rats, we found that the number of TH-positive cells in the daNGO group was higher than that in the PD control group, further indicating that dopaminergic cells were preserved by exposure of these animals to the daNGOs. TH is a rate limiting enzyme of dopamine synthesis and is used as a dopaminergic neuronal marker. Iba-1 is the marker of microglial activation and inflammation. Activated microglia show an increased cell body and amoeboid shape. In the PD control group, the 6-OHDA injections induced significantly increased microglial activation compared to the daNGO group, again suggesting that the neuroprotective effects of the daNGOs resulted from their anti-inflammatory actions. All things considered, we conclude from our current findings that daNGOs have neuroprotective and preventive effects against the neurotoxicity induced by 6-OHDA on dopaminergic neurons, both in vitro and in vivo.The most important cause of PD is the loss of dopaminergic neurons due to neuroinflammation and α-synuclein toxicity. Thus, protecting dopaminergic neurons from these toxic events could be a disease modifying treatment for PD. GO is known to inhibit the aggregation of abnormal proteins that induce neuronal cell death, and recent studies revealed that GO reduces α-synuclein toxicity by preventing α-synuclein amyloid formation (10, 33-35). Our present study confirmed that daNGO has ROS-scavenging and anti-inflammation effects, showing its potential application for PD treatment. Our results also suggest an expanded daNGO application in other neuroinflammation-related neurodegenerative diseases, such as Alzheimer’s disease, multiple sclerosis, and traumatic brain injury as well as abnormal protein aggregation-related diseases. Furthermore, daNGO could be applied to other research fields to elucidate its effects on inflammation processes that occur throughout pain, immune reactions, and degenerative changes.MATERIALS AND METHODSPreparation of deca nano-graphene oxide (daNGO)daNGO preparations were provided by Biogo Co.,LTD (Seoul, Korea), and the graphene oxide was synthesized from graphite using the modified Taylor method . Briefly, the oxidation of bulk graphite with oxidizing agents (KMnO4 and H2SO4) and dispersion were performed and the daNGOs were suspended in deionized water. For measuring the thickness of daNGO particles, samples were prepared on a silicon wafer and measured by atomic force microscopy (AFM) (NX10; Park Systems, Suwon, Korea). Additionally, the lateral sizes of the daNGOs were analyzed by particle size analysis (PSA) (CPS Disc Centrifuge; CPS Instruments, Prairieville, Louisiana).Cell cultureThe human dopaminergic neuroblastoma SH-SY5Y cell line was obtained from KCLB (Korean Cell Line Bank, Seoul, Korea) and grown in DMEM/F-12 (Gibco, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (Gibco) and 1% (100 U/ml) penicillin-streptomycin in humidified incubator with 5% CO2 at 37°C.MTT assayMTT assays were performed to measure cell viability. Briefly, SH-SY5Y cells were seeded at a density of 105 cells per well in a 24-well culture plate (Nunc, Roskilde, Denmark), and grown to confluency. The confluent cells were treated with 6-OHDA (5-400 μM) or/and daNGO (0.3 μg/ml-300 μg/ml) for 24 h. After treatment, the cells were incubated for a further 2 h in 500 μl of MTT solution (0.25 mg/ml of fresh medium) at 37°C with 5% CO2. The growth medium was then replaced with 500 μl of DMSO was added and incubated with shaking for 2-3 min. Absorbance of the converted dye in living cells was detected at a wavelength of 570 nm using an Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland).Measurement of intracellular ROSThe levels of intracellular ROS were quantified using 2’,7’-dichlorofluorescein diacetate (H2-DCF-DA, Invitrogen, Waltham, MA). DCF-DA, a non-fluorescent compound, is deacetylated by ROS within the cell into 2’,7’-dichlorofluorescein (DCF), showing green fluorescence. SH-SY5Y cells were treated with 6-OHDA (Sigma, St. Louis, MO; 10 μM) or/and daNGO (3 μg/ml) for 24 h. After washing, the cells were collected and incubated with 20 μM DCF-DA for 30 min at 37°C in 5% CO2. The cells were then washed twice with PBS, and the relative levels of fluorescence were measured by flow cytometry on a FACSCalibur using CellQuest software (BD Biosciences, Franklin Lakes, NJ).Experimental animalsTwenty three male Wistar rats (Orient Bio Inc., Seongnam, Korea), weighing 300-350 g at the beginning of the experiment, were housed with a 12/12 h light/dark cycle and given free access to food and water. All of the animal procedures used in this study complied with the guidelines of the Institutional Animal Care and Use Committee of the Asan Institute for Life Sciences (Seoul, Korea).Further details are provided in the supplementary information.

PMC

Middle East African Journal of Ophthalmology

PMC10903710

1-22-2024

10.4103/meajo.meajo_89_23

Topography and Choroidal Thickness Measurement in Healthy Asian Indian Subjects using RTVue XR 100 Optical Coherence Tomography

Mansoori Tarannum, Charan Aknoor S. R., Nagalla Balakrishna

Abstract:PURPOSE:The purpose was to study the choroidal thickness and its profile, derived from different point locations in healthy Asian Indian subjects using RTVue XR 100 optical coherence tomography (OCT) and to determine its correlation with age, refractive error, and axial length.METHODS:In this cross-sectional study, 300 eyes of 150 healthy subjects, with no ocular pathology, were scanned in a single session, using a line scan protocol of RTVue XR 100 OCT. Choroidal thickness was measured at the subfoveal region, and six measurements were obtained on either side of the fovea (temporal and nasal) at 500 μm interval apart, up to 3000 μm. The correlation between subfoveal choroidal thickness and age, refractive error, and axial length was assessed.RESULTS:Three hundred eyes of 150 healthy subjects were included in the analysis. Median age of the study participants was 55 years (interquartile range [IQR]: 44–61). The median subfoveal choroidal thickness was 235 μm (IQR: 210–263). The choroidal thickness was minimum at nasal 3000 μm from the fovea, while it was maximum in the subfoveal region. The point zones which were near the fovea showed thicker choroidal thickness than the outer zones, both nasally and temporally (P < 0.00001 at all locations), and at all point locations the choroid were thicker temporally than nasally (All P < 0.00001). Subfoveal choroidal thickness showed negative correlation with age (coefficient = −0.62, P = 0.03) and axial length (correlation = −8.52, P = 0.02). A decrease in subfoveal choroidal thickness of 0.62 μm/year was found by regression analysis.CONCLUSION:Our study provides normative database and topographic profile of choroidal thickness in the normal Asian Indian eyes using RTVue XR 100 OCT.

IntroductionEnhanced depth imaging optical coherence tomography (OCT) provides visualization of choroid and allows noninvasive and in vivo measurement of the choroidal thickness. Choroidal thickness measurement is useful to monitor chorioretinal disease onset and its progression.In the Indian population, choroidal thickness has been reported using the spectral domain (SD)-OCT and swept-source OCT (SS-OCT). Choroidal thickness measurement in the normal population with RTVue XR OCT has not been reported. As there exists a considerable variation in the normal choroidal thickness, it is essential to have a normative database for choroidal thickness among different populations with different OCT machines. Furthermore, choroidal thickness measurements in different macular regions should be known to understand how the variation in thickness changes with various other ocular parameters in healthy adults.Our objectives were to determine the normative database for choroidal thickness and its topographic profile derived from different point locations in the normal Asian Indian eyes using RTVue XR 100 OCT. Furthermore, the correlation between subfoveal choroidal thickness and various ocular factors was assessed.MethodsThis cross-sectional study was conducted at our institute from January 2022 to March 2022. Institute internal review board approved the study protocol, and informed consent was taken from all the subjects.The study cohorts were healthy staff volunteers and patients with no evidence of ocular disease (except cataracts), age range of of 20–80 years. Subjects with a history of intraocular pathology, prior surgery (other than cataract surgery), refractive error >−6 Diopter (D), and >+3 D; retinal pathology or retinal pigment epithelium (RPE) abnormality detected on OCT and poor fixation while performing OCT were excluded from the study.A comprehensive ophthalmic examination, including medical and ocular history, best-corrected visual acuity, refraction, slit-lamp examination, intraocular pressure (IOP) measurement using Goldmann applanation tonometry, and dilated fundus examination, was carried out.Ocular imaging protocolAfter pupillary dilatation with 2.5% phenylephrine hydrochloride and 1% tropicamide eye drops, all the participants underwent High density (HD) Angio Retina scans in a single imaging session under standardized mesopic lighting conditions. All the scans were taken by a retina fellow in training. A single line, enhanced HD scan was performed, centered at the fovea, using the AngioVue HD software (version: A2018,0,0,18) of the Avanti RTVue XR SD-OCT device (OptoVue, Inc., Fremont, California). The 6 mm × 6 mm HD Angio Retina scans, centered on the fovea, were obtained with a rate of 70,000 A-scans per second. Each volume contains 304 × 304 A-scans with two consecutive B-scans captured at each fixed position before proceeding to the next sampling location. The current software uses motion correction technology to reduce motion artifacts and HD scanning mode to improve the resolution of scans. Each OCT scan was reviewed to ensure sufficient clarity in the visualization of the choroid–scleral boundary. Scans with signal strength < 50 or segmentation error were repeated until the image quality obtained was satisfactory. The scans were performed between 2 pm and 4 pm to account for the effect of diurnal variation on choroidal thickness.Measurements were made by the same examiner, using calipers provided within the proprietary software by measuring the distance between the hyperreflective lower border of RPE/Bruch’s membrane complex to the outer hypo reflective choroidal surface, which marks the choriod-scleral interface. Choroidal thickness was measured at 13 locations, consisting of subfoveal choroidal thickness and six measurements on either side of the fovea (temporal and nasal) at 500 μm intervals apart, up to 3000 μm [Figure 1].Figure 1Measurements of choroidal thickness from hyperreflective lower border of retinal pigment epithelium/Bruch’s membrane complex to the outer hypo reflective choroidal surface, which marks the choriod-scleral interface, at subfoveal region and six measurements on either side of the fovea (temporal and nasal) at 500 μm interval apart, up to 3000 μm. OS: Oculus sinister, HD: High densityRefractive error was measured using the automated refractometer (Charops CR1, Korea Huvitz), and axial length measurement was performed using the optical biometer AL scan (Nidek, Gamagori, Japan).Statistical analysis was performed using the SPSS software Version 17 (IBM Corporation, Chicago, IL, USA). The Shapiro–Wilk test was used to assess the normality of distribution. Descriptive statistics included mean and standard deviation for normally distributed variables and median and interquartile range (IQR, first quartile, and third quartile) for nonnormally distributed variables. The correlation between both eyes of the same individual was adjusted using generalized estimating equations. A multivariate regression analysis was performed to determine the correlation between subfoveal choroidal thickness and age, axial length, spherical equivalent, and IOP.ResultsThree hundred eyes of 150 healthy participants were included in the analysis. Eighty subjects were females and 70 were males. The median choroidal thickness was not statistically different between males and females (P = 0.8). Median age of the study participants was 55 years (IQR: 44–61). The mean axial length was 23.08 ± 0.83 mm, and the median spherical equivalent was 0 diopters (IQR: 0–1) [Table 1].Table 1Clinical characteristics of study participants (n=150 subjects, 300 eyes)Parameters measuredValues obtainedAge (years)55 (44–61)Gender (male/female)70/80Number of subjects with diabetes mellitus74Number of subjects with hypertension48Intraocular pressure (mmHg)*12 (12–14)Spherical equivalent (diopters)*0 (0–1)Axial length (mm)§23.08±0.83Anterior chamber depth (mm)*3.23 (3.05–3.54)OCT scan signal strength*62 (61–65)Systolic blood pressure (mm Hg)*130 (120–140)Diastolic blood pressure (mm Hg)*80 (70–90)Phakia/Pseudophakia249/51*Values are given in median (1st–3rd interquartile), §Value is in mean±SD. SD: Standard deviation, OCT: Optical coherence tomographyThe median subfoveal choroidal thickness was 235 μm (IQR: 210–263). The choroidal thickness was minimum at nasal 3000 μm from the fovea, while it was maximum in the subfoveal region. All the point zones that were closer to the fovea showed thicker choroidal thickness than the points away from the fovea, both nasally and temporally (P < 0.00001 at all the locations). At all point locations, the choroid was thicker temporally than nasally (All P < 0.00001) [Table 2].Table 2Choroidal thickness at the various locations from the foveaVariables (mm)Median (1st–3rd quartile)Subfoveal choroidal thickness235 (210–263)Nasal choroidal thickness - 500220 (192–251)Nasal choroidal thickness - 1000210 (179–243)Nasal choroidal thickness - 1500201 (165–229)Nasal choroidal thickness - 2000183 (152–214)Nasal choroidal thickness - 2500164 (131–195)Nasal choroidal thickness - 3000133 (108–164)Temporal choroidal thickness - 500223 (195–251)Temporal choroidal thickness - 1000210 (183–242)Temporal choroidal thickness - 1500201 (175–235)Temporal choroidal thickness - 2000189 (164–223)Temporal choroidal thickness - 2500183 (161–207)Temporal choroidal thickness - 3000171 (149–195)Age-wise choroidal thickness at all the point locations is shown in Table 3.Table 3Age-wise distribution of choroidal thickness* at different point locationsVariables (µm)20–30 years (n=38)31–40 years (n=38)41–50 years (n=68)51–60 years (n=76)61–70 years (n=50)71–80 years (n=30)Subfoveal choroidal thickness238 (217.75–282.5)263 (234.25–277.5)246.5 (223–275.75)237 (209–260)215 (201.75–234.25)203.5 (183–242.5)Nasal choroidal thickness - 500223 (199.5–266)257 (212.25–276)233.5 (202.25–264.75)223 (186–250)207 (176.75–223)192.5 (174–221.75)Nasal choroidal thickness - 1000217 (183–250.75)244 (185.75–266.25)226 (189.75–248)211 (175–235)195 (168.75–215)183.5 (171–218.75)Nasal choroidal thickness - 1500201 (174–247)226 (187.5–250.25)215.5 (184.25–243.75)204 (164–228)179.5 (155–205.75)177.5 (148.25–198)Nasal choroidal thickness - 2000193.5 (158.75–205.5)204 (183–238.25)195 (177.5–219.75)183 (149–217)164 (144.5–186)153.5 (132.75–188.5)Nasal choroidal thickness - 2500173.5 (149.75–199.5)168.5 (139–201.75)174 (146–204)158 (130–201)146.5 (124.75–173.25)127 (115.5–186)Nasal choroidal thickness - 3000139 (121.75–164)139.5 (108.75–170.75)137.5 (114.25–164)139 (110–179)127 (94.5–147.25)109 (91.5–130.75)Temporal choroidal thickness - 500227.5 (195.75–256.25)229.5 (214.75–265.25)233.5 (207–254)223 (196–250)198 (181.5–225.25)202.5 (170.75–245.25)Temporal choroidal thickness - 1000214 (184.5–253.25)224.5 (195–257.25)221.5 (189.75–244)210 (178–243)193.5 (173.75–213.25)196.5 (161.75–220)Temporal choroidal thickness - 1500209 (181.5–243.25)223 (203.25–237.5)209.5 (184.5–235)192 (174–243)181.5 (173–203.25)187.5 (152–206.75)Temporal choroidal thickness - 2000213.5 (183.75–244)213.5 (193.5–240.5)193.5 (171.5–214)186 (163–226)167.5 (155–195)170 (150.75–212.5)Temporal choroidal thickness - 2500202.5 (171.5–237.5)201 (179.25–227.75)186 (165–210)180 (152–203)167 (150.5–176.75)181 (143–211.75)Temporal choroidal thickness - 3000184.5 (165–222.75)182.5 (161–210.25)183.5 (158.5–205.75)163 (149–187)156.5 (134.25–180)150 (127–207.25)*Values are given in median (1st–3rd interquartile)Regression analysis showed a 0.62 μm/year decrease in subfoveal choroidal thickness. A negative correlation was found between subfoveal choroidal thickness and spherical equivalent (correlation = −0.79, P = 0.73), although it was not statistically significant. Subfoveal choroidal thickness showed negative correlation with age (coefficient = −0.62, P = 0.03) and axial length (correlation = −8.52, P = 0.02) [Table 4].Table 4Effect of various parameters on subfoveal choroidal thicknessCoefficientSE P 95% CI Lower boundUpper boundAge (years)−0.6220.2870.032−1.188−0.055Gender1.1875.7380.836−10.12912.503Spherical equivalent (diopters)−0.7882.3260.735−5.3753.798Axial length (mm)8.5233.6670.0211.29115.755Anterior chamber depth (mm)−6.5307.4470.382−21.2168.156Systolic blood pressure (mmHg)−0.1070.3630.768−0.8230.609Diastolic blood pressure (mmHg)0.3440.4460.441−0.5351.223Intraocular pressure (mmHg)−1.3521.8600.468−5.0202.315CI: Confidence interval, SE: Standard errorDiscussionAs choroidal thickness varies among different ethnic populations and also with the type of OCT machine, it is important to establish a normative database in a particular population on a particular OCT machine. In this cross-sectional study, we report the normative database of choroidal thickness in 300 eyes using RTVue XR 100 OCT. To the best of our knowledge, it has not been reported before. We found progressive changes in choroidal thickness across all the point locations within a wide age range. Choroidal thickness showed variation in different point zones in the macula, being thickest in the subfoveal region, followed by the temporal region, and thinnest in the nasal region near the optic disc.Choroidal thickness measurementMean subfoveal choroidal thickness in the normal eyes varies between different studies, and the differences could be due to a difference in the measuring software of the OCT, different light sources in the OCT machine, different population ethnicity, and patient profiles such as variable axial length/refractive error.In our study, the median subfoveal choroidal thickness was 235 μm (IQR: 210–263) with RTVue XR SD-OCT. Choroidal thickness in the normal Indian population has been studied with SD-OCT and SS-OCT Chhablani et al. obtained the HD line raster scans using Cirrus HD-OCT (Carl Zeiss Meditec, Inc., Dublin, CA, USA) in 211 eyes of 115 healthy controls and reported that the mean subfoveal choroidal thickness varied from 294.8 ± 46.5 μm (age range: 20–29 years) to 249.6 ± 36.0 μm (age range: 70–79 years). Another study reported choroidal thickness in 238 eyes (mean age, 28.70 ± 11.28 years) of 119 healthy Indian subjects using SS-OCT (DRI-OCT Triton Plus, TOPCON, Japan). The mean subfoveal choroidal thickness was found to be 299.10 ± 131.2 μm. Previous studies have reported that the choroido-scleral interface is better visualized with SS-OCT than with SD-OCT.TopographyChoroid is a three-dimensional structure and has been shown to exhibit considerable topographic variation. As chorioretinal diseases are not only confined to the fovea but also may involve different regions of the macula, it is essential to understand how the ocular parameters (spherical equivalent, axial length, or age) affect choroidal thickness.In our study, maximum choroidal thickness was noted at the subfoveal region, and a gradual statistically significant decrease in thickness was noted, nasally and temporally, as the distance increased further. This thinning was more prominent on the nasal side of the fovea and the thinnest near the optic nerve head. A previous study reported that the choroidal thickness is greater in the foveal region than in the nasal or temporal retina due to high metabolic demand in the foveal area.Correlation of subfoveal choroidal thickness with other variablesEarlier studies have reported that choroidal thickness varies with age. Ikuno et al. reported a 14 μ/decade decrease in the choroidal thickness. Chhablani et al. reported a negative correlation of age with the choroidal thickness, and a decrease of 1.18 μm/year of choroidal thinning was observed. In our study, there was a significant negative correlation of age with the choroidal thickness, with a 0.62 μm decrease in the subfoveal choroidal thickness with each year. Bhayana et al. found that subfoveal choroidal thickness and age have a negative correlation, which was not statistically significant and they attributed to the fact that the small age range (20–40 years) of the study cohort as the age-related choroidal thinning is mostly seen after 60 years. Some studies have reported no significant correlation between the spherical equivalent and choroidal thickness, while others have reported that choroidal thickness varies with spherical equivalent and axial length.In our study, a negative correlation was found between subfoveal choroidal thickness and axial length, which was statistically significant, similar to the previous study. This means that eyes with longer axial lengths have thinner subfoveal choroid while the eyes with shorter axial lengths have thicker choroid. This should be taken into account while interpreting the subfoveal choroidal thickness in the eyes with high myopia and hyperopia.Limitations of our study were that we did not measure choroidal thickness in the superior and inferior locations, and our study was limited to the subjects of Asian Indian ethnicity.The strengths of this study are the inclusion of a wide age range of participants, and all the OCT scans were performed by a single examiner and in a fixed time period to minimize diurnal variation effect on the choroidal thickness measurement.ConclusionWe have demonstrated that choroidal thickness varies significantly with age and axial length. The choroidal thickness topography varies, based on the different regions of the macula. Our study provides a normative database and topographic profile of choroidal thickness in normal Asian Indian eyes using RTVue XR 100 OCT.Financial support and sponsorshipNil.Conflicts of interestThere are no conflicts of interest.

PMC

Heliyon

PMC10333625

6-20-2023

10.1016/j.heliyon.2023.e17481

Recent advances in CD8

Arenas Valentina Restrepo, Rugeles María T., Perdomo-Celis Federico, Taborda Natalia

Achieving a cure for HIV infection is a global priority. There is substantial evidence supporting a central role for CD8+ T cells in the natural control of HIV, suggesting the rationale that these cells may be exploited to achieve remission or cure of this infection. In this work, we review the major challenges for achieving an HIV cure, the models of HIV remission, and the mechanisms of HIV control mediated by CD8+ T cells. In addition, we discuss strategies based on this cell population that could be used in the search for an HIV cure. Finally, we analyze the current challenges and perspectives to translate this basic knowledge toward scalable HIV cure strategies.

1Introduction HIV cure: a global priorityThe worldwide occurrence of HIV infection poses a significant issue for public health on a global scale, with an approximate 38.4 million individuals affected by HIV in 2021 . In addition to this widespread prevalence, the efforts made by countries to address the HIV epidemic have been inadequate. There exists a delay in diagnosis and a low detection rate [2,3], and only 75% of people living with HIV received antiretroviral therapy (ART) in 2021 . Numerous factors, such as adverse effects of medication, logistical challenges in accessing drugs, and the financial burden associated with lifelong treatment, contribute to suboptimal adherence to ART [4,5]. For instance, individuals receiving ART have demonstrated an increased susceptibility to cardiovascular disease, kidney problems, and bone disorders [6,7]. The clinical status before therapy initiation, virological factors (such as drug resistance mutations), sociodemographic aspects, and inadequate adherence to treatment, all contribute to an ongoing risk of developing resistance to therapy and subsequent treatment failure . Additionally, the absence of a vaccine with satisfactory efficacy in preventing new infections adds to these challenges . Lastly, the persistent social stigma attached to being an HIV carrier exacerbates the situation. These obstacles emphasize the urgent need for finding a cure for HIV, both in terms of public health and global research. A safe, effective, scalable, and cost-effective intervention that promotes sustained virus control in the absence of ART would provide a powerful tool for eventual epidemic control .2Why is it so difficult to find a cure for HIV?Despite the identification of HIV in the mid-80s and important research efforts have been made to understand its pathophysiology and the mechanisms for viral control, there is no effective and scalable strategy to achieve disease remission. Numerous factors contribute to the challenge of finding a cure for HIV infection, linked to the characteristics of viral latency and intrinsic genetic variability of retroviruses. Furthermore, HIV exhibits various mechanisms of immune evasion, such as its location in multiple tissues with limited access to immune effector cells. Of note, antiretroviral drugs block infection in susceptible cells and inhibit active virus replication, but do not act on integrated proviruses in cells with latent infection . The main mechanisms that determine the difficulty in achieving a cure for HIV, and the impact of the CD8+ T cell response against HIV, are described below.2.1HIV latencyHIV belongs to the retroviridae family, which has mechanisms to establish latent reservoirs in host cells. These viral reservoirs are characterized by cells harboring integrated viral DNA (known as provirus) that are transcriptionally silent. However, upon cellular activation, these reservoirs have the capability to generate infectious viral particles . Macaque studies have demonstrated that the lentivirus reservoir is rapidly established upon viral exposure, escaping to innate and adaptive immune effector mechanisms . After reservoir seeding, several mechanisms favor HIV deep latency, including epigenetic repression of HIV transcription , temporary absence of host transcriptional factors [14,15], and provirus integration in non-genic regions of the genome . The low rate of replication in viral reservoirs determines their high stability over time. For example, previous studies have shown that proviruses with replicative capacity can persist free of selective pressure in long-lived CD4+ T cells . In addition, it has been estimated that the viral reservoir has a half-life of 44 months , so it would take more than 70 years to naturally eradicate a reservoir consisting of only 106 cells . Importantly, CD8+ T cell escape and resistance mutations [20,21], as well as resistance to type 1 interferons (IFN) and antibodies [22,23], can be found in the HIV reservoir, determining a further challenge for the design of HIV cure immunotherapies.Intriguingly, CD8+ T cells play a dual role in the immune response against HIV. On one hand, they effectively eliminate HIV-infected cells, while on the other hand, they release soluble molecules that interact with infected CD4+ T cells. This interaction triggers a series of signaling events that lead to non-cytolytic suppression of HIV, inhibition of viral transcription, and promotion of cellular quiescence and stemness. These processes, in turn, may contribute to the establishment of viral latency [24,25]. For instance, the CD8+ T cell antiviral factor (CAF) was reported to inhibit HIV transcription by reducing the association of RNA polymerase II with the HIV promoter . More recently, it has been discovered that CD8+ T cells express ligands that bind to Wnt-frizzled receptors, thereby activating the canonical Wnt/β-catenin signaling pathway. This activation leads to the suppression of HIV transcription in infected cells . In addition, it was reported that this non-cytolytic CD8+ T cell-mediated suppression of HIV replication is independent of the major histocompatibility complex (MHC), and is linked to the silencing of the LTR-dependent viral transcription, and the decrease of CD4+ T cell activation and proliferation [25,28]. These mechanisms explain in part the enhancement of latency reversal mediated by several compounds when accompanied by CD8+ T cell depletion in simian immunodeficiency virus (SIV)-infected macaques [29,30], as well as the loss of ART-mediated viral suppression in SIV-infected macaques when CD8+ T cells are depleted .2.2High viral diversityThe extraordinary genetic diversity in the circulating subtypes of HIV has hampered the development of a cure . There are at least three mechanisms that explain the variability of HIV: i) the high error rate of the viral reverse transcriptase ; ii) recombination phenomena of the strands of the viral genome [34,35]; iii) cellular factors such as APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) and RNA polymerase II, which may induce changes in the viral nucleotide sequence [36,37]. Together, these mechanisms induce a mutation rate estimated at 3.4 × 10−5 substitutions/site/replication cycle , leading to the generation of circulating subtypes and recombinant forms that harbor antiretroviral resistance and immune evasion mutations.2.3Immune evasionIn addition to the antigenic escape product of viral mutations and CD4+ T cell depletion, HIV has additional mechanisms for immune evasion. For example, it has been shown that HIV evades recognition by intracellular receptors such as Cyclic GMP-AMP synthase (cGAS), and Toll-like receptors 7, 8, and 9 inhibiting the production of type I IFN, critical for the control of viral replication . Additionally, the viral protein Nef induces the downregulation of human leukocyte antigen (HLA) class I molecules, preventing recognition by CD8+ T cells . On the other hand, throughout the process of HIV entry into the host cell, the virions can mask specific functional domains of the gp120 glycoprotein, thus eluding detection by antibodies . Taken together, these mechanisms are associated with poor natural control of HIV replication by the host immune system.2.4Localization of the virus in multiple tissues with limited access to immune effector cellsLentiviruses are localized in multiple tissues that are not easily accessible to immune cells. For example, analysis in SIV-infected macaques determined virus localization in tissues such as lymph nodes, intestine, brain, spleen, heart, kidney, lung, and liver . The case of secondary lymphoid organs is relevant considering that lymphoid follicles are the main reservoirs of the virus , and there is poor access of cytotoxic cells to these compartments . Another relevant compartment is the central nervous system, where resident macrophages and astrocytes, can be infected by HIV, persisting for a long time due to the limited access to immune effector cells .2.5Chronic immune activation and immune exhaustionChronic immune activation is one of the main features of HIV infection . The mechanisms that determine persistent immune activation include: i) immunomodulatory functions of viral proteins and immune response against the virus; ii) reactivation of other latent infections in the individual; iii) loss of gastrointestinal mucosal integrity, followed by microbial translocation; iv) alteration in the balance of CD4+ T cells; and v) increased production of proinflammatory cytokines . One consequence of chronic immune activation and antigen persistence is immune exhaustion, particularly in CD8+ T cells, which impairs the functional capacity and viral control, associated with the increased expression of inhibitory receptors such as programmed death (PD)-1 and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), metabolic dysregulation, and poor cell survival .3Models of functional cure of HIV: learning from the exceptionsThe major proportion of untreated people with HIV suffers progressive disease, developing acquired immune deficiency syndrome (AIDS). However, a small proportion of individuals (50% of animals vaccinated [103,104]. Notably, RhCMV/SIV vectors can elicit non-canonical MHC-II and MHC-E-restricted CD8+ T cells that can recognize universal epitopes termed supertopes [105,106], and are essential for RhCMV/SIV vaccine efficacy . This novel mechanism of CD8+ T cell-mediated protection may exploit SIV/HIV immune-evasion adaptations, such as down-modulation of MHC-I molecules and upregulation of MHC-E.In addition to the vaccine-mediated stimulation of CD8+ T cell responses, it was recently shown that the administration of NAb in macaques with Simian-Human Immunodeficiency Virus (SHIV) infection, or in individuals with HIV, promotes the function of virus-specific CD8+ T cells. This effect occurs probably through the modulation of T cell priming, and this vaccinal effect explains, at least in part, the sustained viral control in these individuals [, , ]. In this context, NAb-HIV immune complexes may activate dendritic cells to increase their capacity for conventional and crossed antigen presentation , but new studies are required to elucidate the mechanisms involved. Thus, immunotherapy with monoclonal antibodies offers not only viremia control mediated by virus neutralization, but also enhances the endogenous cellular response, promoting long-lived memory responses.5.2Cytokines and immune adjuvantsSeveral cytokines of the γ−common, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, promote the effector response of CD8+ T cells, as well as the survival of long-lived memory cells . For example, IL-2 promotes the proliferation and accumulation of memory CD8+ T cells ; IL-7 promotes the homeostasis of naïve and memory CD8+ T cells [113,114], while IL-15 and IL-21 promote the survival of HIV-specific CD8+ T cells, the production of IFN-γ, the expression of cytotoxic molecules, and control of HIV in vitro [, , ]. IL-15 also promotes the metabolic plasticity of HIV-specific CD8 + T cells, a characteristic of cells derived from elite controllers .Systemic administration of IL-7 or IL-21 has been explored in macaques with SIV infection. In general, the results showed low toxicity of the treatment, with induction of CD4+ and CD8+ T cell proliferation by IL-7, as well as greater expression of cytotoxic molecules induced by IL-21; however, systemic administration of these cytokines does not induce changes in viral load or viral set point [118,119]. Interestingly, comparative in vitro studies among IL-2, IL-7 and IL-15 showed that the latter cytokine is the most potent to increase Gag-specific CD8+ T cell responses , so it would be a good candidate for immunotherapy. Importantly, IL-15 also promotes in vitro HIV reactivation in latently infected cells . However, while therapeutically-administered IL-15 is rapidly cleared from plasma in vivo, systemic administration of high doses of this cytokine can cause significant adverse effects and toxicity . Therefore, this cytokine could be used as a vaccine adjuvant or as a stimulant for in vitro cell expansion and induction of effector properties. For example, immunization with HIV vaccines in vectors that also express IL-15 induced an increase in memory CD8+ T cells that persisted for several months and showed high proliferative capacity .Considering that, in vivo, IL-15 effects require interaction with the IL-15 receptor alpha (IL-15Rα) [124,125], additional strategies have been devised to exploit and potentiate its function. For instance, N-803 is an IL-15 superagonist designed to increase IL-15 activity. Specifically, N-803 consists of a complex between an IL-15 mutant (containing an asparagine to aspartic acid mutation at position 72, allowing a more stable heterodimeric complex with IL-15Rα) and an IL15Rα: IgG1 Fc fusion protein. These structural properties confer to N-803 higher biological activity and longer half-life than soluble IL-15, which allow potent stimulation of NK and memory T cells [126,127]. An alternative to N-803 is the heterodimeric IL-15 (hetIL-15), a stable native complex of IL-15 and IL-15Rα. Previously, hetIL-15 has been shown to promote proliferation and effector functions of adoptively transferred tumor-specific T cells . Importantly, N-803 and hetIL-15 increase cytotoxic responses and upregulate CXCR5 expression. This latter effect allows the migration of CD8+ T cells into the lymphoid follicles in SIV or SHIV-infected macaques, associated with a transient reduction in viral replication [, , , ]. In line with a role of IL-15 in promoting protective follicular cytotoxic responses, nonpathogenic SIV infection in African green monkeys was associated with enhanced NK cell migration into follicles and the presence of high levels of this cytokine presented in its membrane-bound form on follicular dendritic cells . The safety and virologic impact of N-803 was recently evaluated in a phase 1 study in people living with HIV. Notably, the drug was safe and well-tolerated, with a modest reduction in the circulating inducible reservoir. In addition, N-803 treatment was associated with T cell and natural killer (NK) cell activation . Larger clinical trials in ART-suppressed individuals will elucidate the effectiveness of N-803 for the promotion of follicular cytotoxic responses, latency reversal, and purge of the HIV reservoir.In addition to cytokines of the γ-common family, other cytokines that induce a particular functional profile can also be used in vitro. An example is the induction of follicular-like CXCR5+ cells with the combination of transforming growth factor (TGF)−β and IL-23 , aiming at redirecting CD8+ T cells towards lymphoid follicles, an important viral replication, and reservoirs site.Additionally, various adjuvants that promote the priming of CD8+ T cells by antigen-presenting cells through the activation of pattern-recognition receptors have been explored . For example, agonist ligands of the cGAS-stimulator of interferon genes (STING) pathway have shown effectiveness as adjuvants to increase adaptive immune responses, particularly in pre-clinical trials of immunotherapy against tumors . Through the induction of type I IFN, STING ligands such as cGAMP co-administered to mice together with vaccines promote the expansion and maturation of antigen-specific CD8+ T cells, conferring subsequent protection against tumors or viral infections . Similar strategies could be used in the context of HIV, with the administration of vaccines along with these adjuvants, or through in vitro priming and expansion of virus-specific CD8+ T cells in the presence of cGAMP, followed by adoptive transfer.Another activator of the innate immune response, that in turn would promote adaptive immunity, is the toll-like receptor (TLR)-7 agonist, vesatolimod, which exhibits latency reversal properties . Previous studies in SIV-infected macaques demonstrated that a combination of vesatolimod with neutralizing antibodies or vaccination promotes viral control following ATI. SIV-specific T cell responses and T cell activation were the strongest correlates of virologic control [140,141]. Importantly, in a recent phase 1b clinical trial in people with HIV, vesatolimod was safe and well-tolerated and contributed to a modest delay in viral rebound upon ATI. In addition, vesatolimod induced interferon-stimulated gene expression, cytokine production, and an increase in activated and proliferating T cells .5.3Immune checkpoint blockadeSince CD8+ T cells acquire a state of functional exhaustion due to the persistent antigenic load, the inflammatory environment, and the expression of immune checkpoint receptors, a strategy to promote their function is to block certain inhibitory signals. To date, monoclonal antibodies that inhibit CTLA-4 (ipilimumab), PD-1 (pembrolizumab, nivolimumab, cemiplimab), and PD-1 ligand 1 (PD-L1; durvalumab, atezolizumab, avelumab) receptors have been approved for clinical use . Anti-PD-1 antibodies have been used for therapy in a small number of people with HIV and cancer. In general, good tolerance and safety have been observed. In addition, effectiveness of immunotherapy has been observed to combat different types of cancer, as well as an increase in the CD4+ T cell count after immunotherapy, without viral rebound . Other clinical trials are currently underway to evaluate the effectiveness of these immunotherapies in individuals with HIV who also have various solid cancers and lymphomas , as well as individuals without malignancy [146,147]. Promisingly, the use of antibodies against CTLA-4, PD-1, or PD-L1 induced an increase in the levels of cell-associated unspliced HIV RNA (indicative of latency reversal), as well as a rise in the frequency of Gag-specific CD8+ T cells that produced IFN-γ, TNF-α or CD107a in a subset of participants [147,148]. However, it should be emphasized that these results come from small clinical trials or case reports, and substantial variability is observed between studies and individuals . Moreover, between 10 and 30% of patients receiving immune-checkpoint inhibitors may suffer serious immune-related adverse events , as has been observed in people with HIV . Thus, preliminary data indicate that only a subset of people with HIV will respond to and benefit from immune checkpoint blockade, while there is a high risk of toxicities.Of note, monotherapy with anti-PD-1 for cancer in people living with HIV is insufficient to improve virus-specific responses, probably linked to a terminal exhaustion state of HIV-specific CD8+ T cells . More promising results have been obtained upon the combination of immune checkpoint blockade. As such, enhanced IL-2 and CD107a production by HIV-specific T cells was observed when cells were stimulated ex vivo with viral peptides in the presence of a combination of antibodies against Lymphocyte-activation gene 3 (LAG-3), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), or T cell immunoreceptor with Ig and ITIM domains (TIGIT) . Therefore, blocking multiple signals that inhibit effector immune mechanisms seems promising to promote more effective responses of CD8+ T cells against HIV. Moreover, a combinatory strategy that includes vaccination plus immune checkpoint blockade has shown promising results in ART-suppressed SIV-infected macaques. As such, macaques immunized with a CD40L plus TLR7 agonist–adjuvanted DNA/modified vaccinia Ankara vaccine along with anti-PD-1 therapy exhibited a higher frequency of granzyme B+ perforin+ CD8+ T cells in blood and lymph nodes, particularly within lymphoid follicles. This effect was associated with reductions in the viral reservoir, and a lower viral set point upon ATI . Thus, PD-1 blockade may improve the therapeutic benefits of vaccination, and these combinatorial strategies should be further explored in HIV cure studies.5.4Adoptive therapies of engineered and reprogrammed CD8+ T cellsIn addition to endogenous cells, CD8+ T cells whose TCR antigen receptors have been modified to recognize conserved viral peptides are another option to augment HIV-specific CD8+ T cell responses. One such example is CD8+ T cells with TCRs recognizing the SL9 epitope of the Gag protein, which exhibited potent lytic induction ability and reduced infected cells in vivo in SCID mice . However, these cells can eventually recognize autoantigens, which have been associated with cardiac toxicity, limiting their clinical use . Another option for CD8+ T cells with modification or redirection of their receptors are cells with chimeric receptors, which constitute a promising strategy for the treatment of HIV infection . The use of these cells could overcome the limitations of endogenous CD8+ T cells, such as viral escape, HLA restriction, and immune exhaustion . Previous in vitro and in vivo studies in humanized mice demonstrated that CD4-based CAR T cells containing the 4-1BB costimulatory domain efficiently control HIV replication . More recently, a NAb-based CAR T cell therapy was evaluated in a small number of people living with HIV who underwent ATI. Notably, CAR T cell therapy was safe and well-tolerated, and induced a decrease of cell-associated viral RNA and intact proviruses, delaying viral rebound upon treatment interruption . Another recently described platform is known as convertible CAR T cells. In this platform, an inert form of the human NKG2D extracellular domain was engineered as the ectodomain of the CAR for expression on CD8+ T cells. These CAR T cells were specifically directed to kill HIV-infected cells only in the presence of an activating bispecific antibody based on bNAb and a mutated form of the NKG2D ligand MIC/ULBP. Promisingly, convertible CAR T cells efficiently eliminate the inducible latent reservoir in cells derived from people with HIV in ex vivo assays, hence constitute a promising tool for attacking the latent HIV reservoir . In addition, CAR T cells may also include the expression of receptors such as CXCR5, which directs the cells to lymphoid follicles . In this regard, a recent study in SIV-infected rhesus macaques demonstrated that the adoptive transfer of CAR-T/CXCR5 cells favored the migration of these cells to lymphoid follicles, in proximity to infected cells. This cell adoptive strategy was associated with a reduction in systemic viral load upon ART interruption, without major adverse effects .Parallel to cell engineering, emerging approaches to cell reprogramming by targeting metabolic or transcriptional regulators has demonstrated important therapeutic benefits in tumor and chronic infection models [, , ]. Previous studies have shown that stem-like memory HIV-specific CD8+ T cells expressing the transcription factor T cell factor 1 (TCF-1), endowed with potent antiviral activity and metabolic plasticity, are correlates of natural HIV and SIV control [117,, , ]. Based on this rationale, it was recently explored the potential of reprogramming HIV-specific CD8+ T cells from people with HIV under ART (non-controllers) to acquire properties found in HIV natural controllers. By upregulating the TCF-1 pathway with a glycogen synthase kinase 3 (GSK3) inhibitor, in vitro reprogrammed HIV-specific CD8+ T cells acquired a stem-like memory profile, with enhanced survival, polyfunctionality, metabolic plasticity, and HIV-suppressive activity . These properties of such reprogrammed cells could boost the immunomodulatory potential of other therapies, such as IL-15 or CAR T cells, and these approaches could be combined to promote long-lived memory CD8+ T cells against HIV.6ConclusionsAlthough ART is efficient for the control of HIV replication, in certain cases it is an unsustainable strategy and brings negative consequences for people living with HIV, such as multi-organ disease. A cure for HIV would certainly improve the long-term health of people living with HIV, reducing community-level transmission. Viral latency, its huge diversity, multiple tissue localizations, and immune evasion mechanisms determine major barriers to an HIV cure. Since used alone CD8+ T cell-based strategies have not shown relevant clinical benefit, the combination of such strategies, along with early ART initiation in people with HIV, seems to be the way to achieve more encouraging outcomes in HIV cure studies. The study of the mechanisms for natural and post-treatment CD8+ T cell-mediated HIV control, and the transcriptional and metabolic regulators of their activity, will elucidate novel targets for the design of immune therapies.Funding statementThis work was supported by 10.13039/501100006280Ministerio de Ciencia y Tecnología, Colombia(code 111577757051 to MTR) and Uniremington.Author contribution statementAll authors listed have significantly contributed to the development and the writing of this article.Data availability statementData included in article/supp. material/referenced in article.Declaration of competing interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Health Services Research and Managerial Epidemiology

PMC10664417

11-20-2023

10.1177/23333928231214169

Hospitalization Risk Associated With Emergency Department Reasons for Visit and Patient Age: A Retrospective Evaluation of National Emergency Department Survey Data to Help Identify Potentially Avoidable Emergency Department Visits

North Frederick, Garrison Gregory M, Jensen Teresa B, Pecina Jennifer, Stroebel Robert

BackgroundPatients often present to emergency departments (EDs) with concerns that do not require emergency care. Self-triage and other interventions may help some patients decide whether they should be seen in the ED. Symptoms associated with low risk of hospitalization can be identified in national ED data and can inform the design of interventions to reduce avoidable ED visits.MethodsWe used the National Hospital Ambulatory Medical Care Survey (NHAMCS) data from the United States National Health Care Statistics (NHCS) division of the Centers for Disease Control and Prevention (CDC). The ED datasets from 2011 through 2020 were combined. Primary reasons for ED visit and the binary field for hospital admission from the ED were used to estimate the proportion of ED patients admitted to the hospital for each reason for visit and age category.ResultsThere were 221,027 surveyed ED visits during the 10-year data collection with 736 different primary reasons for visit and 23,228 hospitalizations. There were 145 million estimated hospitalizations from 1.37 billion estimated ED visits (10.6%). Inclusion criteria for this study were reasons for visit which had at least 30 ED visits in the sample; there were 396 separate reasons for visit which met this criteria. Of these 396 reasons for visit, 97 had admission percentages less than 2% and another 52 had hospital admissions estimated between 2% and 4%. However, there was a significant increase in hospitalizations within many of the ED reasons for visit in older adults.ConclusionReasons for visit from national ED data can be ranked by hospitalization risk. Low-risk symptoms may help healthcare institutions identify potentially avoidable ED visits. Healthcare systems can use this information to help manage potentially avoidable ED visits with interventions designed to apply to their patient population and healthcare access.

IntroductionAcute care comprises a large part of ambulatory care and much of it does not take place in an office setting. 1 Emergency departments (EDs) see many acute care visits but there has been a trend toward use of other methods for accessing healthcare including urgent care centers, retail clinics, and telemedicine visits. 2 Access issues may be driving some of the use of ED visits. The National Health Interview Survey showed that barriers to usual care were significantly associated with ED use. 3 Barriers to care such as being “unable to get through on phone,” “couldn’t get an appointment soon enough,” and “not open when you could go” were all associated with ED use.3,4 Consistent with access issues driving ED visits, Weinick et al concluded that many emergency department visits could be managed at urgent care centers and retail clinics. 5 After hours acute care availability within urgent care centers and retail clinics has been associated with some decreased ED use but has not necessarily decreased cost of care.6,7 However, even with alternative forms of healthcare access and extended hours, overcrowding and “clinically unnecessary use of EDs” is still a problem in the US and UK.8,9Healthcare has seen a radical change within the last few years. During the pandemic, there was a need to direct patients with COVID symptoms to the appropriate healthcare setting. Directing all those concerned about COVID to the ED could have resulted in ED overcrowding, and potentially more virus transmission to those in the ED already burdened with other disease. To help reduce ED visits and make COVID care and detection more efficient, Mayo Clinic and the University of California, San Francisco successfully developed online self-triage and self-scheduling systems for COVID that diverted asymptomatic and symptomatic patients without severe symptoms to outpatient testing, thus avoiding ED visits.10–12 These interventions for COVID demonstrated the potential for broader interventions to decrease unnecessary ED visits.There is no consensus on how to identify an unnecessary or avoidable ED visit. Different criteria for identifying avoidable ED visits have surfaced in the literature. 13 Hospital admissions from the ED, 13 procedures and tests done in the ED, 14 medications dispensed, 15 diagnosis codes, 16 and chart review 17 have all been used to assess whether an ED visit was avoidable or not. In this study, we use national ED visit data and associated hospitalization risk to identify potentially avoidable ED visits. Using complex survey analysis on this national data, we can estimate the probability of hospitalization associated with each reason for visit. Our aim was to supply healthcare institutions with a comprehensive list of reasons for visit and their associated risk for hospitalization. Reasons for ED visits associated with low risk of hospitalization could represent opportunities to safely expand care alternatives to the ED such as online self-triage, e-visits, video visits, and self-scheduled office visits. Other interventions such as coaching patients when and how to use various forms of access also need to be explored.MethodsThis was a retrospective analysis of the National Hospital Ambulatory Medical Care Survey (NHAMCS). NHAMCS is a survey conducted yearly by the National Center for Health Statistics (NCHS) division of the Centers for Disease Control (CDC). 18 We used the emergency department surveys of NHAMCS for this study.Data Collection and PreparationData used for this study was collected and curated by the CDC and is available online. 19 Each year over the past several decades, the CDC has collected data from EDs across the US. The survey has a complex sampling design, intended to represent the entire US population's ED visits by utilizing patient weighting factors. The survey results are reported yearly and have data fields that are well-defined and can be combined over multiple years. The years that we used in this study were from 2011 through 2020, for a total of 10 consecutive years. The combined 10 years represented over 220,000 ED visits in national EDs across the US.The national ED dataset consists of fields that can be used in retrospective analysis. The items of interest for this study were the primary reason for visit (presenting symptom), patient age, and the binary outcome of admission to the hospital (coded ADMITHOS). 20 The survey has coded reasons for the ED visit. These reasons for visit have been developed over several decades, going back to the early 1970s. 21 Reasons for an ED visit (coded RFV) can be multiple and are contained in five different fields, labeled RFV1 through RFV5. RFV1 is the primary reason for visit, RFV2 is the secondary reason for visit, and so on up to RFV5, the fifth reason for visit if applicable. All the visits have an associated RFV1 code, with a small number of RFV1 s coded −9, 89970, 89980, or 89990 which had the respective descriptions blank, 0.25% of entries; no reason for visit, 0.03%; not enough information, 0.16%; and illegible, 0.01%. RFV1 codes and their descriptions are downloadable from the NHAMCS website. 20 Also included in the national data are two fields for age. Age by each year is coded (AGE) and includes 94 years from 0 through 93 and a 95th age group representing 94 and above. In addition, there is also an ordinal age variable (coded AGER) for six mutually exclusive age ranges. The six age ranges in the NHAMCS field AGER are: 0–14, 15–24, 25–44, 45–64, 65–74, and ages 75 and up.Outcomes MeasuresOur primary outcome was hospital admissions. Hospital admission was considered an outcome proxy for an unavoidable ED visit. Visits with a very low probability of hospitalization could potentially be handled safely in less intensive settings such as urgent care clinics, retail clinics, or outpatient offices. We determined the proportion of hospital admissions by each primary reason for visit (RFV1) and by age range (AGER).Statistical AnalysisWe used Stata® 18.0 (College Station, Texas). The binary field of hospital admission was used for proportions (percent hospitalizations) and logistic regression. For our analysis, we used the Stata complex survey design module to calculate the proportion of hospitalizations with each reason for visit and with six mutually exclusive age categories within each reason for visit. The six age categories we used were those already in the dataset design of the NHAMCS. Logistic regression analysis was used to evaluate the change in admission odds ratios by age for selected reasons for visits. The age subgroup of 25–44 was used as the base to make logistic regression comparisons with older age subgroups.The NCHS has recommendations concerning best practice for use of the NHAMCS dataset. We followed the recommendations by avoiding use of any subgroup with fewer than 30 ED observations.22,23This study was a secondary data analysis of publicly available, deidentified data from the CDC. Thus, this study did not qualify as human subjects research and was exempt from IRB review.ResultsThe combined 10-year survey sample was 221,027 ED visits. Complex survey analysis of these 221,027 sampled visits resulted in an estimated total of 1.37 billion (1.37B) visits across the US population. There were 23,228 hospitalizations from the surveyed visits for an estimated hospitalization rate for all ED visits of 10.6%, (CI 95%; 9.8% to 11.4%). The risk of hospitalization from an ED visit for females was 10.0% (CI 95%; 9.2% to 10.8%), males were 11.3% (CI 95%; 10.5% to 12.2%).ED visits for those under age 15 were estimated to be 257 million (M) (18.8%), ages 15–24 at 198 M visits (14.5%), ages 25–44 with 381 M visits (27.8%), ages 45–64 with 308 M visits (22.5%), ages 65–74 with 104 M visits (7.6%), and ages 75 and older with 124 M visits (9.1%).Hospitalization by Primary Reason for ED Visits (RFV1, all Ages)The national data had a total of 736 different primary reasons for visits for the 10-year ED visit sample. Of the 736, there were 396 RFV1s that met the criteria of having at least 30 ED visits each. These 396 RFV1s accounted for 1.35B estimated ED visits or 98.5% (1.35B/1.37B) of the total estimated ED visits.Figure 1 is a histogram of the 396 RFV1s by the estimated percent hospitalized for each RFV1. There were 97 RFV1s that each had a probability of hospitalization from the ED visit of less than 2%. These ED visits with less than 2% probability of hospitalization accounted for 16.4% of total estimated ED visits. There were 52 RFV1s associated with ED visits with 2% to 4% probability of hospitalization each (10.7% estimated visits), and 72 RFV1s associated with hospitalization outcomes in 4% to 8% of the ED visits (24% of total estimated visits). There were 175 RFV1s associated with a higher probability of hospitalization: 100 RFV1s, each with 8% to 20% hospitalized (34.6% estimated visits) and 75 RFV1s, each resulting in greater than 20% hospitalized from the ED visits (12.9% of total estimated visits). The entire 396 list of reasons for visits along with the associated hospitalization risk (estimate) is available in supplemental files as “hospital admit percent by primary reason for visit—all ages combined,” sorted by ED visit frequency.Figure 1.Histogram of 396 reasons for NHAMCS ED visits by the estimated hospitalization percent for each RFV1. Reasons for visits included all that met criteria of at least 30 survey occurrences over all ages for the 10-year combined sample.Hospitalization from ED as a Function of AgeProbability of hospitalization associated with ED visits was strongly associated with age. Figure 2 shows the hospitalization percentages by patient age. With a best-fit logit transformation of hospitalization probability (logit = ln [probability hospitalized/probability not hospitalized]), the transformed linear fit was excellent (adjusted r2= .97, P < 0.0001). The best-fit logit model (shown in Figure 2) was −3.91 (CI 95% −4.08 to −3.74) + age (in years) × 0.038 (CI 95% 0.036 to 0.040).Figure 2.Hospitalization percent by each year in age. A best-fit logit model (ln(odds hospitalized) = constant (−3.91) + 0.038 × age (in years)) is superimposed. 0 is birth to 1 year, 1 is from age 1 to 2. The final year on the x-axis is age 94 and up.The relationship between age and hospitalization is also shown in the histograms of reasons for visits by age group (Figure 3). Advancing age groups are generally associated with increasingly higher hospitalization rates for each particular reason for visit. Thus, in Figure 3, we observe a flattening of the histogram with advancing age, with low probability reasons for visit getting fewer in number and more reasons for visit having higher probabilities of hospitalization. This clearly demonstrates that there are fewer and fewer opportunities to identify low-risk reasons for visit with advancing age.Figure 3.Counts of reasons for visits by ED hospitalization risk for that RFV1. Separated by age groups (AGER field in NHAMCS data). Each RFV1 within the age group has a minimum of 30 ED survey visits.Hospitalizations Associated with Reasons for Visit and AgeBecause there were six age categories, not all of the 396 RFV1s with at least 30 surveyed ED visits when counted over all ages had at least 30 visits in each RFV1-age subgroup. However, there were 271 RFV1s of the 396 that had 30 or more surveyed ED visits in at least one of the 6 age subgroups. These 271 RFV1s were represented in a total of 953 unique RFV1-age subgroups that each contained over 30 surveyed ED visits. These 953 unique RFV1-age subgroups accounted for 94% (1.27B/1.35B) of the 10-year estimated ED visits. Both Table 1 and Figure 3 use the 953 unique RFV1-age subgroups. Columns of Table 1 separate the ED visits by risk of hospitalization and show the number of RFV1-age subgroups and estimated ED visits associated with hospitalization probabilities. The supplemental file “hospital admit percent by primary reason for visit and age” contains all 953 unique RFV1-age subgroups with associated admit percent and age group category referred to in Figure 3 and Table 1.Table 1.Hospitalization Percent Categories (Columns) by Age Group (Rows) Showing Estimated ED Visit Volume (in Millions) and Number of Different RFV1-Age Subgroups That are Involved.Hospitalization 0% to 1%Hospitalization 1% to 2%Hospitalization 2% to 4%Hospitalization 4% to 8%Hospitalization 8% to 20%Hospitalization Over 20%Age range, in yearsED visits (% across age)Number of different RFV1sED visits (% across age)Number of different RFV1sED visits (% across age)Number of different RFV1sED visits (% across age)Number of different RFV1sED visits (% across age)Number of different RFV1sED visits (% across age)Number of different RFV1s 0–14 74.6 (31.5)5742.1 (17.7)2376.5 (32.3)2015.5 (6.5)2128 (11.8)230.5 (0.2)2 15–24 51.1 (28.5)5826.6 (14.8)2039.6 (22.1)2649.5 (27.6)319.7 (5.4)162.9 (1.6)8 25–44 52.4 (14.5)6051.3 (14.2)2572.7 (20.1)3263.5 (17.6)43115 (31.8)526.2 (1.7)16 45–64 8.2 (2.8)2219.1 (6.6)1228.7 (9.9)2555.5 (19.2)28107.1 6771 (24.5)47 65–74 3.0 (3.4)80.4 (0.4)12.4 (2.7)75.1 (5.7)827.1 (30.3)2951.4 (57.5)50 75up 1.4 (1.3)61.7 (1.5)31.8 (1.6)42.4 (2.2)619.1 (17.4)2583.6 72 Total 190.7 (15.1)211141.2 (11.1)84221.7 (17.5)114191.5 (15.1)137306 (24.2)212215.6 195Changes in RFV1 Frequency Rank with AgeThe frequency rank of the primary reasons for visit changes with age as shown in Figure 4. For example, sore throat is a very common reason for visit in younger ages and ranks 11th overall in national ED visit frequency, but in the 75 and older group drops to 109th. Figure 4 shows that skin rash as a reason for visit also drops in relative frequency with age while vertigo and back pain as reasons for visit increase with age.Figure 4.Twelve most frequent reasons for ED visits (over all ages) showing within age group relative frequency rank as age increases. In parentheses are the NHAMCS RFV codes for each of the twelve most frequent reasons for visit.Other less frequent overall reasons for visit (not shown in Figure 4) such as neurologic symptoms of arm weakness or speech problems have higher frequencies with age. Alcohol intoxication is more common as a reason for visit among younger adults but not in children or older adults. Our supplemental files contain estimated ED visit counts, thus allowing readers to consider frequency of ED visits along with hospitalization risk in determining what interventions may be most effective.Changes in Hospitalization Risk by Age for Most Frequent Individual Reasons for VisitFigure 5 shows the same 12 top ED visits have differing amounts of change in hospitalization percent with age. Figure 5 shows that within each RFV1 there is generally an increase of percent hospitalization with increasing age, which we have examined in more detail in the next section.Figure 5.Twelve most frequent reasons for ED visits (over all ages) showing the ED visit percent hospitalized as age increases. In parentheses are the NHAMCS RFV codes for each of the twelve most frequent reasons for visit.Odds Ratios of Hospital Admission for Adults Following Emergency Department VisitThe association of hospitalizations with age in Figure 2 shows that adults in the age range 25–44 have lower overall hospitalization risk compared to older age groups. In comparison to a baseline group of ages 25–44, those aged 45–64 had a hospitalization odds ratio (OR) of 2.52 (CI 95%; 2.34 to 2.71). Compared to the 25–44-year-old base, those aged 65–74 had a hospitalization OR of 5.12 (CI 95%; 4.62 to 5.67), and those 75 years and up had hospitalization OR of 7.10 (CI 95%; 6.49 to 7.76).We were also able to examine the change in hospitalization odds ratio with individual RFV1s using logistic regression in the complex survey design. There were 90 reasons for visits that met criteria for 30 ED visits in each of four adult age groups.Of the 90 RFV1s fulfilling criteria for analysis, there were 5 RFV1s in the base age of 25–44 that had no hospital admissions among the surveyed visits. The RFV1s with no admissions in ages 25–44 were: suture insertion/removals (0 admissions/250 survey ED visits, 1.4 M estimated ED visits); shoulder injury (0 admissions/126 survey visits, 721 K est. ED visits); urinary frequency and urgency (0/75, 437 K); other urinary dysfunction (0/53, 333 K); and nosebleeds (0/54, 394 K). Lacking a single hospital admission for those 5 RFV1 in the age base range, we eliminated them from further OR analysis, leaving 85 RFV1s, all of which had at least the 30 requisite ED survey counts in all 4 upper age groups and at least one hospital admission in the comparison base of ages 25–44. For those 85 RFV1s we calculated the odds ratio of hospital admission of each of the three older age groups (ages 45–64, 65–74, and 75 and up) compared to the base of those aged 25–44. The complete list of 85 RFV1s with odds ratios and confidence intervals are in supplemental files as “hospital admit odds ratios of reasons for visit by age group compared to the 25–44 age group.”Of the 85 RFV1s meeting the above criteria, none had a statistically significant lower hospitalization OR (CI 95% upper limit less than 1) for any of the 3 older comparison age groups. For the 45- to 64-year-olds, 41% (35/85) of the reasons for visit had significantly higher odds of hospitalization than the base group of 25- to 44-year-olds (CI 95% lower limit OR greater than 1). The same analysis for 65- to 74-year-olds showed that 68% of examined reasons for visit (58/85) had significantly higher hospitalization odds compared to the 25- to 44-year-olds. For those 75 and older, 78% (66/85) of RFV1s had significantly greater hospitalization odds compared to the 25- to 44-year-olds. No hospital admissions were reported in the surveys for three additional RFV1-age subgroups (above the 25- to 44-year base age): insect bites in 2 age subgroups (65–74, and 75 and up) and lacerations (head and neck) in the 65–74 age group. Odds ratios could not be calculated for these subgroups and are left blank in the supplemental file.Reasons for Visit with Low Probabilities of Hospitalization from EDTable 1 shows that there were 191 M emergency department visits from 211 RFV1-age subgroups that had extremely low hospitalization rates (less than 1%). An additional 141 M visits from 84 RFV1-age subgroups had hospitalization rates between 1% and 2%.) These low-risk subgroups (<2% hospitalizations) accounted for 332 M ED visits (26.2% of the 1.27B estimated visits). Using the supplemental file labeled “hospital admit less than 1% by primary reason for visit and age,” readers can examine reasons for ED visit sorted by <1% hospitalization risk and age group. Although the level of detail in the supplemental files is intended to help those wanting to look for specific healthcare opportunities to decrease avoidable ED visits, a few generalizations can be stated. In the age group 0 through 14, the extremely low-risk RFV1s (<1% hospitalizations) include injuries, lacerations, extremity/joint pain, ear and eye concerns, skin rashes, sore throat, and bites. There were 57 of these low hospitalization risk reasons for visit in this age group that accounted for 75 M national ED visits (Table 1). For the 15–24 age group, there were 58 individual RFV1s that were associated with hospitalization in less than 1% of the 51 M ED visits (Table 1). This older age group also had extremity injuries, ear and eye concerns, skin rashes, sore throat, and extremity/joint pain accounting for the ED visits with low hospitalization risk. In addition, the 15–24 age group also had low-risk ED visits concerning exposure to sexually transmitted disease, unconfirmed pregnancy, vaginal symptoms, wheezing, cough, sore throat, and urinary tract infection.The supplemental file labeled “hospital admit percent by primary reason for visit and age” contains the complete information on hospitalization risk (and 95% confidence intervals) for each of the 953 RFV1-age subgroups that met the criteria for inclusion (at least 30 ED visits in each subgroup of RFV1 and age range).DiscussionPrincipal FindingsThe hospitalization risk of an ED visit varies widely with primary reason for visit and age. National data is available to help determine reasons for ED visits that are unlikely to result in a hospitalization. These lower-risk reasons for visits may be opportunities to intervene with alternatives to the ED. Use of this national data may help guide those who are developing interventions to decrease potentially avoidable ED visits.Practice ImplicationsOur analysis shows that there are millions of ED visits with very low probability of hospital admission. It has been suggested that many of these ED visits are avoidable and may be safely handled in other practice settings, both in the US and internationally.24–26 A number of interventions to decrease avoidable ED visits have been tried with varying degrees of success. 27 Interventions have used strategies such as improving primary care access, 6 and ED diversion either before or at the time of ED visit.28,29 In addition, there has been evidence of increasing utilization of non-ED options for receiving acute care such as urgent care centers for treatment of low-acuity conditions. 2 Identification of potentially avoidable ED visits is an important issue to consider when addressing healthcare costs and quality. 13 Hollander and Sharma in a commentary in the NEJM Catalyst categorize some potential approaches to decrease ED visits by distinguishing acute health concerns that are unlikely to need ED, reasonable to evaluate before deciding if they need to go to the ED, and highly likely to need in-person evaluation in ED. 30 The datasets we supply in this manuscript can help do this, identify those unlikely to need the ED as well as those who very likely need to be evaluated in the ED. Institutions can use the hospitalization risk tables herein to tailor interventions to specific reasons for visit that pertain to their unique healthcare system. For example, we found that younger patients with extremity injuries were low risk for hospitalization. Our institution's online self-triage self-scheduling intervention took advantage of this finding as well as the Ottawa x-ray rules 31 to direct patients reporting ankle injuries to self-schedule an outpatient visit with a pre-scheduled x-ray. Likewise, Mayo Clinic developed an online self-triage and self-scheduling tool for ear and hearing symptoms. 32 Mayo Clinic outcomes data from online ear and hearing self-triage supports that these symptoms are low risk, similar to the findings of national ED visits found in this study. 32 In addition to a lack of concerning outcomes such as increased hospitalizations, the makeup of diagnoses following the ear and hearing self-triage use were similar to those from ED visits, suggesting that using self-triage might be an intervention that could decrease avoidable ED visits. 33 It should be noted that outcomes research for self-triage is difficult and evaluation of self-triage quality has often been done using “patient vignettes” or “simulated patients.” 34 One of the more recent actual outcomes studies of self-triage was limited to ear and hearing concerns as noted above 32 and more research needs to be done. 34 Patient ImplicationsNational ED data shows that elderly patients presenting to the ED often end up admitted to the hospital. Our supplemental tables may be helpful to consult when deciding about interventions for potentially avoidable ED visits and whether there should be some age limits for those interventions.Perceived seriousness of medical problem was the reason for an ED visit for 77% of adults ages 18–64 based on the National Health Interview Survey. 35 It is possible that online resources such as self-triage could help direct patients to healthcare that was more consistent with the actual need for acute medical attention rather than the patient-perceived seriousness. Additional research is needed to determine how best to do this. Although online self-triaging (available 24/7) would seem to be an excellent modality to triage and advise low-risk patients for alternative care options to the ED, Mayo Clinic self-triage experience tells us that many patients with ear and hearing concerns still went to the ED with diagnoses that could have been treated in the office despite no online triage recommendation to go to the ED. 32 Clearly there are a number of factors in addition to perceived seriousness of medical problem that impact the patient decision to go to the ED. Patients now have the opportunity to have telemedicine visits 24/7 with providers via services such as Teladoc, Virtuwell, and others.36,37 Patients also have the opportunity to self-schedule face-to-face or other visits with providers through Zocdoc, Lybrate, or with their own providers.38–42 Many patients also now have the ability to message their provider for advice about acute care. Social determinants of health also appear to play a significant role in ED visits, 43 and Medicaid expansion may also. 44 Low health literacy, lack of access to online resources, and illness anxiety can limit the potential impact of telemedicine visits, patient messaging, and self-triage for those in need. Many patients who visit the ED do not have the financial means for online care or do not have a digital connection. Thus, potential interventions involving patient messages and other forms of connected care are not currently feasible for many ED patients. Identification of avoidable ED visits can help guide selection of patients for interventions, but the interventions themselves will need to take into account the myriad reasons why patients elect to go to the ED and the resources available to these patients.Comparison with Other StudiesOur findings are consistent with the findings of Yang et al who found that non-avoidable ED visits were associated with increasing age. 45 Ginsburg et al also suggested that age be used in the adult triage process in the ED. 46 Reasons for visit stated as injury and psychiatric symptoms also influence predictability of an avoidable visit. 45 LimitationsThe NHAMCS datasets have several limitations. It is a challenging task to obtain a representative sample of ED visits across diverse populations and locations in the United States. Advances in data collection from interoperability and accessibility of electronic health records have resulted in the CDC discontinuing the NHAMCS survey methodology that was used for the data we are using here. The last public user files using the methodology in the current datasets will be for 2022 data to be released in 2024. 47 The RFV1 categories are subject to possible patient and ED staff interpretation. It is unknown how well the patient articulates the reason for ED visit and how well it gets translated into the survey dataset.Hospital admission has been a de facto standard as a measure for illness severity. However, a number of factors identified by survey such as ED busyness, time of day, waiting times, patient social support, and ED culture all have been found to influence the decision to admit. 48 In addition, reasons for visit with a low probability of admission might quite reasonably require monitoring in an ED setting; for instance, anaphylaxis, which might be coded as “shortness of breath” or “rash” requires ED-level care, but may not necessitate hospital admission.Observation admissions are also used in EDs instead of hospital admission for a variety of reasons. The NHAMCS datasets have a field labeled OBSDIS which are observations then dismissals. However, observation admissions both for adults and children are highly variable depending on the ED and hospital.49,50 We did not include observation admissions as a primary measure. However, for readers who are interested, we did include the proportion of ED visits where there was either a hospital or observation unit admission (either ADMITHOS =1 or OBSDIS =1). These risks are found in our tables in supplemental files.We also did not examine other potential ways of looking at “avoidable” ED visits. For example, Hsia et al and Yang et al considered avoidable ED visits from a “conservative definition” as those who not only were dismissed from the ED but also did not have any diagnostic tests, screening, procedures or medications before dismissal.15,45 We also acknowledge that algorithms identifying low-acuity emergency department visits are “imperfect predictors of visit acuity” as noted by Chen et al. 16 Rosano et al published a systematic review of the impact of access on ED visits which gives context to studies such as this one. 51 The NHAMCS dataset does not have data that pertains directly to available healthcare access in the locations surrounding the sampled national EDs. Thus, our study lacks the ability to assess the role access has in national ED visits. In addition, there are medical conditions whose hospital admission rates through the ED are affected by local healthcare factors. The terminology “ambulatory care-sensitive conditions” (ACSCs) has been used to describe this.52,53 An example of an ACSC is asthma. A patient with asthma who is unable to readily get treatment for exacerbations, lacks appropriate immunizations, or whose asthma is not well controlled due to factors in the healthcare system, may have a higher likelihood of hospital admission. The NHAMCS does not contain specific data to assess how different healthcare systems might alter the hospital admission rates for ambulatory care-sensitive conditions.The NHAMCS dataset was not designed to contain data on all the possible reasons for the ED visit other than the “reason for visit” data field. Lack of primary care access, lack of after hours care, lack of transportation, and lack of financial resources are all possible reasons for patients to go the ED, regardless of the presenting symptom contained in the “reason for visit” data field. As such, the NHAMCS dataset was not designed to help us answer exactly why the patient presented to the ED for care rather than seek care somewhere else. What the national data does show is that there are large numbers of younger patients being seen across a national ED sample who have very low risk for hospitalization.Our included supplemental files examine in detail the risk of hospitalization associated with the ED reason for visit and patient age. Those planning interventions to decrease ED utilization, whether through triage, 24 h walk-in clinics to improve access, or other interventions, can use our supplemental files to help assess the potential risk of ED diversion. For example, use of our hospitalization risk tables can help those planning interventions to reduce ED overcrowding. A concern for ED diversion interventions is the potential risk when patients do not go to the ED for a specific symptom. Those planning interventions to address ED overcrowding can cite the national data we supply in the supplemental files. Using the supplemental tables, those planning the interventions can identify patients with lower risk.ConclusionFurther interventions aimed at decreasing avoidable ED visits are necessary to reduce ED overcrowding and optimize the use of limited healthcare resources. Our research found that although hospitalization risk by ED visit varies with primary reason for visit and age, there remain millions of ED visits at low risk for hospitalization and thus may represent a possibility for intervention. The data we have collected regarding the primary reason for visit frequency and subsequent hospitalization risk may assist those in developing interventions specific to their patient populations and healthcare systems.Supplemental Materialsj-xlsx-1-hme-10.1177_23333928231214169 - Supplemental material for Hospitalization Risk Associated With Emergency Department Reasons for Visit and Patient Age: A Retrospective Evaluation of National Emergency Department Survey Data to Help Identify Potentially Avoidable Emergency Department VisitsClick here for additional data file.Supplemental material, sj-xlsx-1-hme-10.1177_23333928231214169 for Hospitalization Risk Associated With Emergency Department Reasons for Visit and Patient Age: A Retrospective Evaluation of National Emergency Department Survey Data to Help Identify Potentially Avoidable Emergency Department Visits by Frederick North, Gregory M Garrison, Teresa B Jensen, Jennifer Pecina and Robert Stroebel in Health Services Research and Managerial Epidemiologysj-xlsx-2-hme-10.1177_23333928231214169 - Supplemental material for Hospitalization Risk Associated With Emergency Department Reasons for Visit and Patient Age: A Retrospective Evaluation of National Emergency Department Survey Data to Help Identify Potentially Avoidable Emergency Department VisitsClick here for additional data file.Supplemental material, sj-xlsx-2-hme-10.1177_23333928231214169 for Hospitalization Risk Associated With Emergency Department Reasons for Visit and Patient Age: A Retrospective Evaluation of National Emergency Department Survey Data to Help Identify Potentially Avoidable Emergency Department Visits by Frederick North, Gregory M Garrison, Teresa B Jensen, Jennifer Pecina and Robert Stroebel in Health Services Research and Managerial Epidemiologysj-xlsx-3-hme-10.1177_23333928231214169 - Supplemental material for Hospitalization Risk Associated With Emergency Department Reasons for Visit and Patient Age: A Retrospective Evaluation of National Emergency Department Survey Data to Help Identify Potentially Avoidable Emergency Department VisitsClick here for additional data file.Supplemental material, sj-xlsx-3-hme-10.1177_23333928231214169 for Hospitalization Risk Associated With Emergency Department Reasons for Visit and Patient Age: A Retrospective Evaluation of National Emergency Department Survey Data to Help Identify Potentially Avoidable Emergency Department Visits by Frederick North, Gregory M Garrison, Teresa B Jensen, Jennifer Pecina and Robert Stroebel in Health Services Research and Managerial Epidemiologysj-xlsx-4-hme-10.1177_23333928231214169 - Supplemental material for Hospitalization Risk Associated With Emergency Department Reasons for Visit and Patient Age: A Retrospective Evaluation of National Emergency Department Survey Data to Help Identify Potentially Avoidable Emergency Department VisitsClick here for additional data file.Supplemental material, sj-xlsx-4-hme-10.1177_23333928231214169 for Hospitalization Risk Associated With Emergency Department Reasons for Visit and Patient Age: A Retrospective Evaluation of National Emergency Department Survey Data to Help Identify Potentially Avoidable Emergency Department Visits by Frederick North, Gregory M Garrison, Teresa B Jensen, Jennifer Pecina and Robert Stroebel in Health Services Research and Managerial Epidemiology

PMC

Advances in Medical Education and Practice

PMC10290466

6-20-2023

10.2147/AMEP.S406537

Can Non-Virtual Reality Simulation Improve Surgical Training in Endoscopic Sinus Surgery? A Literature Review

Stan Constantin, Vesa Doiniţa, Tănase Mihai Ionuț, Bulmaci Mara, Pop Sever, Rădeanu Doinel Gheorghe, Cosgarea Marcel, Maniu Alma

AbstractSimulation in endoscopic sinus surgery allows residents to learn anatomy, to achieve the correct handling of various rhinological instruments, and to practice different surgical procedures. Physically or non-virtual reality models are the main items in endoscopic sinus surgery simulation. The objective of this review is to identify and make a descriptive analysis of non-virtual endoscopic sinus surgery simulators which have been proposed for training. As a new state of the art, surgical simulators are developed continuously, so they can be used to learn basic endoscopic surgery skills by repetitive maneuvers, permitting detection of surgical error and incidents without risk for the patient. Of all training physical models, the ovine model stands out because of the similarities of the sinonasal pathways, the wide availability, and the low costs. Considering the similar nature of the tissues involved, the techniques and surgical instruments can be used almost interchangeably with minimal differences. Every surgical technique studied until now has a degree of risk and the only aspects that consistently reduced the number of complications are training, repetition, and hands-on experience.

IntroductionSimulation has been a key factor in the evolution of medical learning and practice since the 20th century, leading to higher precision and quality in medical training. A surgical procedure that is commonly used for reducing and improving symptoms of sinus infections and nasal obstruction is known as endoscopic sinus surgery. In order to provide superior patient care with minimal risk, the training of surgical residents and young specialists has become increasingly important. As a result, various methods have been researched to enhance their surgical techniques.1–3Endoscopic sinus surgery provides multiple opportunities for instructing young doctors outside of the operating room. By utilizing simulation in rhinology surgery, residents can gain knowledge of anatomy, perfect the correct application of various rhinology instruments, and practice performing different surgical procedures.4–6Residents must attain several fundamental surgical skills before they can conduct endoscopic sinus surgery. The camera and monitor system makes it challenging to comprehend the three-dimensional (3D) image of structures. Residents must create in their mind a 3D image of the sinus pathway anatomy from two-dimensional (2D) computed tomography (CT) scan images.7 Endoscopic sinus surgery simulators can vary from cheap, low-fidelity physical models constructed from inexpensive products, to expensive high-fidelity virtual reality simulators.8A simulator must be validated in different domains before it is found useful for training surgical skills. The European Association for Endoscopic Surgery (EAES) developed guidelines to define the principles of simulator validation.9 Face validity describes the ability of a simulator to create a realistic environment that is comparable to the actual surgical method. This can be assessed using an instructor and a group of beginners through a structured questionnaire.10 Content validity is the assessment of the simulator’s ability to deliver what is required to be obtained. This is demonstrated by the achievement of pre-established teaching objectives. In surgical simulators, the content can be grouped globally, such as hand-eye coordination, manual dexterity, and specific content, which examines the importance of the model in learning a distinct task, such as foreign body removal from the airways or nasal packing. The basis for using any simulator for evaluation and feedback is construct validity. This type of validity is the simulator’s ability to make a measurable difference between the different levels of expertise between beginners. Predictive validity is the ability of the simulator to estimate the actual performance of skills11 and concurrent validity is the simulator’s ability to resemble another simulator that has already been validated or is considered the gold standard.7Despite the increasing popularity of virtual reality simulators at the beginning of the century, physically, or non-virtual reality models are the main items in surgical simulation because technology has not evolved the haptic capabilities of real-life simulation yet.The primary aim of this review article is to comprehensively explore the various non-virtual endoscopic sinus surgery simulators that have been proposed and utilized for the purposes of training. The central focus of this analysis will be to provide a detailed and descriptive examination of these simulators, with the objective of identifying their unique features and characteristics. The ultimate goal of this investigation is to offer a more comprehensive understanding of the current state of non-virtual endoscopic sinus surgery simulators and to provide a basis for further research in this area.Materials and MethodsWe performed a general analysis of the studies published before 2023 about the models proposed and used for training in endoscopic sinus surgery. In this way, we have collected data from the MEDLINE, EMBASE, and OVID databases. Key terms used in the search for each database included “endoscopic sinus surgery”, “rhinology”, “simulation”, “training”. Also, the reference list of included studies was searched for other additional articles.Were included articles that studied non-virtual physical simulators, task trainers, and cadaveric animal models proposed for training in endoscopic sinus surgery. Non-English language studies, conference abstracts and those not related to training using physical simulators were excluded. The literature review is presented in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A flow chart showing the literature review process is shown in Figure 1. Figure 1Flow chart of the literature review process.ResultsA total of 647 studies were identified and screened, of which, 314 articles were assessed as relevant. Of those, 262 were excluded after the full-text screen and 16 articles were excluded during data extraction. In final, a total of 36 studies were identified describing 20 non-virtual physical simulators for training in endoscopic sinus surgery skills. All the 36 studies are part of primary literature.These were classified according to their design, physical bench models, 3D printed models, ovine models and vegetal models. Also, their evaluation was based on fidelity, the evaluation methods used, and the validation studies. Of these, 15 simulators have at least one validation study. Face validity was evaluated by 12 studies, 10 studies assessed content, 9 studies attempted to show construct, and only 2 concurrent validities.The literature search for non-virtual reality surgical simulators for training in endoscopic sinus surgery procedures was resumed in Table 1. Table 1Summary of the Identified StudiesNo.DateStudyType of SimulatorValidation11996Gardiner et al12Sheep head–22007Briner et al13Physical bench modelFace32008Leung et al14Physical bench modelUnable to show construct validity42008Nogueira et al15Physical bench model–52009Stamm et al16Physical bench modelFace62010Acar et al17Sheep head–72011Rowan Valentine et al18Live sheep+physical bench model–82011Mladina et al19Lamb head–92012Steehler et al20Physical bench modelFace, content, and construct102012Burge et al21Physical bench model–112012Wais et al22Physical bench model–122013Steehler et al23Physical bench model–132013Touska et al24Sheep head–142013Mladina et al25Lamb head–152014Awad et al26Sheep headFace and content162015Fortes et al27Physical bench modelFace172015Awad et al28Sheep headConstruct182015Skitarelić et al29Lamb head–192016Mallman et al30Lamb head–202016Delgado-Vargas et al31Sheep head–212016Chang et al323D printed modelFace and content222017Harbison et al33Physical bench modelFace, content, and construct232017Alrasheed et al343D printed modelContent and construct242017Mosaad el Sissi et al35Sheep head–252017Fernandes de Oliveira et al36Lamb head–262018Mladina et al37Lamb head–272018Luis Macias-Valle et al38Live sheep–282018Hsieh et al393D printed model–292019Yoshiyasu et al403D printed modelConstruct302019Ding et al413D printed model–312019Zhuo et al423D printed modelFace and content322020Alwani et al433D printed modelFace, content, concurrent, and construct332020Dong et al443D printed modelFace, content, and construct342021Gallet et al453D printed modelFace, content, and construct352021Rosenbaum et al46Physical bench modelFace, content, construct, and concurrent362021Tikka et al47Vegetal model–Abbreviations: 3D, three-dimensional; 2D, two-dimensional; CT, computed tomography; EAES, European Association for Endoscopic Surgery; PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses; SIMONT, Sinus Model Otorhino Neuro Trainer.Ovine ModelsSeveral studies have presented the ovine model as a good alternative to surgical training in functional endoscopic sinus surgery. Gardiner et al, to our knowledge, described for the first time a series of endoscopic surgical maneuvers performed on the sheep’s head, ranging from simple steps such as endoscope maneuvering to much more complex maneuvers. Although the anatomy of the sheep’s head is different from that of the human head, the maxillary, ethmoidal and frontal sinuses have approximately the same orientation. On top of that, the model itself is cheap, accessible and with good tissue quality.12Awad et al proposed the sheep’s head for use as a step in the simulation scale, previous to in‐vivo exercises, as it expresses a chance to concentrate on basic endoscopic sinus surgery procedures, to learn on virtual reality training models and cadaveric simulators. The training was evaluated by a task distinctive checklist and global evaluation instrument. The simulator proved an obvious connection between surgical skills and competence efficiency.26,28Mladina et al19,25,37 chose the lamb’s head instead of the sheep’s head, because it has nasal cavities that do not correspond to the dimensions of the standard endoscopic sinus surgery instruments, the lamb’s head has smaller dimensions than the sheep’s head, closer to the human head. Also, due to the absence of a lacrimal sac, dacryocystorhinostomy training could not be performed on the lamb’s head, proved by endoscopic and macroscopic dissection.19 In another study, Mladina et al demonstrated that training in cerebrospinal fluid leak repair could be done by elevation of a nasoseptal flap using lamb’s head model, but only just as a previous step before starting practice on a human cadaver.25Thus he developed a training plan defined by 10 surgical steps performed on each nasal fossa of the lamb’s head, training that proved to be useful for the 10 participating beginners, by improving working times and rapidly gaining obvious progress, which allows moving on to the next step in training, training on the human cadaver head.37Rowan Valentine et al18 used eight sheep under general anesthesia and underwent internal carotid artery isolation followed by arterial pressure monitoring and placement of a rapid infuser.18 The Sinus Model Otorhino Neuro Trainer (SIMONT) nasal model was modified to allow placement of the internal carotid artery in the sphenoid sinus, which could allow the simulation of a catastrophic internal carotid lesion. This is the first model that simulates the endoscopic endonasal injury of a large blood vessel like the internal carotid artery and was useful for the management of such critical situations.15The ovine head model, presented previously in the literature12,17,24,29–31,35,36,38 with good results, offers a rational choice for training in endoscopic sinus surgery. The benefits of using the sheep model for learning consists in validity, effectiveness, and pertinence of acquired skills in endoscopic procedures. The tissue feel and endoscopic view makes it a superior model for young trainees who are about to start executing endoscopic sinus procedures.In-vivo animal models and cadaveric simulation offer the best handling fidelity for training in endoscopic sinus surgery. Having fresh tissue offers the bleeding possibility, which is variable depending on whether the cadaver is embalmed or fresh frozen. In-vivo animal simulation is not allowed in some countries and consequently, animal cadaveric simulation can be more accessible.48–50 The ovine sinus model has allowed the acquisition of endoscopic surgical skills using video endoscopic techniques. This model is cheap, portable, requires reduced equipment, and can be used anywhere.26 Also, the use of the ex-vivo ovine model for training does not require the approval of an institutional review committee and may raise fewer ethical concerns than the use of live ovine model. The sheep’s head model as a sinus simulator for the aim of endoscopic sinus surgery learning proved to have face, content, and construct validation.26,28Physical Bench ModelsPhysical bench models are low-fidelity trainers with a low level of anatomy with the potential to provide different tasks for training in basic endoscopic sinus surgery.Nogueira et al showed the development of a physical bench model for training in endoscopic sinus surgery called SIMONT, with the capability to practice basic endoscopic sinus surgery procedures.15 Stamm et al in their study, used four SIMONT sinus models to determine the feasibility of balloon dilatation of the paranasal sinus ostium, thus one experienced surgeon performed all dilatations. The SIMONT sinus model proved to be feasible in training for sinus ostia balloon dilatation.16 In another study, Fortes et al evaluated the same SIMONT sinus model, 111 otolaryngologists (60 residents and 51 otolaryngologists with experience) performed uncinectomy, the main ostium of maxillary sinus identification, ethmoidal bulla opening, frontal recess opening, sphenopalatine artery identification, posterior ethmoidal cells opening, sphenoid intersinus septum resection, orbital decompression, and pedicled nasoseptal flap creation. The evaluation included three subjective questionnaires. The SIMONT sinus model proved to bring benefits for the training of otolaryngology surgeons.27A low-cost sinus surgery task trainer was developed by Steehler et al, so 10 otolaryngologists, 15 residents and 52 medical students performed 5 simulated tasks-recess probing, targeted injections, removal of superior sutures, extraction of posterior beads, and antrostomy of an egg with the removal of contents using this low-cost sinus surgery task trainer. 18-question Likert-type scale survey and video records of tasks were used to evaluate this low-cost sinus surgery, task trainer. The model was able to show face, content, and construct validity.20 Steehler et al in another study, evaluating the same low-cost sinus surgery task trainer with 52 medical students with no experience in sinus surgery, performed each one recess probing, targeted injections, removal of superior sutures, extraction of posterior beads, and antrostomy of an egg with the removal of contents. Training videos, checklists, and global rating scale were used for evaluation. This low-cost sinus task trainer proved that can be a valuable training tool for basic skills in endoscopic sinus surgery.23Wais et al randomized 14 residents into 2 groups. The first group had a pretraining session with 5 different training modules with tasks to complete as quickly as possible with a low number of mistakes. The day after the pretraining, both groups participate in a cadaveric endoscopic sinus surgery training. Video recordings, a global rating scale, and a task-specific checklist 5-point Likert scale were used for evaluation.The findings of the study suggest that participants who underwent a pretraining session with low fidelity training modules were able to acquire the necessary skills for endoscopic sinus surgery. On the following day, these participants outperformed the group who did not have a pretraining session. The low fidelity training modules proved to be effective in providing basic surgical skills that are essential before transitioning to cadaver training. Furthermore, the use of these low fidelity modules does not raise any ethical concerns, making them a practical and ethical training tool for young doctors. Overall, the study highlights the importance of pretraining sessions and the use of low fidelity training modules for developing basic surgical skills in endoscopic sinus surgery.22Other low-cost endoscopic sinus surgery task trainers showed face, content, construct, and concurrent validity.33,46Three-Dimensionally Printed Model3D printing technology is increasingly used recently to create surgical simulators useful in training. It offers the possibility to create complex anatomical models of high fidelity, being able to simulate any anatomical or pathological variation based on CT images, so that any surgical maneuver can be simulated on such a model. There are several studies in the literature that have proposed the use of 3D printed models for training in endoscopic rhinosinusal surgery and have benefited from validation in this regard.Alrasheed et al developed an anatomical model of the osteomeatal complex and frontal sinus based on CT images of a patient that they segmented using 3D visualization software, then printed this model using a different material for the mucosa and bone tissue. Certain endoscopic procedures were simulated on this model, being evaluated by 20 participants with different levels of training, receiving content and construct validation.34Hsieh et al created a specific patient, 3D printed sinus and skull base model for which he demonstrated anatomical accuracy and a good haptic sense with participants of different degrees of training.39Zhuo et al developed a model using CT data of a pediatric patient without rhinosinusal changes, which was printed using a desktop-level 3D printer. Subsequently, a series of tasks were performed by a group of rhinologists and residents for validation. This is according to the authors the first study to evaluate a pediatric rhinosinusal anatomical model for surgical training in endoscopic sinus surgery.42In another study by Alwani et al,12 residents performed uncinectomy, maxillary antrostomy, anterior ethmoidectomy, posterior ethmoidectomy, sphenoidotomy, and frontal sinusotomy on the PHACON Sinus Trainer (Leipzig, Germany), then they performed the same maneuvers on a cadaver head.43A five-item Likert scale questionnaire, a modified fifteen-item questionnaire, a modified global rating scale and video recordings were used for evaluation. This 3D printed model demonstrated face, content, concurrent, and construct validity.Dong et al used a simulated secretion that mimics mucus and blood that accumulates at the rhinosinusal level during endoscopic surgery and a dissolvable dressing pack. Using a 3D printed head model, simulated secretion was injected in paranasal sinuses and the simulated dressing was also injected in the nasal cavity. 46 residents performed pledget insertion, nasal packing, and postsurgical debridement on this 3D printed head model. A checklist and global rating scale were used for evaluation and face, content, and construct validity were proved.44Gallet et al evaluated 30 participants (10 beginners, 11 intermediate, and 9 expert level) by stability and the accuracy of endoscopic camera handling, train accuracy, dexterity and workspace, precision in the use of the surgical instrument and dissection abilities, using a 3D printed head model as a support for learning modules. These learning modules were able to show face, content, and construct validity with good relevance in endoscopic sinus surgery training.45Other studies have evaluated low-cost 3D printed models with good results and have shown that they are useful tools in acquiring basic surgical techniques in endoscopic rhinosinusal surgery without raising ethical issues.32,40,41Vegetable Model-Capsicum and Tomato ModelAlso, a vegetable model useful in rhinosinusal surgical training has recently been described in the literature. In a study conducted by Tikka et al, Capsicum and tomato were introduced as models for basic endoscopic sinus surgery training. The study involved 10 fellows from the same academic year, who were tasked with performing the proposed maneuvers on these models. The results of the study showed that the participants were able to acquire basic surgical skills, such as hand-eye coordination and maneuvering instruments specific to endoscopic rhinosinusal surgery, with good results.The participants reported that the exercises on the Capsicum and tomato model helped improve their abilities to begin training on the cadaveric model and subsequently on human patients. The use of these models in training has the potential to enhance the learning process, allowing trainees to become more comfortable and proficient with surgical instruments and techniques before operating on actual patients.Notably, the use of Capsicum and tomato models in surgical training is an innovative approach that has gained attention in recent years. These models offer a safe and cost-effective way to provide hands-on experience to trainees, allowing them to gain confidence and familiarity with the instruments used in endoscopic sinus surgery. Additionally, the use of these models in training can potentially reduce the risk of complications and improve patient outcomes by ensuring that trainees are well-prepared and proficient in their surgical skills.Overall, the study conducted by Tikka et al highlights the potential benefits of using Capsicum and tomato models in basic endoscopic sinus surgery training. As such, it is a promising option that warrants further exploration and consideration in the development of surgical training programs.47DiscussionFor aspiring doctors, becoming competent in endoscopic sinus surgery can be an immense challenge, as it requires mastering a variety of complex skills and knowledge beyond the proper surgical procedures. In addition to these procedures, young doctors must also gain a deep understanding of the complex 3D anatomy of the nasal cavity and paranasal sinuses. This knowledge is critical to successfully performing endoscopic sinus surgery, as it enables the surgeon to navigate the anatomical structures, avoid damaging vital tissues, and achieve optimal surgical outcomes.Another significant challenge in mastering endoscopic sinus surgery is the need to translate 2D views into 3D anatomical space. This requires an understanding of how the instruments are positioned in relation to the anatomy, as well as spatial orientation and bimanual dexterity in working with endoscopes and surgical tools. In addition, the surgeon must have the ability to make the right choice in choosing the surgical instruments that are best suited to complete the intended task.Overall, the demands of endoscopic sinus surgery training are multifaceted and require a comprehensive approach to achieve competency. Mastery of these skills requires a combination of didactic education, hands-on training, and experience in surgical procedures. With proper training and guidance, young doctors can acquire the necessary skills and knowledge to become proficient in endoscopic sinus surgery and provide high-quality care to their patients.48The use of surgical simulators has become increasingly important for young doctors to acquire and practice the necessary skills for endoscopic sinus surgery. This is because surgical simulators offer a safe and controlled environment for trainees to repeatedly practice procedures and techniques without putting patients at risk. By providing opportunities for hands-on experience and repetition, surgical simulators have become a valuable tool for young doctors to gain confidence and proficiency in performing complex endoscopic sinus surgeries. This ultimately leads to better patient outcomes and a higher quality of care.51 Moreover, the trainee can objectively evaluate surgical skills achieved and the progress in a measurable way.52 As a new state of the art, surgical simulators are developed continuously, so they can be used to learn basic endoscopic surgery skills by repetitive maneuvers, permitting detection of surgical error and incidents without risk for the patient.53All of these simulators apply themselves to learning endoscopy skills and hand-eye coordination. The capacity of task achievement ameliorates with exercise. They do not support surgical decision‐making and do not have the quality to learn surgical anatomy. Given the relief of construction, low cost, and total performance element, these low‐fidelity physical simulators may be brought into practice for learning the basic skills in endoscopic sinus surgery.48While non-virtual reality simulators have been used for a very long time, they are still popular because they may have several good features. The ideal surgical simulator is accessible, safe, available, reusable, realistic in the handling of surgical instruments, and has the ability to show progress with repetition.Several studies demonstrate that the surgical skills developed on low‐fidelity physical simulators might confer the same manner of profit as high‐fidelity surgical simulators, such as cadavers. This is possible because the teaching method was considered to be superior to the physical substrate.25Endoscopic sinus surgery simulation on cadavers is better than physical models, but it depends on cadaver availability and ethical considerations. The main advantage is that the cadaver constitutes the only true anatomical model of human paranasal sinuses.48,50Cadaveric simulators, despite being considered as the gold standard over physical and virtual reality surgical simulators, are yet to establish if their use increases operating efficiency. Nevertheless, novices considered the experience of learning on cadavers to be highly useful in everyday practice.54Animal tissue is one of the popular non-virtual simulators because of the realistic tissue feel and anatomy likeness. Despite that, it is the growing difficulty in using human cadavers for learning that has driven the quest for other possibilities.ConclusionMoreover, it is worth noting that the ovine model has been extensively utilized in various research studies and surgical training programs, and has demonstrated promising results in preparing trainees for actual surgical procedures. As a result, it has become a popular choice for medical institutions and rhinology centers worldwide.One of the key advantages of the ovine model is its cost-effectiveness. Compared to other training models, such as cadavers or computer simulations, the ovine model is significantly less expensive, making it accessible to a wider range of medical trainees. Additionally, the ovine model is readily available in many countries, making it an excellent option for aspiring rhinology doctors worldwide.Furthermore, the ovine model offers a safe and controlled environment for trainees to practice surgical procedures. Since the model is not a live human patient, there are fewer risks associated with the training process. Additionally, the ovine model enables trainees to focus on specific surgical techniques, such as endoscopic navigation, dissection, and hemostasis, without the added stress of operating on a human patient.In conclusion, while the ovine model is not a perfect replica of human rhinosinusal anatomy, it offers a cost-effective and safe training option for aspiring rhinology doctors to hone their surgical skills. With its widespread availability and ability to simulate real-life surgical scenarios, the ovine model remains a valuable asset in the training and education of future endoscopic sinus surgeons.

PMC

Journal of Asthma and Allergy

38469567

PMC10926854

3-07-2024

10.2147/JAA.S451911

A Case of Type I Food Allergy Induced by Monosodium Glutamate

Osada Reeko, Oshikata Chiyako, Kurihara Yuichi, Terada Kosuke, Kodama Yuka, Yamashita Yuga, Nakadegawa Ryo, Masumitsu Hinako, Motobayashi Yuto, Takayasu Hirokazu, Masumoto Nami, Manabe Saki, Zhu Yingyao, Tanaka Ryo, Kaneko Takeshi, Sasaki Aya, Tsurikisawa Naomi

AbstractMonosodium glutamate (MSG), a salt form of a non-essential amino acid, is widely used as a food additive, particularly in Asian cuisines, due to its unique flavor-enhancing qualities. Type I allergic reactions to MSG have not previously been reported. Our patient, a 21-year-old woman, was 14 years old when she first noticed swelling of her tongue (but no oral itching, diarrhea, or abdominal pain) after eating various snack foods. Current skin prick testing elicited a weak positive reaction to MSG. We then performed an oral challenge test during which our patient ingested potato snacks. Subsequent histology showed telangiectasia of the buccal mucosa, interstitial edema in the subepithelial submucosa, and mast cell infiltration. Oral mucosal challenge tests using sodium glutamate confirmed oral swelling in this patient. This report is the first to confirm a case of type 1 allergy to MSG by combining pathology findings with the results of challenge testing.

IntroductionMonosodium glutamate (MSG), a salt form of a non-essential amino acid, is widely used as a food additive, particularly in Asian cuisines, due to its unique flavor-enhancing qualities. First reported in 1968 as Chinese restaurant syndrome,1 some people have described symptoms including a burning sensation at the back of the neck and on the forearms and chest; headache; chest pain; numbness at the back of the neck and radiating to the arms and back; nausea; and palpitations after consuming MSG. Using results from an evaluation conducted by the American Federation of Experimental Biology Societies, the United States Food and Drug Administration reaffirmed the safety of MSG as a food additive in 1995.2MSG is intentionally added to foods as a flavoring agent. One of its components, glutamic acid is found naturally in almost all foods, including vegetables, meat, fish, and breast milk. In general, protein-rich foods such as breast milk and meat contain large amounts of protein-bound glutamate, whereas vegetables (particularly peas, tomatoes, and potatoes), fruits, and mushrooms contain high concentrations of free glutamate. In addition, glutamate is particularly abundant in some cheeses, including parmesan.2The symptoms complex due to MSG consumption is considered to begin within one hour of ingestion of more than 3 g of MSG as an oral bolus. Typically, a dose of MSG of 2.5 g or less causes no symptoms in nonallergic people; larger doses can cause a burning sensation, facial pressure, headache, and drowsiness, but these symptoms usually dissipate within 4 h.3 However, the information was not communicated accurately overseas, where the use of MSG remained restricted. Descriptions of MSG-induced asthma, urticaria, angioedema, and rhinitis have prompted suggestions that MSG causes an allergic reaction in patients presenting with these conditions. However, none of the reports of MSG-induced asthma have proven reproducible, and MSG-induced asthma was not replicated in a double-blind placebo-controlled study.4 Our current report is the first to confirm a case of type 1 allergy to MSG by combining pathology findings with the results of challenge testing.CaseThe 21-year-old woman whose case we present was diagnosed with allergic rhinoconjunctivitis when she was 17. Beginning when she was 14 years old, she noticed that her tongue swelled after eating various snack foods but had no oral itching, diarrhea, or abdominal pain. The frequency of swelling of the tongue increased once she turned 20. Swelling of the tongue did not occur after eating either homemade meals without additives, items from conveyor-belt sushi restaurants, or at any of the meals in restaurants during a trip to Europe. In contrast, her tongue swelled after she ate at a chain family restaurant and after having fast-food hamburgers, French fries, potato snacks, parmesan cheese, and consommé-flavored (but not light-salt) potato chips. In addition, she did not develop any adverse symptoms after ingesting potatoes prepared otherwise than mentioned or after eating any type of fruit.We performed a skin prick test and measured antigen-specific serum immunoglobulin E (IgE) levels. Total IgE (IU/mL) and antigen-specific IgE (IU/mL) in serum were measured by using enzyme-linked immunosorbent assay (ELISA) according to the nephelometry method (BN II, Dade Behring Inc, Deerfield, IL).5 Our patient’s total serum IgE level was 36.0 IU/mL (normal, ≤173 IU/mL), with antigen-specific serum IgE levels of 0.15 IU/mL each for house dust and Japanese cedar pollen (positive reaction defined as specific IgE > 0.35 IU/mL) but undetectable for wheat, potato, and pollens other than cedar. We then measured wheals and flares at 15 min after skin pricking with these antigens;6 we defined a positive allergen reaction as eliciting a mean wheal diameter ≥3 mm larger than that produced by the negative control (saline)7,8 or at least 50% larger than that due to the positive control (histamine).9 We obtained sodium glutamate as MSG (Ajinomoto, Ajinomoto Co., Inc, Tokyo, Japan). Our patient had a positive reaction to 100 mg/mL of MSG (wheal size, 4×4 mm; saline, 0×0 mm; histamine, 6×5 mm) but negative reactions to 10 mg and 1 mg/mL of MSG, potato, wheat, gluten, and omega-5-gliadin. We confirmed the lack of nonspecific reaction (0 × 0 mm) to 100 mg/mL of MSG on the skin of six people without MSG sensitivity.After obtaining written informed consent from our patient, we then performed an oral challenge test during which she ingested potato snacks that contained MSG. In the oral challenge test, this patient continued to consume snack foods until oral symptoms appeared. After ingesting 4 sticks (1.5 g each), she experienced only a tingling tongue; after 23 sticks (ie, about 20 min after starting to eat), she had a sore throat, painful tongue, itchy palate, and buccal mucosal swelling and bleeding. Because of severe pain, she could not ingest any more than 31 sticks; at this point (ie, about 30 min after she began eating), we obtained a biopsy of the buccal mucosa. Routine histology showed telangiectasia of the buccal mucosa (Figure 1a) and interstitial edema in the subepithelial submucosa (Figure 1b). In addition, c-kit immunohistochemistry revealed mast cell infiltration (Figure 1c). Given these findings, we diagnosed a type I allergic reaction in our patient and confirmed that her oral bleeding was from biting her tongue, which became edematous due to her allergic reaction. Figure 1Buccal mucosal pathology after challenge testing. We biopsied our patient’s buccal mucosa immediately after intraoral bleeding developed due to her ingestion of potato snacks. Hematoxylin and eosin staining shows telangiectasia (black arrows) (a) and edema (black arrows) (b) of the subepithelial submucosa. Immunohistochemistry for c-kit reveals mast cells (red arrows) (c). The images in panels (b and c) are of the same section.To further characterize our patient’s food allergy, we performed oral mucosal challenge tests using common components of snack foods, specifically powdered fats and oils, salt containing umami seasoning, and a commercial product that is 97.5% sodium glutamate (Ajinomoto), with saline as a control. We performed a single-blind oral mucosal challenge test using saline and confirmed a negative reaction. Powdered fats and oils did not elicit any adverse responses, but both the umami-seasoned salt and commercial 100 mg/mL MSG product caused pain in the oral cavity, swelling of the tongue, and facial erythema (flushing), with the MSG product causing stronger reactions. We used the dose of 100 mg/mL MSG because it yielded a reaction in the skin-prick test, whereas 10 and 1 mg/mL did not. We therefore further characterized our patient’s food allergy as a type 1 allergic reaction to MSG.Many foods contain MSG, and it is difficult to avoid consuming it when eating out. Our patient learned to be careful not to bite her tongue when she experiences oral discomfort during meals. Since her diagnosis of MSG allergy, our patient has not had any more episodes of oral bleeding, and her quality of life has improved.DiscussionFirst categorized by Gell and Coombs, hypersensitivity reactions are now classified according to nine types: antibody-mediated (types I–III), cell-mediated (IVa–c), tissue-driven mechanisms (V, VI), and direct response to chemicals (VII).10 Type I (IgE-mediated) reactions occur in patients with allergic rhinitis, conjunctivitis, asthma, atopic dermatitis, pollen-induced food and drug allergies and allergies to house dust mites and other animals, various foods, materials (eg, latex), and drugs.10 The allergen-specific IgE in type I reactions is produced by mast cells and basophils. Although our patient had considerable numbers of mast cells in the subepithelial submucosa, skin-prick testing elicited only a token IgE reaction. Because food allergies also manifest through non-IgE-dependent mechanisms,11 we consider that allergy to food additives may develop through a type I allergy mechanism with low IgE reactivity. In the case we present, our patient experienced no allergic symptoms after ingesting MSG-containing snacks before the age of 13, and she developed hay fever when she was 17 years old; these features suggest that her sensitization to MSG was acquired after provocation. This congenital sensitization likely explains her low total IgE level and modest responses to skin prick tests.Reported allergic reactions to MSG include urticaria, angioedema, allergic rhinitis, bronchial asthma, and exercise-induced anaphylaxis, but these reports are associated with discrepancies in the time between MSG intake and appearance of the allergic reaction and a lack of reproducibility in provocation tests using a placebo.4,12 The evidence to support MSG as a cause of urticaria or angioedema was insufficient, because reported cases have been limited by inadequate blinding, small sample sizes, and the potentially confounding withdrawal of antihistamines before MSG challenges. The possibility that MSG may induce acute rhinitis symptoms and contribute to chronic rhinitis has been raised. Future studies addressing a possible relationship between the ingestion of MSG and the development of rhinitis will need to differentiate MSG allergic rhinitis from incidentally occurring chronic rhinitis.In regard to diagnosing food allergy, a food provocation test under double-blind placebo-controlled conditions is considered the gold standard.13 We instead opted to perform a single-blind placebo-controlled challenge provocation test because we anticipated that our patient would experience oral swelling and severe pain after ingesting food containing MSG. Because we had previously confirmed that the placebo test using saline was negative, we decided that a single-blind design was our best option in terms of patient safety during the provocation test. In addition, we quickly responded to and treated the severe allergic reaction that occurred during the provocation test.The actual dose of MSG in the snack food that caused our patient’s symptoms was unknown, so she continued to eat the snack food during the provocation test until symptoms actually appeared. The dose of 100 mg/mL MSG used in the oral challenge test was determined according to the prick test threshold, but we consider that this dose exceeds that when MSG is used as an actual seasoning. Our patient ingested the MSG-containing food for 20 to 30 min before symptoms began to appear. We therefore consider it likely that the symptoms caused by a 3-g oral bolus differ from those due to consumption of commercially available foods.Anaphylaxis due to poly-γ-glutamic acid reportedly develops relatively late (ie, 5 to 14 h) after the ingestion of fermented soybeans.14 Although natto and MSG have similar structural formulas, their sensitization pathways seem to differ.15 We speculate that, given the widespread use of this food additive, MSG allergy may be more prevalent than appreciated, with many cases unreported. In the case we report here, the skin prick test for MSG was positive, challenge testing led to the appearance of mast cells in our patient’s oral submucosa, and the reproducibility of her symptoms supported the diagnosis of a type I allergy to MSG. In addition, this report is the first to describe the use of pathology findings with the results of challenge testing to confirm MSG type 1 allergy.ApprovalInstitutional approval of the manuscript was not required to publish the case details.ConclusionWe here present a patient who showed oral allergy induced through the ingestion of MSG. This report is the first to confirm a type 1 allergy to MSG by combining pathology findings with the results of challenge testing.

PMC

Environment international

37331181

PMC10519343

8-01-2023

10.1016/j.envint.2023.108009

Prenatal exposure to polycyclic aromatic hydrocarbons and cognition in early childhood

Sun Bob, Wallace Erin R., Ni Yu, Loftus Christine T., Szpiro Adam, Day Drew, Barrett Emily S., Nguyen Ruby H.N., Kannan Kurunthachalam, Robinson Morgan, Bush Nicole R., Sathyanarayana Sheela, Mason Alex, Swan Shanna H., Trasande Leonardo, Karr Catherine J., LeWinn Kaja Z.

Background:Epidemiological evidence for gestational polycyclic aromatic hydrocarbon (PAH) exposure and adverse child cognitive outcomes is mixed; little is known about critical windows of exposure.Objective:We investigated associations between prenatal PAH exposure and child cognition in a large, multi-site study.Methods:We included mother–child dyads from two pooled prospective pregnancy cohorts (CANDLE and TIDES, N = 1,223) in the ECHO-PATHWAYS Consortium. Seven urinary mono-hydroxylated PAH metabolites were measured in mid-pregnancy in both cohorts as well as early and late pregnancy in TIDES. Child intelligence quotient (IQ) was assessed between ages 4–6. Associations between individual PAH metabolites and IQ were estimated with multivariable linear regression. Interaction terms were used to examine effect modification by child sex and maternal obesity. We explored associations of PAH metabolite mixtures with IQ using weighted quantile sum regression. In TIDES, we averaged PAH metabolites over three periods of pregnancy and by pregnancy period to investigate associations between PAH metabolites and IQ.Results:In the combined sample, PAH metabolites were not associated with IQ after full adjustment, nor did we observe associations with PAH mixtures. Tests of effect modification were null except for the association between 2-hydroxynaphthalene and IQ, which was negative in males (βmales = −0.67 [95%CI:−1.47,0.13]) and positive in females (βfemales = 0.31 [95%CI:−0.52,1.13])(pinteraction = 0.04). In analyses across pregnancy (TIDES-only), inverse associations with IQ were observed for 2-hydroxyphenanthrene averaged across pregnancy (β = −1.28 [95%CI:−2.53,−0.03]) and in early pregnancy (β = −1.14 [95%CI:−2.00,−0.28]).Significance:In this multi-cohort analysis, we observed limited evidence of adverse associations of early pregnancy PAHs with child IQ. Analyses in the pooled cohorts were null. However, results also indicated that utilizing more than one exposure measures across pregnancy could improve the ability to detect associations by identifying sensitive windows and improving the reliability of exposure measurement. More research with multiple timepoints of PAH assessment is warranted.

1.IntroductionAccumulating epidemiological evidence suggests that prenatal exposure to polycyclic aromatic hydrocarbons (PAHs), a prevalent environmental toxicant resulting from the incomplete combustion of fossil fuels and other organic materials, is associated with adverse neurocognitive development. Predominant sources of PAH exposure include inhalation of ambient air pollution, smoking, and from diet through consuming grilled or barbequed meats(Agency for Toxic Substances and Disease Registry (ATSDR), 1995). Neurodevelopmental deficits in children associated with prenatal PAH exposure have included differences in attention, behavior, and cognition(Jedrychowski et al., 2015; Perera et al., 2014). Hypothesized mechanisms for neurotoxic effects include disruption of pathways that regulate neuronal differentiation, synapse formation, and synapse plasticity(McCallister et al., 2008; Deanna D Wormley et al., 2004), epigenetic modifications (Herbstman et al., 2012), endocrine disruption(Takeda et al., 2004), oxidative stress and neuroinflammation(Saunders et al., 2006), and impairment of long-term potentiation of hippocampus, critical to learning and memory (D. D. Wormley et al., 2004).Several previous studies have specifically investigated associations between prenatal PAH exposure and childhood IQ(Edwards et al., 2010; Perera et al., 2012, 2009). Two of these studies, conducted in New York City, and Krakow, Poland, demonstrated lower childhood IQ (Perera et al., 2009) or nonverbal reasoning ability (Edwards et al., 2010) associated with prenatal PAH exposure measured in persona. In contrast a third study in Chongqing, China of prenatal PAHs measured in cord blood showed no association with child IQ (Perera et al., 2012). Measures of prenatal PAH exposure via personal air monitoring have the benefit of capturing longer periods of exposure than PAHs measured in urine, are limited to airborne sources, and do not capture exposure by routes such as dietary intake. This is important, as diet is estimated to contribute up to 70–90% of total PAH exposure, but with substantial geographic and temporal variability(Agency for Toxic Substances and Disease Registry (ATSDR), 1995; Skupinska et al., 2004; Suzuki and ´ Yoshinaga, 2007). Furthermore, previous work largely aggregates measures of various PAH compounds into a single exposure assessment, whether by constructing a composite of PAH concentrations in air, or by measuring PAHs in cord blood at birth. This does not allow for evaluating associations of individual PAH metabolites, or co-exposure to multiple PAH metabolites, which may have different neurodevelopmental toxicities that are not apparent in single-pollutant models. Further, the use of a single exposure metric may be prone to misclassification error as it does not account for intraindividual variability and may not reflect patterns of exposure over pregnancy; nor does it allow for the exploration of windows of vulnerability to prenatal PAH exposure.Another gap in the current literature is the limited research on child or pregnancy-related factors that might modify adverse associations between PAH and child cognition or other neurodevelopmental endpoints. Studies of prenatal exposures to similar toxicants such PM2.5 and child IQ show differences in outcomes by child sex, with inverse associations more prominent in boys than girls (Chiu et al., 2016; Lertxundi et al., 2019). Brain development differs between males and females and sex hormones are believed to interact with the neuroendocrine and immune systems in modulating the fetus’ response to stressors (McCarthy et al., 2017). We also hypothesized that maternal body composition could interact with PAH exposure on fetal brain development. PAHs are lipophilic and accumulate in fatty tissue(Pastor-Belda et al., 2019), and studies of other lipophilic environmental contaminants demonstrate that they are transferred from adipose tissue to placenta (Kapraun et al., 2022), potentially exposing fetuses of women with higher body mass to higher exposure to PAHs even given the same level of environmental exposure.Our study adds to the existing literature by estimating the associations between prenatal exposure to PAH and childhood cognition at age 4–6 years in a large, diverse, multi-site U.S. sample, with robust adjustment for potential confounders. We measured PAHs in mid-pregnancy urine, which captures all routes of PAH exposure. We hypothesized that higher prenatal PAH exposure would be associated with lower childhood IQ. We also use contemporary mixtures methods to estimate associations between mixtures of PAH metabolites and early childhood cognition. We assessed whether sex or pre-pregnancy body mass index modified associations between prenatal PAH and childhood IQ. Finally, in a subset of participants, we evaluated multiple time points of prenatal exposure to PAH in relation to childhood IQ to better estimate associations across pregnancy and to explore periods during which the fetus may be more susceptible to the impacts of PAH exposure.2.Subjects and methods2.1.Design and participantsThe current investigation is part of the National Institutes of Health Environmental influences on Child Health Outcomes (ECHO) PATHWAYS Consortium, which pooled data from three pre-existing cohorts (LeWinn et al., 2022). Two cohorts of the ECHO PATHWAYS Consortium had mid-pregnancy PAH data and were included in this analysis: the Conditions Affecting Neurocognitive Development and Learning in Early Childhood (CANDLE) study, in Memphis, TN; and The Infant Development and Environment Study (TIDES) in San Francisco, CA, Minneapolis, MN, Rochester, NY, and Seattle, WA. Design, recruitment, and data collection for CANDLE and TIDES have been previously described(LeWinn et al., 2022). In brief, CANDLE enrollment of pregnant mothers occurred between 2006 and 2011. Participant follow-up included two prenatal study visits in mid and late pregnancy, biospecimen collection, and ongoing postnatal follow up of mothers and offspring. Mother-child pairs were followed prenatally and at regular intervals with clinic and home visits and telephone surveys, including clinic visits at approximately 4 years of age. The TIDES study recruited mothers between 2010 and 2012, with three prenatal study visits occurring in early, mid, and late pregnancy that included biospecimen collection and maternal surveys. Mother-child pairs were followed with questionnaires and a clinic visit at approximately 6 years of age. For the current study, CANDLE and TIDES participants were included if they were singleton births with a mid-pregnancy prenatal urinary PAH measure and a measure of IQ. We excluded children born less than 34 weeks of pregnancy, as well as children of mothers who smoked during pregnancy, as the relationship between children born prematurely and of mothers who smoke and child IQ is well-established in the literature (Herrmann et al., 2008; Soleimani et al., 2014). Maternal prenatal smokers were defined using self-reported smoking at any point in pregnancy, or had urinary cotinine levels of greater than 200 ng/ml measured in mid or late pregnancy(Schick et al., 2017). The CANDLE study was approved by the institutional review board (IRB) of the University of Tennessee Health Sciences Center. TIDES was approved by the IRBs of the University of California, San Francisco, University of Minnesota, University of Rochester Medical Center, and University of Washington. The current analysis was conducted by ECHO PATHWAYS and was approved by the University of Washington IRB.2.2.Exposure assessmentFor CANDLE participants, urine collection occurred in mid-pregnancy, at a mean of 23.0 weeks (SD = 3.0). In TIDES, collection occurred once each in early-, mid-, and late-pregnancy, with mid-pregnancy samples used in the main analyses. Early pregnancy urinary measures were taken, on average, at 10.9 weeks’ gestation (SD = 2.4); mid-pregnancy, at a mean of 20.8 weeks (SD = 3.8); and late-pregnancy, at a mean of 32.7 weeks (SD = 3.1). The methods for the extraction and processing of urinary hydroxy-PAHs (OH-PAHs) has been published previously(Guo et al., 2013). Briefly, extraction of OH-PAHs from urine was performed by liquid–liquid extraction followed by LC-MS/MS analysis (Guo et al., 2013). A Waters Acquity I-Class UPLC system (Waters; Milford, MA, USA) was used for chromatographic separation of PAHs, connected with an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm, Waters; Milford, MA, USA). The identification and quantification of PAH metabolites was performed on an ABSCIEX 5500 triple quadrupole mass spectrometer (Applied Biosystems; Foster City, CA, USA). The laboratory participated in several external quality assurance schemes to validate OH-PAHs assay successfully(Kannan et al., 2021). Individual urinary OH-PAH metabolites were included in this analysis if they were detected in at least 70% of the pooled TIDES and CANDLE study sample. These include 1-hydroxypyrene, 1- and 2-hydroxynaphthalene, 1/9-, 2-, and 3-hydroxyphenanthrene, and 2/3/9-hydroxyfluorene. In addition, we analyzed the molar sums of the hydroxynaphthalene and hydroxyphenanthrene metabolites for separate analyses. For OH-PAH measured below the limit of detection (LOD), we substituted the value of LOD/2.2.3.OutcomeChild cognition was assessed using a validated measure of IQ in each cohort. In CANDLE, IQ was measured at approximately age 4 years using the Stanford-Binet Intelligence Scales, Fifth Edition (SB-5). The SB-5 included 10 subtests addressing five cognitive factors, including knowledge, fluid reasoning, quantitative reasoning, visual-spatial processing, and working memory. The IQ score in the SB-5 has excellent internal consistency and test–retest reliability (0.98 and 0.92, respectively)(Sattler, 2018). In TIDES, the Wechsler Intelligence Scale for Children, Fifth Edition (WISC-V), was administered at approximately 6 years(Raiford, 2018). For the WISC-V, IQ was derived from five subtests and included Vocabulary (Verbal Comprehension), Block Design (Visual Spatial), Matrix Reasoning (Fluid Reasoning), Digit Span (Working Memory), and Coding (Processing Speed). This combination represents one primary subtest from each index scale. IQ was calculated using the Tellegen and Briggs formula(Wechsler, 2003). Reliability and validity coefficients for this five-subtest version are 0.945 and 0.934 respectively (Sattler, 2018). This approach is comparable to prior studies(Mollon et al., 2018) which have utilized an abbreviated four-subtest version of the WISC-IV to assess child cognition. IQ scores from both the SB-5 and WISC-V were standardized to age in 3-month intervals to a mean score of 100 and a standard deviation of 15. While the specific tests used to capture the different cognitive domains may vary between SB-5 and WISC-V, performance on the instruments is highly correlated in typically developing children and they provide a standardized metric of overall cognitive performance (Roid, 2003). This approach is similar to prior studies which sought to examine child IQ across different instruments (Kullar et al., 2019; Lanphear et al., 2005; Ni et al., 2022). All examiners were thoroughly trained on the administration and scoring of the SB-5 or WISC-V by licensed psychologists. They participated in didactic instruction and guided practice, inter-rater reliability exercises, as well as weekly supervision by psychologists post-training. Overall reliability equal to or greater than 90% was required for the examiners to administer the measure independently. To identify potential data entry and scoring errors, we used logic checks (e.g., comparing raw scores to expected standardized scores) and reviewed outliers (i.e., scores significantly above or below the normative mean).2.4.Other variablesMothers reported on their own and their child’s characteristics at prenatal and postnatal study visits, including child race, marital status, pre-pregnancy body mass index (BMI), maternal education attainment, household size, smoking and tobacco use during pregnancy and in the postnatal period, length of breastfeeding and family income. Family income was adjusted for regional price differences and inflation using established econometric methods from the U.S. Bureau of Economic Analysis (BEA)(Bureau of Economic Analysis, 2021). Medical record abstraction was used to collect information on infant sex, gestational age, and birthweight. Child secondhand tobacco smoke exposure was reported at the child’s age 4–6 visit by maternal survey. Mothers were asked if they, the child’s father, child’s caregiver or care provider, or any other person living in the home with the child smoked (Yes/No). Child opportunity was measured using the Education and Social and Economic domains of the Child Opportunity Index, linked to maternal address at enrollment(Acevedo-Garcia et al., 2014). Maternal IQ was assessed at the age 4–6 visit using the Wechsler Abbreviated Scale of Intelligence (WASI) short form in both cohorts(Axelrod, 2002).2.5.Statistical analysisWe described the demographics of the study sample overall as well as for the component CANDLE and TIDES cohorts. For descriptive summaries of OH-PAH metabolites, we standardized raw PAH values to the median study sample urinary specific gravity (SG) to account for urinary dilution, as follows: PAHstandard=PAHraw×SGmedian−1SGobserved−1 Where PAHstandard represents the standardized PAH value, PAHraw represents the raw PAH value, SGmedian represents the median specific gravity for the PAH metabolites, and SGobserved represents the specific gravity for the PAH metabolite(Levine and Fahy, 1945). To assess the relationship between OH-PAH and IQ, we used multivariable linear regression, with separate models for each individual urinary metabolite (and the hydroxynaphthalene and hydroxyphenanthrene sums). Unless specified, all analyses were restricted to participants without missing covariate data. Consistent with prior studies of urinary measures of prenatal PAH exposure and health outcomes(Ferguson et al., 2017) (Abid et al., 2014; Ferguson et al., 2017), we used raw, log-transformed OH-PAH metabolite concentrations in the multivariable linear regressions and an adjustment term for specific gravity was included to account for urinary dilution. Estimates and 95% CI were multiplied by ln to calculate change in IQ per 2-fold increase in OH-PAH metabolite or parent compound sum. We took a staged approach to covariate adjustment to examine the influence of increasing adjustment on effect estimates. Potential confounders were determined a priori and defined as factors either directly or indirectly associated with prenatal PAH exposure and child IQ. We also identified selected precision variables with well-established relationships with child IQ although not likely to be associated with prenatal PAH exposure(Lewinn et al., 2020). Supplemental Fig. 1 shows a Directed Acyclic Graph for covariate selection and regression model development. A “minimal” model adjusted for study site (five categories), child sex (binary), child age (continuous), analysis batch (four categories), specific gravity (continuous), and an interaction between specific gravity and cohort to account for urinary dilution in each cohort’s OH-PAH measures. A “full” model, considered the main analysis, additionally adjusted for maternal education (four categories), maternal IQ (continuous), maternal age (continuous), marital status (binary, married or living as married vs. not), birth order (binary, first born vs. not), prenatal urinary cotinine (continuous), secondhand smoke exposure (binary, any vs. none), breastfeeding practice (binary, none vs. any), an interaction between household income and household count, each of the education and economics domains of the Child Opportunity Index (continuous), and child race (three categories). Race and ethnicity are conceptualized as important social constructs that, in this analysis, may capture unmeasured confounding due to exposures inequitably distributed by race and associated with both exposure to air pollution and EF (e.g., other toxic exposures like lead). Finally, an “extended” model additionally included gestational age (continuous) and birthweight (continuous), potential confounders that were not included in the full model because they may also lie on the causal pathway.2.6.Sensitivity analysesWe conducted a number of sensitivity analyses. To evaluate alternate methods of accounting for urinary dilution, we standardized individual OH-PAHs as well as urinary cotinine to SG via the above formula rather than adjusting for SG as a covariate. We substituted below LOD OH-PAH values by censored likelihood multiple imputation (CLMIClick or tap here to enter text.)(Boss et al., 2019), using fixed effects meta-analysis rather than our primary pooling approach since differences in OH-PAH distributions across cohorts precluded fitting pooled CLMI models. To compare with our primary analysis’s exclusion of participants with missing covariates (i.e. complete case analysis), we imputed missing covariates using the method of multiple imputation by chained equations. To assess whether our results might be driven by heterogeneous associations across study sites or cohort, we performed a series of “full” regressions leaving one study site out in turn as well as repeating the main analyses by study cohort. To exclude the influence of participants with intellectual disability, who may have underlying neurodevelopmental differences linked to their level of cognition, we repeated our analyses after restricting our sample to participants with IQ above 70. As a final sensitivity analyses, we repeated analyses using more parsimonious adjustment models with covariate adjustment comparable to prior cohort studies of prenatal PAH exposure and IQ in New York and Krakow (Edwards et al., 2010; Perera et al., 2009).2.7.Secondary analysesAs a secondary analysis, we examined the associations between mixtures of OH-PAH metabolites and early childhood IQ by weighted quantile sum (WQS) regression(Carrico et al., 2015; Czarnota et al., 2015). OH-PAH levels were standardized to specific gravity via the formula above and transformed into deciles. We estimated weighted sums of individual OH-PAH metabolites to comprise the WQS score. To improve the sensitivity to detect the associations between the WQS score and IQ in multivariable linear regression models, we used bootstrap resampling methods to estimate index weights, using 1000 bootstrap samples for each analysis. We separately tested for positive or negative associations between the OH-PAH mixture and IQ. Estimates were adjusted for all covariates in the “full” model. To correct for potential Type 1 error, for any full sample WQS analysis that resulted in a 95% CI that did not include the null, we estimated a permutation test p-value (Day et al., 2022).To investigate possible effect modification by child sex and maternal pre-pregnancy BMI, we used multivariable linear regression and added an interaction term between each modifier and OH-PAH metabolite. We estimated stratum-specific associations and corresponding 95% CI in each group and evaluated evidence of effect modification using p-values for interaction with a significance threshold of 0.05. Pre-pregnancy BMI was dichotomized as overweight/obese (≥25 kg/m2) vs. not (less than25 kg/m2). All estimates were adjusted for covariates in the “full” model.2.8.Analyses across pregnancyIn the study sample from the TIDES cohort that had OH-PAH measures from all three periods of pregnancy, we examined associations of pregnancy-averaged OH-PAH exposures and IQ as well as estimating period-specific associations to explore the possibility of differing susceptibility to PAH exposure at different points in pregnancy. 1-Hydroxypyrene and 2/3/9-hydroxyfluorene were excluded from these TIDES-only analyses due to the high proportion of samples below LOD in the participant sample. We calculated pregnancy-average OH-PAH metabolite concentrations over the three periods of pregnancy and examined associations with IQ. In addition, for each of the three periods in pregnancy, we separately performed multivariable linear regression. We then examined OH-PAHs during each period in pregnancy with and without mutual adjustment for the other two periods to assess the independent association of OH-PAH at a particular window in pregnancy on childhood IQ. All regressions were performed with the “full” model described above.All analyses were completed in R version 3.6.5 (R Development Core Team). P-values less than 0.05 were deemed statistically significant.3.ResultsThe inclusion of enrolled CANDLE and TIDES participants in the analytic sample is illustrated in Fig. 1. Of the 2,403 participants enrolled in CANDLE and TIDES, 1,375 had both prenatal PAH measures and child IQ. After excluding 118 prenatal smokers and 34 children born prior to 34 weeks, the study sample comprised 838 CANDLE participants and 385 TIDES participants, for a total of 1,223. Participants in the analytic sample were similar across sociodemographic and maternal characteristics to the study population at enrollment (Supplemental Table 1). Descriptive characteristics of the analytic sample are presented in Table 1. Compared to child participants in CANDLE, those in TIDES were more often reported to be White (69% TIDES, 29% CANDLE), more likely to ever breastfeed (93% TIDES, 68% CANDLE), and less likely to be exposed to second hand smoke (2% TIDES, 25% CANDLE); they had higher income households on average ($105 k ± 58 k TIDES, $42 k ± 29 k CANDLE) with fewer household members. Mothers in TIDES were more educated (80% with some college or beyond, 46% in CANDLE), more likely to be married (87%, 59% CANDLE), and less often overweight or obese (44%, 61% CANDLE) (Table 1).3.1.Distribution of prenatal urinary OH-PAHs and IQThe distribution of OH-PAH values in the analytic sample and by cohort, standardized to SG, are described in Supplemental Table 2. The proportion of sample below LOD was highest for 1-hydroxypyrene and 2/3/9-hydroxyfluroene and lowest for 2-hydroxynapthalene (Supplemental Table 2). Urinary OH-PAH concentrations were higher in CANDLE than TIDES participants for all metabolites with the exception of 3-hydroxyphenanthrene and 1-hydroxypyrene (Supplemental Table 2). OH-PAHs exhibited moderate-to-strong pairwise correlations (Supplemental Table 3). Child IQ averaged 102.9 (standard deviation 15.6) in the pooled study sample.3.2.Associations between urinary OH-PAHs and IQIn minimally adjusted models, we observed significant inverse associations between several individual OH-PAH metabolites and IQ, most notably for 2-hydroxynapthalene, 1-hydroxypyrene, and 2/3/9-hydroxyfluorene. None of these associations persisted after full or extended adjustment for covariates and there were no meaningful differences between the results of the full and expanded adjustment models (Fig. 2).3.3.Sensitivity analysesEstimates were similar when OH-PAH values were standardized to specific gravity to account for urinary dilution rather than by including specific gravity as a covariate (Supplemental Fig. 2); 1-hydroxynaphthalene was positively associated with IQ (β = 0.61, 95% CI: 0.02, 1.22; p = 0.04). Other sensitivity analyses, including using CLMI to impute OH-PAH values below LOD, restricting the study sample to those with IQ over 70, imputation of missing covariates, leave-one-out analyses (exclusion of individual study sites one at a time) and by study cohort, and using more parsimonious adjustment were also similar to the main analyses (Supplemental Figs. 3–7).3.4.Secondary analysesIn secondary analyses of OH-PAH mixtures using WQS, we did not observe any association, either positive or negative, between a mixture of OH-PAHs and IQ (Table 2). Estimates of effect modification by child sex are provided in Fig. 3. There was no evidence of association between OH-PAHs and IQ in either males or females and there was no statistical evidence for effect modification by sex with the exception of 2-hydroxynapthalene, which was associated with lower IQ in males (βmales = −0.67, 95% CI: −1.5, 0.13), but not associated with IQ in females (βfemales = 0.31, 95% CI: −0.52, 1.1) (p-value for interaction = 0.04) (Fig. 3). We observed no evidence for effect modification by maternal pre-pregnancy BMI (Fig. 3).3.5.Associations between urinary PAHs and IQ across pregnancyThere were 356 TIDES participants who had prenatal OH-PAHs measured in all three periods of pregnancy and IQ data in childhood. The distribution of prenatal PAH metabolite concentrations averaged over all of pregnancy and by period of pregnancy are provided in Supplemental Table 4. Intraclass correlation coefficients for PAH metabolites across pregnancy ranged from 0.29 to 0.47 (Supplemental Table 5). Associations between prenatal OH-PAH and IQ averaged across three periods of pregnancy are shown in Fig. 4. 2-hydroxynapthalene (β = −1.28 [95%CI: −2.53, −0.03]) and sum of hydroxynapthalenes (β = −1.56 [95%CI: −2.87, −0.26]) were associated with lower IQ in fully adjusted models (Fig. 4). Associations of prenatal OH-PAH exposure and IQ stratified by pregnancy period with and without mutual adjustment for exposure in the other periods are shown in Supplemental Figures 6 and 7, respectively. In period-specific analyses, we observed an association between early pregnancy 2-hydroxynaphthalene and lower IQ that persisted with full (β = −1.14 [95%CI: −2.00, −0.28]) and extended (β = −1.15 [95%CI: −2.01, −0.29]) adjustment for covariates (Supplemental Figure 6) but was not statistically significant after adjustment for OH-PAHs in the other two pregnancy periods (Supplemental Figure 7).In additional secondary analyses we explored effect modification by child sex and pre-pregnancy BMI on pregnancy-averaged and early pregnancy associations of 2-hydroxynapthalene and IQ. While none of the tests for interaction were significant, patterns suggest interesting areas for future research in larger samples. We observed inverse associations between pregnancy-averaged 2-hydroxynapthalene and IQ in males (βmales = −1.59 [95% CI: −3.11, −0.06]) but not females (βfemales = −0.97 [95% CI: −2.84,0.89] (pinteraction = 0.60). The pattern was similar for early pregnancy 2-hydroxynapthalene exposure, with lower IQ in males (βmales = −1.50 [95% CI: −2.55,−0.45]), but no association in females (βfemales = −0.68 [95% CI:−1.85,0.48]) and no significant interaction (pinteraction = 0.24). Analyses of effect modification by pre-pregnancy BMI showed inverse associations between pregnancy-averaged 2-hydroxynapthalene and IQ in normal weight and underweight women (βnormal= −2.06 [95% CI:−3.73,−0.38]), with no evidence of associations in overweight or obese women (βobese = −0.30 [95% CI: −1.98, 1.37]) and without evidence of interaction (pinteraction = 0.12). Associations were similar for early pregnancy 2-hydroxynapthalene and IQ (βnormal = −1.67, [95% CI: −2.98, −0.35]; (βobese = −0.75, [95% CI: −1.71, 0.21]) pinteraction = 0.21).4.DiscussionTo the best of our knowledge, this is the largest prospective study to date of prenatal PAH exposure and preschool and early school-aged cognition and the first to examine associations using exposure measures across three windows of pregnancy. For our primary analyses, we examined associations of mid-pregnancy OH-PAH and IQ using a multi-center pooled cohort, taking advantage of a relatively large and diverse sample spanning several regions of the U.S. In this pooled sample, we did not observe evidence of associations between individual OH-PAH metabolites nor OH-PAH mixtures with child IQ. Moreover, we did not observe evidence of effect modification by child sex or maternal obesity, with the exception of potential sex-specific associations of 2-hydroxynapthalene, which was associated with lower IQ in boys but not girls.In secondary analyses, we leveraged the availability of multiple OH-PAH measures across pregnancy in the smaller cohort, TIDES, to address two specific gaps in the existing literature: 1) potential measurement error resulting from single time point OH-PAH assessment, and 2) examination of sensitive periods during pregnancy. OH-PAH metabolites in a spot urine sample reflect exposure only in the hours prior to urine collection, due to the short half-lives of OH-PAHs(Li et al., 2012). While it is standard for epidemiological studies to include a single measure of prenatal PAH exposure (Edwards et al., 2010; Perera et al., 2009), this approach is likely vulnerable to measurement error and may bias measured associations toward the null(Cathey et al., 2018). To address this limitation, we calculated pregnancy-average exposures using measures in early, mid and late pregnancy in a subset of our study sample with multiple OH-PAH measures available. These pregnancy-average OH-PAH metabolites should better approximate exposure across pregnancy. We observed that pregnancy-average 2-hydroxynapthalene as well as the sum of naphthalene mono-hydroxylated metabolites were both associated with lower IQ. If these reflect true adverse impacts of PAHs upon fetal neurodevelopment, it may be that such associations are more easily detectable by averaging multiple measures of PAHs across pregnancy than using a single pregnancy assessment. In addition, we used the multiple measures of OH-PAHs available in TIDES to explore evidence for sensitive windows of exposure. These analyses indicated that adverse associations between IQ and OH-PAH metabolites were most evident for early pregnancy exposures, in particular for 2-hydroxynapthalene. This suggests that early pregnancy may be a period of greater sensitivity, which is consistent with evidence for some other chemical exposures(Zhu et al., 2020). However, our study was not specifically designed to test sensitive periods of PAH exposure during pregnancy, which would require a larger sample size sufficiently powered to enable formal testing of differences across timing of exposure (Sánchez et al., 2011); thus, these preliminary findings need to be replicated in other study populations.Epidemiological evidence of adverse associations between PAHs and child cognitive development has been somewhat inconsistent. In a New York City cohort of non-smoking Black and Dominican-American mothers, prenatal PAH exposure measured in personal air in the third trimester of pregnancy was associated with developmental delay in their children at age 3(Perera et al., 2006) and reduced IQ at age 5(Perera et al., 2009). In a second cohort of mother–child dyads in Krakow, prenatal PAH exposure measured by personal air in the second or third trimester of pregnancy was associated with lower nonverbal reasoning skills at age 5(Edwards et al., 2010). In contrast, a third prospective cohort of mother–child dyads in Chongqing, China showed no association between prenatal PAHs measured in DNA adducts and IQ at age 5, and associations of PAH with IQ only in the presence of interaction with environmental tobacco smoke(Perera et al., 2012). Likewise, a cross-sectional study using data from the National Health and Nutrition Examination Survey found no evidence of associations between urinary PAH metabolites and the need for educational assistance in middle childhood, though their PAH measures were contemporaneous with their outcome rather than a reflection of the in utero environment(Abid et al., 2014).There are several possible explanations for heterogeneity in the literature, including variability in magnitude of exposure between populations. For example, the ambient air PAH concentrations measured in the Krakow cohort were very high relative to measurements from other urban settings in Europe(Binková et al., 1995) and Canada (Nethery et al., 2012), and more than five times that observed in the New York cohort(Jedrychowski et al., 2005). Differences in the method used to measure PAHs (e.g., urine, cord blood, personal air), furthermore, preclude direct comparison of PAH concentrations across cohorts. The PAH metabolites captured in our urinary measures were limited to low molecular weight PAHs, which reflect both dietary and air pollution sources (Agency for Toxic Substances and Disease Registry (ATSDR), 1995). High molecular weight PAHs are largely excreted in feces (Ramesh et al., 2004), not urine, and nearly all of the high molecular weight PAHs measured in our study were excluded from analyses due to a large proportion of samples being below the limit of detection. The urinary OH-PAH metabolites concentrations measured in this study were similar to other studies of pregnant women and women participating in NHANES(Woodruff et al., 2011). The largely null findings of mid-pregnancy PAH in our full cohort could be explained by relatively low PAH exposures. The composition of prenatal PAH mixtures also varies across study regions and may lead to inconsistency across studies. For example, in the Chongqing cohort, the major source of PAH was a coal-fired power plant that operated the winter before child participants were born, which may have led to a substantially different profile of PAH exposure(Perera et al., 2012). In comparison, a major source of airborne PAHs in New York City is thought to be vehicular traffic, which may be more similar to the sources and thus the composition of PAH mixtures encountered in our cohort(Perera et al., 2009).Between-study heterogeneity in results could also stem from differences in methodological approach. We observed that several individual OH-PAH metabolites were negatively associated with IQ in the full cohort analysis but only in the model with minimal covariate adjustment, and our associations were greatly attenuated with full covariate adjustment. The New York and Krakow studies(Edwards et al., 2010; Perera et al., 2009) utilized less robust adjustment models, including using maternal education alone as a proxy for socioeconomic status, while we controlled for multiple domains and levels of socioeconomic status. We considered whether the results of prior studies were affected by residual confounding; to explore this, we conducted additional sensitivity analyses utilizing similar covariate adjustment as in the New York and Krakow studies. Even with this less robust adjustment, we saw no evidence of associations between prenatal OH-PAH metabolites and IQ in our dataset, which suggests that covariate adjustment cannot entirely explain the discrepancies between studies.Effect modification may also contribute to the inconsistency in findings across studies. Based on possible mechanisms for neurotoxicity of prenatal PAH, we hypothesized that associations may vary by participant characteristics, including infant sex and maternal obesity. In both our full cohort and TIDES-only analyses, 2-hydroxynapthalene was associated with lower IQ in boys but not girls. This is notable as studies in animals and humans suggest that the neurodevelopmental consequences from environmental toxicant exposure differ between males and females(Gade et al., 2021). PAHs in particular are linked to neuroinflammation, (Saunders et al., 2006; Deanna D Wormley et al., 2004) which trigger sex-specific responses in the fetus,(McCarthy et al., 2017) and are theorized to underlie differences in the frequency and severity of neurodevelopmental disorders between sexes(McCarthy et al., 2017). PAHs also accumulate in fatty tissue (Pastor-Belda et al., 2019), potentially exposing fetuses of overweight or obese women to a greater burden of PAH exposure even at similar environmental exposures (Vizcaino et al., 2014). We saw no evidence for differences in associations by maternal obesity in the full cohort analyses. In our TIDES-only analyses we observed inverse associations with IQ for 2-hydroxynapthalene in normal and underweight women and not overweight or obese women, but without statistical evidence for effect modification. Given the mixed results and exploratory nature of some of these analyses, future studies that are well-powered to detect effect modification and with larger variation in exposure are needed to characterize the populations most vulnerable to PAH exposure.Despite primarily null findings in this analysis and others, a growing body of toxicological evidence provides biological plausibility for neurotoxic effects of prenatal PAH exposure. A number of pathophysiological mechanisms have been proposed for how PAHs harm the developing brain. PAHs are transferred across the placenta, easily cross the blood–brain barrier, and are accumulated and metabolized in the brain(Rouet et al., 1981). PAH exposure is associated with endocrine disruption(Takeda et al., 2004) and alterations to global methylation, implying epigenetic modification (Herbstman et al., 2012), with subsequent effects on neurodevelopment. Toxicological studies also point to oxidative stress, suggesting that PAH metabolism increases intracellular reactive oxygen species and inhibits the brain antioxidant scavenging system(Saunders et al., 2006), as well as initiating p38MAP kinase pathways and activation of inflammatory microglial cells and bystander neuronal death(Dutta et al., 2010). Other suggested mechanisms include downregulation of developmental ionotropic glutamate receptor sub-unit expression, which is critical for synaptic formation and the maintenance of synaptic plasticity mechanisms during cortical and hippocampal development(D. D. Wormley et al., 2004; Deanna D Wormley et al., 2004).There are important limitations to our study. Our primary analyses leveraged the large sample size of a multi-cohort sample but relied on a single measure of prenatal PAH exposure. As discussed above, this is an important limitation because urinary OH-PAH metabolites reflect exposure in approximately the prior 12 h(Jongeneelen et al., 1990) and have exhibited moderate to high intraindividual variability in some (Cathey et al., 2018) but not all(Dobraca et al., 2018) prior analyses of urinary PAHs measured repeatedly. Notably, we were able to reduce the measurement error associated with using a single timepoint in our analysis using our subsample of TIDES participants with three measures across pregnancy. However these three spot urinary PAH measures reflect exposure over a relatively short window of time and as a result limited our ability to further investigate window-specific effects. Second, we were interested in both the associations of individual PAH compounds as well as their potential effects as a mixture; we therefore had a large number of prospectively defined analyses, increasing the opportunity for chance findings. A third possible limitation in our study sample is the pooling of two cohorts that have different demographics, average age at outcome assessment, and measures of IQ. Our “leave one site out” sensitivity analyses supported robustness of the study results to cohort and site inclusion or exclusion. Although the SB-5 and WISC-V IQ measures were standardized to age and estimates were adjusted for age at assessment, the children administered the WISC-V were older on average than those given the SB-5, which may have contributed to the differences in IQ scores, and we cannot exclude the possibility of residual confounding by child age. Fourth, all of our PAH measures were subject to left-censoring due to batch-specific LOD, which could be a source of bias in measured associations.(Hewett and Ganser, 2007). Our method of imputing PAH measures using the value of LOD/2 is a limitation as it centers all imputed values on a single value without variability as opposed to a true distribution of low exposures. It is reassuring that our results were not sensitive to using likelihood-based CLMI in place of the primary approach of substituting the value of LOD/2, but this is still a limitation since CLMI assumes a common distribution of PAHs across batches, which was not observed in our data (Supplemental Table 2). Finally, because we excluded children of mothers who smoked during pregnancy, our results are only generalizable to a non-smoking population.In summary, this study represents the largest and most robustly adjusted epidemiological analysis of prenatal PAH exposure and childhood cognition, including examination of multiple measurements of PAH across time in a subsample. In our primary analysis of mid-pregnancy PAH exposure in the overall, multi-cohort sample, we observed no associations. However, in secondary analyses in the TIDES cohort, we found that average exposure across pregnancy and early pregnancy exposure to 2-hydroxynapthalene were each associated with lower IQ. Better-powered studies assessing multiple prenatal PAH measures as well as early pregnancy exposures to PAH are needed to confirm these findings and advance our understanding of PAH impacts on child neurodevelopment.Supplementary Material1

PMC

Pediatrics

36756736

PMC10039668

3-01-2023

10.1542/peds.2022-058330

Overuse of Reflux Medications in Infants

Wolf Elizabeth R., Sabo Roy T., Lavallee Martin, French Evan, Schroeder Alan R., Huffstetler Alison N., Schefft Matthew, Krist Alex H.

Gastroesophageal reflux (GER) occurs in up to 2/3 of healthy infants1 and usually resolves by 1 year of life. In contrast to GER, gastroesophageal reflux disease (GERD) is reflux that can involve (a) poor weight gain, (b) pain or (c) mucosal injury on endoscopy.2 Acid suppressants such as histamine-2 receptor antagonists (H2RA) and proton-pump inhibitors (PPI) reduce gastric acidity but are not effective against GER.2–4 Acid suppressants have been linked to a higher risk of serious infections5,6 and fractures.7 Weighing the risks of these medications against their unclear benefit, Choosing Wisely and the American Academy of Pediatrics have recommended against their use in infants with GER. Our objective was to determine the individual and health care system characteristics associated with acid suppressant overuse using a statewide all-payers claims database from the most recent years available with newer International Statistical Classification of Diseases, 10th Revision (ICD-10) codes that can distinguish GER from GERD.METHODSThe data were obtained from the Virginia All-Payers Claim Database. The patient’s ZIP code was classified on the basis of the Education Demographic and Geographic Estimates Program.8 Infants 0 to 11 months of age with at least 30 days of continuous enrollment between 2016 to 2019 were included. We excluded infants who had been diagnosed with a Pediatric Complex Chronic Condition at any point in their patient record.9 We converted other exclusion criteria used in earlier claims-based studies,10 such as esophagitis, ulcers, and weight loss (full list available in Supplemental Table 2) from International Statistical Classification of Diseases, 9th Revision (ICD-9) to ICD-10 codes, and these were applied if they occurred 0 to 1 days before the prescription of a H2RA or PPI. We conducted 2 separate analyses: 1 in which GERD was an exclusion criterium and 1 in which it was not. We modeled the rate of H2RA or PPI use using a multivariable binomial regression model including sex, insurance type, rurality, and indicators for low birth weight and prematurity. All statistical analyses were performed at a 5% statistical significance level by using R (version 4.1.0). Ethical approval was given by the Virginia Commonwealth University institutional review board with an exempt determination.RESULTSWe identified 270 437 total infants (68% of live births in Virginia from 2016–2019). There were 16 910 (7%) nonmedically complex children who were prescribed a H2RA or PPI (Fig 1). There were 5196 (2%) infants excluded with a diagnosis of GERD. There were 5433 (2%) unique children without one of the exclusion criteria who were prescribed a H2RA or a PPI. The odds of being prescribed H2RA or PPI were higher (adjusted odds ratio [aOR] 1.9; 95% confidence interval [CI] 1.8–2.0) for infants with commercial insurance compared to those with public insurance (Table 1). Children from rural settings had higher odds of being prescribed H2RA or PPI compared to children from both urban (aOR 1.2; 95% CI 1.1–1.3) and suburban (aOR 1.6; 95% CI 1.5–1.7) settings. Removing GERD as an exclusion factor did not meaningfully or substantially alter the estimates (Supplemental Table 3).DISCUSSIONIn our statewide analysis, nearly 7% of all infants and 2% of infants without one of the exclusion criteria were prescribed an acid suppressant. Earlier studies found that 2% to 6% of infants were prescribed an acid suppressant,10 although the change from ICD-9 to ICD-10 codes and lack of 1:1 mapping makes comparisons with older data challenging. Our estimate being at the lower end of this range may be a result of the 2014 Choosing Wisely recommendation, the growing recognition of the harms of acid suppressants, or the updated exclusion criteria that we employed. Providers often have difficulty distinguishing GER from GERD. Observations of poor weight gain and mucosal changes on endoscopy are relatively straightforward and free from provider interpretation. However, the assessment of pain is more subjective since an infant may cry for many different reasons and the peak age for reflux overlaps with that of normal developmental crying patterns. Furthermore, some providers may feel compelled to “upcode” from a diagnosis of GER to a diagnosis of GERD to justify their medical management. In conclusion, acid suppressant overuse remains a persistent problem particularly among commercially insured infants and those residing in rural areas. Reduction efforts should include providers and patients outside of urban academic children’s hospitals to achieve maximal benefit.Supplementary MaterialAppendix

PMC

Animal Nutrition

38357575

PMC10864210

11-14-2023

10.1016/j.aninu.2023.11.001

The application of protease in aquaculture: Prospects for enhancing the aquafeed industry

Chen Shiyou, Maulu Sahya, Wang Jie, Xie Xiaoze, Liang Xiaofang, Wang Hao, Wang Junjun, Xue Min

Low-fishmeal and protein-saving diets are two prominent nutritional strategies utilized to address challenges related to the scarcity and sustainability of protein sources in aquaculture. However, these diets have been associated with adverse effects on the growth performance, feed utilization, and disease resistance of aquatic animals. To mitigate these challenges, exogenous protease has been applied to enhance the quality of diets with lower protein contents or fishmeal alternatives, thereby improving the bioavailability of nutritional ingredients. Additionally, protease preparations were also used to enzymatically hydrolyze fishmeal alternatives, thus enhancing their nutritional utilization. The present review aims to consolidate recent research progress on the use of protease in aquaculture and conclude the benefits and limitations of its application, thereby providing a comprehensive understanding of the subject and identifying opportunities for future research.

1IntroductionAquaculture is a burgeoning sector within the food production industry and occupies a critical position in the provision of nutrition (FAO, 2020). The consumption of aquatic foods has been increasing at a rate of 3.0% annually between 1961 and 2019, outpacing the corresponding growth rate of the global population and nearly doubling it within the same timeframe (FAO, 2020). However, despite this growth, the application of fishmeal obtained from wild forage fish as aquatic feed remains a significant challenge. The global fishmeal production consumed by the aquaculture sector jumped from 33% to 66% between 2000 and 2016 (Naylor et al., 2021) and grew to 78% after 2019 (European Commission, 2021). Harvesting forage fish always brings about overexploitation that adversely impacts aquatic ecosystems because these fish play a vital role in the conversion of plankton into sustenance for species at higher trophic levels (Cury et al., 2000). Furthermore, fishmeal production is heavily influenced by weather patterns, such as the El Niño-Southern Oscillation phenomena (Maulu et al., 2021; Naylor et al., 2009). The long-term global fishmeal production is expected to stabilize at around 5 million metric tons (Bachis, 2022; FAO, 2020), which is insufficient to meet the projected expansion of aquaculture (Hua et al., 2019; Shepherd and Jackson, 2013). Additionally, the price of fishmeal seems to be rising much faster than its production, varying from 657 USD per metric ton in Jul 2002 to 1,610 USD per metric ton in Jul 2022 (Index Mundi, 2022). Therefore, minimizing the use of fishmeal in aquafeeds is imperative (Tacon and Metian, 2008). Two main nutrition strategies, i.e., low-fishmeal diets (LFD) and protein-saving diets (PSD), could be conducted to cope with the above problem. LFD involves substituting fishmeal with alternative proteins including fishery and terrestrial animal by-products, plant-based meals, single-cell proteins, and insect meals in the formulated feed to satisfy fish's standard protein requirements (Agboola et al., 2020; Galkanda-Arachchige et al., 2020; Kaiser et al., 2022; Wang et al., 2022; Yan et al., 2023). A growing amount of fishmeal from fishery by-products is reportedly being used and was estimated at over 27% of the global fishmeal used in aquaculture in 2020 (FAO, 2020). Insects and single-cell proteins are a promising alternative to conventional feedstuffs because they have a short life cycle, their production does not require huge arable land, and they have high digestible protein with amino acids profile similar to fishmeal (Li et al., 2021; Maulu et al., 2022; Wang et al., 2022). Plant-based proteins, such as cottonseed protein concentrate, soybean meal, and rapeseed meal, have been widely studied as a substitute for fishmeal recently (Kaiser et al., 2022; Wang et al., 2020; Xie et al., 2023). The protein-saving diet is a nutritional regimen characterized by a lower content of dietary protein, which is complemented with essential nutrients supplementation, including amino acids, high lipids, and high sugar (Dong et al., 2013; Lee et al., 2019; Li and Robinson, 1998; Shiau et al., 1990; Yu et al., 2022).While LFD and PSD have been recognized to offer cost-saving advantages and reduce dependence on fishmeal, studies have also linked them to adverse effects on fish and shrimp (Panigrahi et al., 2019; Willora et al., 2022). Currently, plant-based proteins dominate fishmeal substitution in aquafeed (Chen et al., 2022a, 2022b); however, these proteins have several drawbacks, including anti-nutritional factors (ANFs), low digestibility, poor bioavailability, and palatability issues (Shomorin et al., 2019). Previous research has indicated that diets incorporating plant proteins may impede the activity of digestive enzymes. This hindrance is attributed to the presence of protease inhibitors in plant proteins, which bind to the active sites of endogenous proteases (Francis et al., 2001; Gatlin et al., 2007; Kamel et al., 2015; Segobola, 2016; Xu et al., 2022). Adding exogenous enzymes to aquatic feed is a proven nutritional approach that can improve the quality of diets that include plant-based proteins or other undesirable proteins (Dalsgaard et al., 2012; Li et al., 2016; Liang et al., 2022; Zheng et al., 2020). Proteases, as a dietary enzyme additive, can improve protein utilization by addressing endogenous enzyme deficiencies and hydrolyzing macromolecular proteins (Saleh et al., 2022). Additionally, they can enhance other nutrient absorption (Cowieson and Roos, 2016; Zaworska-Zakrzewska et al., 2022). However, a significant challenge in the commercial application of exogenous protease additives exists; proteases are heat-sensitive additives that undergo a reduction in efficacy during feed processing, and the extrusion process is primarily responsible for the degradation of thermosensitive nutrients (Espinosa et al., 2020). The feed industry relies on this process to gelatinize the starch, inactivate ANFs, and destroy pathogenic microorganisms (Drulyte and Orlien, 2019; Glencross et al., 2012; Shi et al., 2016); consequently, the optimal method of feed protease supplementation necessitates careful consideration. Presently, protease preparations are also creatively applied to the enzymatic hydrolysis of protein sources (Boyd et al., 2020). Protease-treated proteins always show higher crude protein digestibility and more bioactive substances, while the ANFs present in the enzymatically hydrolyzed proteins can be reduced (Caine et al., 1998; Rooke et al., 1998; Wu et al., 2020). The present review concludes the application of protease in aquafeed, encompassing its advantageous attributes as well as inherent limitations, aiming to facilitate a thorough comprehension of the topic and suggest prospective avenues for further investigation in order to fully exploit all potential outcomes.2Feed proteasesProteases, which were initially identified in 1903, are enzymes responsible for breaking down complex proteins into smaller units through hydrolysis (Rawlings, 2013; Vines, 1903). Despite this, exogenous proteases have only been used as a mono-component commercial enzyme additive for the past 10 to 15 years (Cowieson and Adeola, 2005; Fru-Nji et al., 2011; Li et al., 2012). As an emerging product, its global market share is multiplying, valued at $3,454.3 million in 2020, and current projections indicate that it will reach $5,762.7 million by 2030 (Allied Market Research, 2022). Commercial feed proteases, such as alcalase, papain, flavourzyme, neutrase, and trypsin, can be classified into three categories based on their source: animal, plant, and microorganisms (Islam et al., 2022a). Also, they can be divided into acid, neutral, and alkaline proteases based on working pH values (Flores et al., 2019). pH and temperature always influence protease activity. Certain studies propose that these variables may alter protease structure, consequently impacting their activity (Awad et al., 2020; Buchholz et al., 2020). Hence, the outcomes may fluctuate based on the pH of the animal's gastrointestinal tract and the cultivation temperature. Considering the variability in pH ranges within aquatic animals' gastrointestinal tracts, it is imperative to account for these factors when employing proteases in aquafeed. Nevertheless, it appears that current applications in aquafeed do not take this into consideration. Information on some commercial feed proteases used in aquaculture is provided in Table S1. Most of them are derived from microorganisms' fermentation, making up nearly two-thirds of the market. The primary reason for the prevalence of microbial proteases is their characteristic as extracellular enzymes, which makes them easy to extract and cost-effective to produce without sacrificing their high catalytic activity (Beg and Gupta, 2003). Additionally, various microorganisms can produce industrial proteases, which can be modified using advanced technologies (Ali et al., 2016; Beg and Gupta, 2003). Proteolytic enzymes can break down proteins into peptide fragments composed of 2 to 20 amino acids or free amino acids (Islam et al., 2022b). Of note, the protease activities are highly specific in their cleavage. For instance, trypsin and alcalase are two different proteases with distinct substrate specificities. Trypsin targets peptide bonds that are located at the C-terminal side of lysine and arginine residues, whereas alcalase exhibits a broader specificity, preferring to hydrolyze peptide bonds located at the C-terminal side of hydrophobic residues, as shown in Fig. 1 (Vogelsang-O'dwyer et al., 2022). Peptides consist of a particular sequence of amino acids released from bulk protein and show different activities such as antioxidative, antibacterial, immunoregulation, and anti-inflammatory profiles (Jia et al., 2021). In aquaculture, the use of proteases can be categorized into two distinct applications. Firstly, as a feed additive, and secondly, for pre-treating fishmeal alternatives. The effects of feed protease preparation in aquaculture can be summarized as follows (Fig. 2): supplementing the deficiency of endogenous proteases and hydrolyzing complex proteins into simpler units. Supplementing endogenous digestive enzymes benefits the digestibility of nutrients by farmed animals, with a particular emphasis on proteins. Consequently, it effectively reduces nitrogen (N) emissions. Moreover, the breakdown of large protein molecules promotes the formation of bioactive peptides and the degradation of certain proteinaceous antinutrients. These effects not only positively influence the health and growth of farmed fish but also yield economic benefits (Mohammadigheisar and Kim, 2018; Schneider and Lazzari, 2022; Zheng et al., 2020).Fig. 1Schematic representation of the enzymatic hydrolysis of proteins into peptides or free amino acids by alcalase and trypsin.Fig. 1Fig. 2An illustration of the benefits of exogenous protease in fish diets. N = nitrogen.Fig. 23Effects of protease application on aquatic animals3.1Growth performancePrevious research has consistently demonstrated the growth-enhancing benefits of protease supplementation in diets, as outlined in Table 1. In the case of omnivorous fish, a dosage of 500 mg/kg of protease supplementation in PSD diets has been found to significantly improve various growth performance indicators of Nile tilapia Oreochromis niloticus, including final body weight (FBW), weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER), and feed conversion ratio (FCR) (Saleh et al., 2022). Similarly, a study on blue tilapia O. niloticus × Oreochromis aureus reported that the supplementation of 175 mg/kg of protease in fishmeal-free diets could significantly improve FBW, WG, and FCR parameters (Li et al., 2019). Furthermore, a study conducted on the carnivorous fish, European seabass Dicentrarchus labrax, suggested that dietary supplementation of high levels of high-protein distiller's dried grains (HPDDG) with protease inclusion increased growth performance, as well as improved FCR (Goda et al., 2020). For crustacean species, multiple studies conducted on Pacific white shrimp (Litopenaeus vannamei) demonstrated that the addition of protease in LFD diets proved to be an effective nutritional strategy for improving shrimp growth (Yao et al., 2019; Li et al., 2016). Similar positive outcomes were observed in a study on the Chinese mitten crab Eriocheir sinensis (Chowdhury et al., 2018). However, Wu et al. reported that high-dose protease could inhibit growth performance in Nile tilapia. Other research also indicated that the excessive supplementation of exogenous protease always induced negative growth-promoting benefits (Liu et al., 2018; Wu et al., 2020). It is speculated that this may be due to the excessive addition of protease, leading to metabolic disorders of other nutrients in compound feed. According to Guan et al. , supplementing with high levels of protease resulted in a 42.1% reduction in abdominal fat by regulating glucose and lipid metabolism. Meanwhile, this supplementation also led to a decrease in protein efficiency ratio (PER) and hindered the growth performance of largemouth bass Micropterus salmoides. Additionally, Song et al. proposed that excessive protease inclusion could cause damage to the intestines by hydrolyzing mucosal proteins when there are insufficient substrates available for hydrolysis. The effect of protease addition on aquatic animal growth is also influenced by various pelleting methods. Shi et al. supplemented LFD with exogenous protease at 125, 150, and 175 mg/kg levels, and processed them using either pelleting or extruding technology. The study findings indicated that gibel carp (Carassius auratus gibelio) showed significantly better growth performance when fed pelleted diets at all three supplementation levels, as opposed to extruded diets supplemented with protease. Additionally, the growth-promoting action of protease supplementation is significantly affected by the compound feed ingredients. For example, a study on common carp Cyprinus carpio found that supplementing 175 mg/kg protease in 20% fishmeal-based diets did not significantly impact fish growth (Leng et al., 2008). However, when the fishmeal inclusion level in diets was reduced to 10% or 6% with protein being replaced with soybean meal, the WG of fish increased significantly compared to groups without protease (Leng et al., 2008). In aquaculture production, diets are formulated with a nutrition strategy that enables an animal to achieve the best growth performance with minimum costs. Suppose the experimental diets' nutritional requirements for the animal being studied are not reduced to the minimum optimal level, any increase in nutrient utilization or fish growth caused by exogenous proteases cannot reflect the actual animal response (Son and Ravindran, 2011).Table 1The effects of dietary protease as a mono-component enzyme additive on growth and physiological parameters of aquatic animals.Table 1Aquaculture speciesInclusion level(s)Effects on the growth and physiological parametersReferencesOmnivorous fishNile tilapia or GIFT(Oreochromis niloticus)Four diets with two controls: high protein (30%) and low protein (29%). The third diet contained 500 mg/kg protease, the fourth contained 250 mg/kg proteaseDietary supplementation of protease improved the productive performance of the fish besides sparing the protein inclusion and producing economical dietsSaleh et al. 2,500 U/kg diet protease supplementationProtease supplementation can improve growth, nutrient assimilation, and hematology and alter gene expression of growth hormone and insulin-like growth factor I of Nile tilapiaHassaan et al. 500 mg/kg of protease inclusion in the dietsGrowth performance and feed utilization, including highest goblet cells, the thickness of muscularis, mucosal folds, and enterocytes. Furthermore, the immune parameters of the fish were improvedHassaan et al. Protease supplemented in plant-based diets at the levels of 0, 1.38, 2.76, 5.52, and 11.04 U/g diet5.52 U/g protease supplementation could promote growth performance, intestinal physical barrier function, innate immunity, and the fish's resistance against Streptococcus agalactiaeWu et al. Blue Tilapia(Oreochromis niloticus × O. aureus)Compressed (CD) or extruded (ED) diets containing 30 g/kg or 90 g/kg fishmeal were supplemented with or without proteaseWeight gain was improved and the feed conversion ratio decreased significantly with the supplementation of protease in 30 g/kg fishmeal CDLi et al. Protease supplemented 0 (control), 1,000, and 1,500 mg/kg in dietsSignificantly improved growth performance and feed utilization of the fishLin et al. Supplementation of protease to the diets at 175 mg/kgImproved growth performance with no significant effect was observed on the whole-body composition and protein retentionHuan et al. Protease supplemented in fishmeal-based diets at 175 mg/kg dietImproved the growth and nutrient utilization, higher intestinal villus length, and promoted the retention of crude protein and phosphorousLi et al. Gibel carp(Carassius auratus gibelio)Four diets: 75, 150, 300, and 600 mg/kg protease in the dietImproved growth performance. 150 or 600 mg/kg of protease led to foregut muscular thickness thinner, and protease activities in hepatopancreas and foregut were higher in the fish fed 150 or 300 mg/kg proteaseLiu et al. 500 mg/kg protease added in low fishmeal diets (LFD)Improved the growth and immune response of the fishXu et al. Pelleted LFD containing 30 g/kg fishmeal (60 g/kg fishmeal replaced by soybean meal) supplemented with 125, 150, and 175 mg/kg exogenous protease150 to 175 mg/kg protease supplementation in a pelleted LFD improved the growth performance, crude protein, and retention of protein and lipid of the fishShi et al. Common carp(Cyprinus carpio)The fish diets were supplemented with 0.0%, 0.1%, 0.2%, 0.3%, and 0.4% of exogenous protease papainEnhanced growth performance in terms of length gain, weight gain, and specific growth ratePatil et al. Rohu(Labeo rohita)Poultry by-product meal-based diets supplemented with exogenous protease at the levels of 150, 300, 450, 600, and 750 mg/kg dietGrowth performance, whole-body composition, and blood biochemistry were enhanced in the fish fed with protease-supplemented dietsMaryam et al. Carnivorous fishAfrican catfish(Clarias gariepinus B.)Four artificial diets formulated and enriched with protease enzyme at levels of 0 (control), 750, 1,000, and 1,250 U/kg dietImproved larval growth and survival compared with controlKemigabo et al. European seabass(Dicentrarchus labrax)Protease supplemented at 30%, 40%, and 50% of high protein distiller's dried grains supplemented with 1,000 mg/kg of protease as a replacement for soybean mealProtease supplementation at 50% significantly increased growth performance, feed utilization, and better feed conversion ratio. Further, hematology and serum biochemistry, humeral immune parameters (total protein, globulin, cholesterol, lysozyme activity), and total antioxidant capacity significantly increasedGoda et al. Crustacean speciesPacific white shrimp(Litopenaeus vannamei)A high fish meal diet (HFD) with 250 g/kg fishmeal and an LFD with 225 g/kg fishmeal. 175 mg/kg protease supplemented to LFDEnhanced growth, including higher weight gain and lower feed conversion ratio than the fish fed with diets without protease additionLi et al. Protease supplemented in LFD at 125, 150, and 175 mg/kgEnhanced total superoxide dismutase and polyphenol oxidase contents in both serum and hepatopancreas were higher and serum malondialdehyde content and the cumulative mortality during disease challenge tests were significantly reducedSong et al. Protease complex (175 mg/kg) in LFD (10% fishmeal)Improved the growth performance and nutrient utilization of the shrimp. However, no differences in whole-body proximate composition, intestinal villi width, and hepatopancreatic lipase activityYao et al. Chinese mitten crab(Eriocheir sinensis)Diets supplemented with 125, 150, and 175 mg/kg of a dietary protease175 dietary proteases enhanced the fish's protein and lipid retention efficienciesChowdhury et al. CD = compressed diets; ED = extruded diets; LFD = low fishmeal diets; HFD = high fishmeal diets.Enzymatic hydrolysis of protein under protease treatment to replace an appropriate proportion of untreated proteins or fishmeal is also beneficial for the growth of fish. According to Pfeuti et al. , incorporating protease-treated feather meal into the diet of rainbow trout Oncorhynchus mykiss contributed to a growth rate increase of 10.5% to 11.5% compared to the untreated counterparts. Similarly, Cao et al. recorded that keratinase-treated feather meal (KFM) could promote an 81% higher growth rate of juvenile turbot Scophthalmus maximus than the steam-processed group. Replacing around three-quarters of un-hydrolyzed pre-mixed protein with pre-mixed protein hydrolysates in the diet of larval snakehead Channa argus led to a 38.1% increase in their FBW (Sheng et al., 2023). A study on juvenile barramundi Lates calcarifer showed that raw alga could only replace 20% fishmeal in diets based on the WG indicator, while the replacement level could be increased up to 40% when supplemented with protease-treated alga (Van Vo et al., 2020). For soy protein with protease treatment (SPP), Song et al. confirmed that replacing up to 85% of fishmeal protein with SPP negatively affected the growth of starry flounder Platichthys stellatus. However, the low to moderate levels of replacement (15% to 50%) could significantly improve growth parameters compared with full fishmeal protein diet groups. Moreover, manifold research investigating the effects of dietary protease-treated proteins on larval fish suggested that these enzymatically hydrolyzed products can facilitate rapid growth by serving as a highly digestible source of essential amino acids (EAA) and protein in diets (Delcroix et al., 2015; Kvale et al., 2009; Ovissipour et al., 2014; Sheng et al., 2023; Srichanun et al., 2014). Unlike adults, fish larvae show a weak ability to fully utilize conventional formulated feeds due to the immature digestive tract and lack of functional digestive systems. Compared to bulky proteins, protein hydrolysates (equal to be pre-digested) owning low molecular weight are more comfortably absorbed by the intestinal epithelial cells. However, overconsumption of protein hydrolysates in the diet also negatively influences larval fish growth (Kolkovski and Tandler, 2000). As feed additives, protease-based hydrolysis of proteins has successfully been used in PSD and LFD to mitigate growth inhibition caused when high levels of fishmeal are replaced with plant-based proteins in aquafeeds. In herbivorous fish, Xiao et al. found that supplementing grass carp Ctenopharyngodon idella diet with 10 g/kg of protease-treated soybean protein increased their growth performance significantly. The difference in growth performance between the fish under the high-protein diet (34% CP) and those receiving the protease-treated soybean protein supplement (added to a 32% CP diet) was not statistically significant. In carnivorous fish, European seabass, the addition of 30 g/kg of anchovy and jumbo squid hydrolysates demonstrated the ability to reduce the negative effects of LFD (Costa et al., 2020). Also, 33.4 g/kg shrimp hydrolysate, 28.8 g/kg tilapia hydrolysate, and 31.2 g/kg krill hydrolysate supplemented in LFD of juvenile olive flounder Paralichthys olivaceus yielded better results on the fish's growth performance (Khosravi et al., 2018). Supplementation of protein enzymatic hydrolysate partially alleviated the amino acid deficiency caused by the PSD or LFD, leading to improved growth by meeting the animals' requirements (Wang et al., 2021). Moreover, some protein hydrolysates have shown superior attractiveness to aquatic animals (Barroso et al., 2013; Kolkovski et al., 2000a; Leal et al., 2010). For example, Cheng et al. (2019a) suggested that the diets containing cottonseed meal protein hydrolysate (CPH) were more attractive compared to those including squid extract, yeast nucleotides, betaine, and allicin in Chinese mitten carb Eriocher sinensis; consequently, 0.6% of CPH was recommended to be added in the diets as attractants. In a further study, dietary supplementation of CPH at 6 g/kg was proved to stimulate the appetite and increase the feeding rate of carp via the target of rapamycin (TOR) signaling pathway, which finally contributed to enhanced growth performance (Cheng et al., 2019b).3.2Degradation of ANFs and hydrolysis of complex proteins into simpler unitsThe promotion of growth in aquatic animals by protease is also partly attributed to its positive effects on the hydrolysis of proteinaceous antinutrients which are known to be harmful (Han et al., 2020; Hart et al., 2010; Zhu et al., 2021). Studies have demonstrated that protease pretreatment could significantly degrade ANFs (Table 2). For example, Yu et al. suggested that keratinase treatment could effectively degrade nearly 60% β-soybean globulin and 37% soybean globulin in soybean meal. Tan and Sun investigated the hydrolysis ability of different proteases from the Chinese market on degrading ANFs of soybean meal in vitro and found that protease DP100 could significantly eliminate 73.3% and 52.1% of glycinin and β-conglycinin, respectively. Moreover, it was reported that the addition of protease in diets could also reduce the ANFs during feed production. For instance, Wu et al. investigated the effect of four graded levels of protease supplementation in plant-based diets containing soybean globulin (16.65 g/kg) and β-conglycinin (15.85 g/kg) on Nile tilapia. The study findings revealed a significant dose-dependent reduction in the concentration of these two ANFs.Table 2Beneficial effects of protease pretreatment on the nutrient profiles of proteins.Table 2ProteinsIncubation conditionNutritional profilesReferencesDefatted soy flourFlavourzyme, novozym and alcalase; material-water ratio 1:20; pH 7.0Each enzyme degraded both β-conglycinin and glycininHrčková et al. Soybean protein isolateAlkaline protease and papain mixture; material-water ratio 1:20; 50 °C; pH 8.5; for 4 hDecreasing glycinin and β-conglycininCao et al. Soybean meal1.0 mg/g of Bacillus subtilis subtilisin-protease; 50 °C; pH 4.5; for 16 hIncreasing soluble matter and soluble crude protein, and decreasing the level of soybean trypsin inhibitorsCaine et al. Soybean meal10 mg/kg protease DP100Decreasing 73.3% glycinin and 52.1% β-conglycininTan and Sun Soybean meal6 mg/g keratinase; material-water ratio 5:4; for 24 hDecreasing 37% glycinin and 60% β-conglycininYu et al. Cottonseed mealSubtilisin; material-water ratio 1:25; 45 °C; pH 7.0; for 5 hIncreasing soluble crude protein and amino acids, and increasing peptide contentsLiu et al. Cottonseed mealAS1.398 protease; 45 °C; pH 70.0; for 5 hIncreasing the soluble protein by 125%, amino acids (74 to 180 Da) by 59.4%, and the small peptides (180 to 1,983 Da) by 605.3%Gui et al. (2010a)Soybean meal and cottonseed meal mixture (1:1)A multiple enzyme complex including neutral protease; material-water ratio 1:5; 50 °C; pH 7.5; for 6 hIncreasing water-soluble nitrogen and decreasing molecular weightSong et al. Perilla meal protein7% alcalase; material-water ratio 1:22; 61.4 °C; for 4 hIncreasing the soluble peptide or protein concentration, and increasing the DPPH scavenging capacityZhang et al. Antarctic krill8% trypsin; 45 °C; pH 7.9; for 8.5 hIncreasing amino nitrogen content, decreasing molecular weightLiu et al. Da = Dalton; DPPH = 2,2-diphenyl-1-picrylhydrazyl.Another main function of protease is believed to be the enzymatic hydrolysis of protein into individual amino acids and peptides (Table 2). Song et al. used neutral protease to break down a blend of soybean meal and cottonseed meals (1:1) and found that water-soluble nitrogen contents increased from 8.3% to 42.7%. In a more detailed analysis of molecular mass distribution, results showed that the contents of four different ranges (5,000 Da) all increased significantly (Song et al., 2016, 2018). Small peptides are known to be more easily absorbed than high molecular weight proteins, partly explaining the growth-promoting effects of protease. It is well known that the small peptides and free amino acids show distinct mechanisms for absorption, which reduces the antagonism caused by the competition for common absorption sites of free amino acids (Gilbert et al., 2008). Additionally, small peptides can be fully absorbed into the circulatory system and utilized by the liver to directly create proteins, yielding a higher rate of protein synthesis compared to the utilization of amino acids alone (Gilbert et al., 2008). However, a study involving spotted seabass Lateolabrax maculatus found that replacing 50% of the fishmeal in their diet with protease-based hydrolyzed soybean protein isolates resulted in a decrease in both WG and SGR (Cao et al., 2022). Another study on totoaba Totoaba macdonaldi, replacing dietary fishmeal at 40% with SPP also indicated that the fish was significantly affected negatively (Villanueva-Gutiérrez et al., 2022). This outcome can be largely attributed to the factor that the resulting increased levels of luminal peptides and free amino acids may saturate intestinal peptides and amino acid transporters. This could cause rapid peptide influx, which may in turn accelerate amino acid oxidation and endogenous excretion (Zhang et al., 2002). Consequently, the excessive amino acids in diets containing protease-treated proteins may be excreted as intact molecules through urine or gills, which could partially account for the observed impairment of growth (Berge et al., 1994). As shown in the work of Yuan et al. (2019a, 2019b), substituting high levels of cottonseed meal protein hydrolysate for fishmeal resulted in a decrease in fish growth performance. Specifically, the replacement caused a decrease in amino acid metabolism and activated the AMPK/SIRT1 pathway while inhibiting the TOR signaling pathway.3.3Apparent digestibility coefficient (ADCs) of nutrientsThe growth improvement of aquatic animals by protease is also mainly accredited to the enhancement of nutrient digestibility. The ideal amount of exogenous protease added to feed shows a considerable impact on nutrient digestibility, particularly crude protein (ADCCP), as evidenced by Table 3. Hassaan et al. confirmed that feeding Nile tilapia diets including protease led to an improvement in ADCs for essential amino acids (ADCEAA). Lee et al. examined the impact of dietary protease inclusion on the digestibility of amino acids in rainbow trout that were fed 17 different feed ingredients. The findings validated a boost in ADCEAA and non-essential amino acids (ADCNEAA). Apart from crude protein and amino acids, an optimal dose of exogenous protease inclusion could also effectively improve the digestibility of other nutrients: crude ash (ADCAsh), crude lipid (ADCCL), gross energy (ADCGE), and dry matter (ADCDM) (Table 3). For example, Maryam et al. found that 150 to 750 mg/kg protease supplementation in a diet based on poultry by-products significantly enhanced ADCAsh, ADCCL, and ADCDM in rohu Labeo rohita. Parallelly, Drew et al. reported that supplementing canola: pea mixtures-based diets with moderate protease led to significant increases in ADCCL, ADCGE, and ADCDM. In terms of trace elements and macroelements, one investigation conducted on tilapia demonstrated that dietary protease supplementation in LFD did not yield statistically significant improvements in ADCs for calcium (ADCCa), phosphorus (ADCP), iron (ADCFe), and copper (ADCCu) (Huan et al., 2018). However, other studies indicated that ADCP levels increased as a result of protease supplementation (Dalsgaard et al., 2012; Li et al., 2019). Furthermore, Ayhan et al. and Cho and Bureau reported that supplementing gilthead sea bream Sparus aurata diets with protease contributed to a significant increase in the ADCs of nitrogen (ADCN), a crucial factor affecting water quality.Table 3The effects of dietary protease as a mono-component enzyme additive on aquatic animals' nutrient digestibility and digestive enzyme activity.Table 3SpeciesFish size, gExperimental diets and protease information 1Nutrient digestibility and digestive enzymes activities 2ReferenceOmnivorous fishGenetically improved farmed tilapia GIFT(Oreochromis niloticus)18.5Protease (EC3.4.23.18 with 13,830 U/g) supplementation at 0, 100,200, 400, and 800 mg/kg in the basal diets↑ADCCP at 200 and 400 mg/kg, but ↓ADCDM and ADCCP at 800 mg/kg;↑Protease at all doses in all distal, mid, and proximal intestineWu et al. 7.56500 and 1,000 mg/kg protease (CAS No. 9001-927) addition in low fishmeal diets (LFD) with different levels of malic acid↑Chymotrypsin, trypsin, and lipase at both protease supplementation levels with malic acid supplementationHassaan et al. 8.76Exp. 1) Measurement of in vitro digestibility of several raw materials with or without protease (pineapple waste extract) supplementation; Exp. 2) Adding 1%, 2%, and 3% of pineapple waste extract in basal dietsExp. 1) ↑ADCCP of fishmeal (65%, 60%, and 53% CP) and soybean meal (51% and 48% CP) ingredients in vitro; Exp. 2) →ADCCP at all levels in vivo, but ↑ADCCP in all supplementation levels of protease in vitroYuangsoi et al. 11.6Addition of 500 mg/kg of protease (5,000 U/g) in LFD with partial dietary fishmeal (FM) replacement with cottonseed meal (CSM; FM:CSM = 2:1, 1:1, and 1:2)↑ADCCP, ADCCL, ADCDE, and ADCDM in all diets: except for →ADCDM in FM:CSM = 1:2 diets; furthermore, ↑ADCEAA in all diets: except for →ADCThr and ADCVal in FM: CSM = 1:2 dietsHassaan et al. Blue tilapia(Oreochromis niloticus × O. aureus)1.70Inclusion of 175 mg/kg AG175™ (35,000 U/g) in either 30 g/kg or 90 g/kg FM-based diets, exposed to compressing or extruding processing↑ADCCP and ADCDM at 30 g/kg FM diets but not 90 g/kg FM dietsLi et al. 7.70175 mg/kg AG175™ addition in LFD↑ADCCP, ADCDM, and ADCP, while →ADCCaLi et al. 15.0175 mg/kg PT125™ (25,000 U/g) added in diets containing 40 g/kg cork↑ADCDM and →ADCCP;↑Protease and amylase in the intestineYang et al. 7.00Supplementation of 175 mg/kg AG175™ in LFD (FM was replaced with meat and bone meal)→ADCCP, ADCDM, ADCCa, ADCP, ADCFe, and ADCCuHuan et al. Gibel carp(Carassius auratus gibelio)8.0875, 150, 300, and 600 mg/kg protease (neutral protease) supplementation in protein-saving diets (PSD)↑ADCCP at 150 mg/kg supplementation diets, and ↑ADCCL by 75 to 300 mg/kg protease addition, while →ADCDM by all the inclusion levels of protease;↑Protease at 600 mg/kg diets both in hepatopancreatic and foregut tissuesLiu et al. 17.2500 mg/kg protease (20,000 U/g) added in LFD→ADCDM, ADCCP, and ADCP;↑Chymotrypsin and trypsin in the liver, meanwhile, ↑Trypsin and amylase in the intestineXu et al. 35.0125, 150, and 175 mg/kg AG175™ supplemented in the LFD (60 g/kg FM was isonitrogenous replaced by soybean meal) subjected to either pelleting or extruding processing↑ADCCP at all the inclusion levels and ADCDM at 150 and 175 mg/kg in pelleting process diets, however, →ADCCP and ADCDM at all inclusion levels in extruding processing dietsShi et al. Common carp(Cyprinus carpio L.)11.9; 48.7Exp. 1: 175 mg/kg Aquagrow (alkaline protease) added in three diets with different FM contents (10%, 15%, and 20%); Exp. 2: 175 mg/kg of the same protease added in an LFD containing only 6% of FMExp. 1) ↑Protease at 10% FM diets but not 15% and 20% FM diets; Exp. 2) ↑Protease at the PSD dietsLeng et al. Jian carp(Cyprinus carpio var. Jian)52.5175 mg/kg AG175™ supplemented in LFD↑ADCCP and ADCCa, and →ADCPZhang et al. Rohu(Labeo rohita)11.3150, 300, 450, 600, and 750 mg/kg CIBENZA DP® (600,000 U/g) included in LFD (FM was replaced with poultry by-product meal in all diets)↑ADCAsh, ADCCP, ADCCL, and ADCDM at all levels; except for ADCCP at 750 mg/kg;↑Amylase, protease, and lipase are both in the hepatopancreas and intestine at all supplementation levels, except for protease at 750 in the intestineMaryam et al. Sterlet(Acipenser ruthenus)37.010 and 20 g/kg of papain added to the basal diets↑Amylase at 20 g/kg in the posterior intestine, ↑Lipase at 20 g/kg in the anterior intestine, ↓Trypsin at both inclusion levels in the anterior intestine, and ↑ LAP at 20 g/kg in the posterior intestineWiszniewski et al. 56.01,000 and 2,000 mg/kg of bromelain (Sigma-Aldrich with 900 U/g) added to a commercial diet↑Pepsin in the stomach and lipase in the gastrointestinal tract at both inclusion levels, however, ↓Trypsin and LAP in the gastrointestinal tract at both levels, and ↓Amylase at 2,000 mg/kg in the gastrointestinal tractWiszniewski et al. Carnivorous fishBlack carp(Mylopharyngodon piceus)3.030.05%, 0.1%, 0.2%, and 0.3% of protease (neutral protease with 8,000 U/g) included in basal diets↑ADCCP, and →ADCDM↑Protease in the hepatopancreas but not in the intestine improved by 1%, 2%, and 3% inclusion of protease in the diets, however, →Amylase in the hepatopancreas and intestine by all the inclusion levelsChen et al. African catfish(Clarias gariepinus B.)10.1Larvae and fingerlings diets contained 0, 750, 1,000, and 1,250 U/kg protease↑ADCCP at all supplementation levelsKemigabo et al. Catfish(Pangasius hypothalamus)2.23Basal diets were supplemented with 0, 2,000, 4,000, 6,000, and 8,000 mg/kg of crude papain↑ADCCP at all supplementation levelsRachmawati and Prihartono Rainbow trout(Oncorhynchus mykiss)190Exp. 1: 250 mg/kg Poultrygrow-250 (5,840 U/g) added in coextruded flax:pea mixtures- and canola:pea mixtures-based diets. Exp. 2: 250 mg/kg of the same protease added in dehulled flax-based dietsExp. 1) ↑ADCCP, ADCGE, and ADCDM in canola:pea-based diets but not in flax:pea diets; Exp. 2) →ADCCP, ADCGE, and ADCDM in dehulled flax-based dietsDrew et al. 88.01,000 and 2,000 mg/kg of RONOZYME ProAct (75,000 U/g) added to basal diets→ADCCP and ADCCL at all inclusion levelsYigit et al. 110; 106; 73.0228 mg/kg protease was added in three kinds of LFD; soybean meal-based, sunflower meal-based, and rapeseed meal-based↑ADCCP, ADCAsh, ADCCL, ADCDM, and ADCP in soybean meal-based diets, but →ADCCP, ADCAsh, ADCCL, ADCDM, and ADCP in sunflower meal-based and rapeseed meal-based diets;Dalsgaard et al. 110237 mg/kg protease (a bacterial mono-component) added in LFD↑Xylose, mannose, and uronic acidsDalsgaard et al. 250175 mg/kg protease (a protease extracted from bacteria) added in 17 feed ingredients to evaluate the digestibility;↑ADCDM in feather meal-2 and soybean meal, ↑ADCGE in feather meal-2, single cell protein, and poultry by-product-2, and ↑ADCAA (at least one) among these 17 ingredientsLee et al. Largemouth bass(Micropterus salmoides)31.9300 and 500 mL/t neutral proteases included in LFD (FM was replaced with cottonseed protein concentrate)↑Protease at both levels of inclusion in the foregut and hindgutGuan et al. Gilthead sea bream(Sparusaurata)89.52,000 mg/kg protease added in LFD (FM was replaced with soybean meal)→ADCCP, ADCDM, and ADCP, while ↑ADCNAyhan et al. Olive flounder(Paralichthys olivaceus)5.26175 mg/kg protease supplemented in LFD↑Trypsin in the intestineBae et al. Yellow perch(Perca flavescens)0.590.1% pancreatin in basal diets→Trypsin, chymotrypsin, and pepsinKolkovski et al. (2000b)Crustacean speciesRed swamp crayfish(Procambarus clarkii)8.180, 100, 200, 400, 800, and 1,600 mg/kg of protease (20,000 U/g) added to LFD (FM were replaced by plant protein)↑Protease both in the intestine and hepatopancreas at 200 and 400 mg/kg protease inclusion levels, ↑Amylase in hepatopancreas at all levels and in the intestine at 200 mg/kg, and ↑Lipase in both intestine and hepatopancreas at all levels, except for at 1,600 mg/kg in the intestineYang et al. Chinese mitten crab(Eriocheir sinensis)0.730, 125, 150, and 175 mg/kg AG175™ added in LFD (40 g/kg FM replaced with 20 g/kg soybean meal and 40 g/kg CSM)↑Trypsin in hepatopancreatic tissues at 150 and 175 mg/kgChowdhury et al. Pacific white shrimp(Litopenaeus vannamei)2.12175 mg/kg AG175™ included in LFD (10% FM was replaced with a combination of soybean meal and meat-and-bone meal)↑ADCCP and ADCDM;↑Protease in hepatopancreatic tissues, and →Amy and LipYao et al. 3.30175 mg/kg AG175™ included in PSD (reducing FM contents)↑Protease in the hepatopancreas but not in the intestineLi et al. 1.711,000 mg/kg of four kinds of protease (Cenzyme; Enzeco protease 180; Multizyme AK; and Fungal protease) supplemented in basal diets↑ADCCP in four kinds of protease supplementation diets (in vitro)Divakaran and Velasco 0.33125, 150, and 175 mg/kg of a commercial protease in LFD (FM was replaced with soybean meal and peanut meal)↑Trypsin at all inclusion levels, ↑Lipase at 150 and 175 mg/kg inclusion levels, as well as ↑Amylase at 175 mg/kg level of proteaseSong et al. 2.96175 mg/kg of AG175™ added to LFD (FM was replaced with soybean meal)→ ADCCP and ADCDM;↑Protease in the hepatopancreas, while →Amylase and Lipase in the hepatopancreasYao et al. 4.55175 mg/kg of protease (alkaline serine protease) supplemented in basal diets↑Protease in the intestine and hepatopancreas, and ↑Lipase in the hepatopancreas, meanwhile, →Amylase in the hepatopancreasTan et al. ReptileChinese pond turtle(Chinemys reevesii)5.70Adding 0, 150, and 200 mg/kg of protease in basal diets→Pepsin, amylase, and lipase in the stomach; →Amylase and lipase in the liver and intestine; ↑Trypsin at all supplementation levels in the liver and 150 mg/kg in the intestine, but ↓Trypsin at 200 mg/kg in the intestineLiang et al. ‘↑’ represents a significant rise (P 0.05) compared to the group without protease supplementation.1Abbreviations in the column of ‘Experimental diets and protease information’: LFD = low fishmeal diets; PSD = protein saving diets; Exp = experiment; FM = fishmeal; CSM = cottonseed meal. Any unmarked information about proteases refers to information that is not explicitly stated in the references.2Abbreviations in the column of ‘Nutrient digestibility and digestive enzymes activities’: ADCCP = apparent digestibility coefficients of crude protein; ADCDM = apparent digestibility coefficients of dry matter; ADCCL = apparent digestibility coefficients of crude lipid; ADCDE = apparent digestibility coefficients of digestible energy; ADCEAA = apparent digestibility coefficients of essential amino acid; ADCAsh = apparent digestibility coefficients of crude ash; ADCCa = apparent digestibility coefficients of calcium; ADCP = apparent digestibility coefficients of phosphorus; ADCFe = apparent digestibility coefficients of iron; ADCCu = apparent digestibility coefficients of copper; ADCGE = apparent digestibility coefficients of gross energy; ADCAA = apparent digestibility coefficients of amino acid; and ADCN = apparent digestibility coefficients of nitrogen; CP = crude protein; Exp = experiment; FM = fishmeal; CSM = cottonseed meal; Thr = threonine; Val = valine; LAP = leucine aminopeptidase.Studies have partly attributed the improved nutrient digestibility by protease to the improvement of the intestinal structure, which contributed to the thinning of intestinal muscular mucosa (Hassaan et al., 2020; Maryam et al., 2022). It was suggested that this thinning would dwindle the peristalsis speed and consequently prolong digestion and absorption time (Liu et al., 2018). Current research on terrestrial animals confirmed that incorporating moderate protease levels into their diets can enhance the morphology of intestinal mucosa (Cowieson et al., 2017; Ding et al., 2016; Peek et al., 2009; Xu et al., 2017). In aquatic animals, a study on grass carp reported that the height and width of folds in three intestinal segments increased when fed diets including protease (Feng et al., 2023). A study in tilapia elucidated that optimal levels of protease supplementation could enhance the development of villi and improve intestinal morphology (Wu et al., 2020). Similarly, Saleh et al. observed that 500 mg/kg protease in PSD diets and 250 mg/kg in a diet with optimal protein requirement both could significantly improve intestinal health in Nile tilapia. The health of the intestines could be influenced by the decrease in antinutrient factors (as mentioned in Chapter 3.2) and the release of bioactive peptides through protease hydrolysis of substrates (López-Barrios et al., 2014). The release of peptides or free amino acids facilitates absorption and provides energy for intestinal cells (Gilbert et al., 2008). Contrariwise, a previous study found that supplementing 75 to 600 mg/kg protease in gibel carp diets did not result in any significant changes in the villi length and width of the intestine besides not affecting the growth performance (Liu et al., 2018). The discrepancies in the reported outcomes may be partially explained by variations in the species, protease type, experimental settings, and dietary compositions.Furthermore, nutrient digestion is aided by digestive enzymes (Wang et al., 2022). Numerous research studies supported the advantageous impact of incorporating dietary proteases in enhancing endogenous digestive enzyme secretion (Table 3). For example, Feng et al. stated that the inclusion of a moderate amount of protease K in the diets of grass carp can enhance the activities of intestinal brush border enzymes. Also, Maryam et al. discovered that administering a diet containing 450 mg/kg protease resulted in a significant improvement in the activities of amylase, lipase, and protease in rohu. Nevertheless, it should be emphasized that the incorporation of proteases is not invariably advantageous, as evidenced by several investigations documenting the restricted efficacy of this approach in enhancing certain nutrients' digestibility (Drew et al., 2005; Farhangi and Carter, 2007; Liu et al., 2018; Xu et al., 2022; Yigit et al., 2016). One possible explanation for these findings is that the inclusion level of the exogenous protease was not optimized in these experiments, and as a result, excessive dosages may have made a negative impact on the fish. In addition, feed ingredient composition could potentially affect the efficiency of dietary protease to some extent. Hassaan et al. revealed that replacing fishmeal diets with cottonseed meals resulted in a decrease in the improvement of ADCDM, ADCCL, ADCCP, and ADCGE, as the replacement ratios increased. Furthermore, Drew et al. found that protease supplementation could significantly increase ADCDM, ADCCP, ADCCL, and ADCGE of rainbow trout fed with canola-pea-based diets but not flax-pea diets. Interestingly, protease supplemented in the same formula diets with various granulation processes showed different effects on the digestibility of nutrients as well: pelleted diets showed higher ADCDM and ADCCP than the extruded diets (Shi et al., 2016). In the context of optimizing nutrient digestibility, the appropriate level of protease inclusion and diet processing method are critical factors that determine the beneficial results. Moreover, the selection of the appropriate protease type, in accordance with the properties of the protein substrate, is also necessary to achieve optimal outcomes. These considerations are of utmost importance for the development of effective and sustainable aquaculture practices.3.4Physiological functionRecently, some studies have confirmed the beneficial effects of proteases on the physiological function of aquatic animals, which may potentially promote growth performance (Table 1). In fish, reductions in blood aspartate aminotransferase (AST) and alanine aminotransferase (ALT) have been reported in Nile tilapia (Hassaan et al., 2019, 2020), gibel carp (Liu et al., 2018), and largemouth bass (Guan et al., 2021), fed with adequate protease supplementation levels. Also, Yang et al. reported that dietary 200 to 800 mg/kg protease supplementation could significantly decrease the contents of AST and ALT in red swamp crayfish Procambarus clarkia fed plant-based diets. Blood ALT and AST are crucial indicators of liver injury in aquatic animals, and their low levels always indicate liver health (Chen et al., 2021, 2022a, 2022b). The aforementioned findings substantiated the assertion that the incorporation of protease in LFD exerted a hepatoprotective effect on aquatic animals. The utilization of LFD, specifically dietary formulations substituting plant-based proteins for fishmeal, has been observed to considerably predispose fish or shrimp to oxidative stress-induced damage. The supplementation of protease has been verified to exert a positive protective effect against oxidative stress-induced damage. For example, a study on gibel carp showed that 500 mg/kg protease supplementation in LFD could significantly increase hepatic total antioxidant capacity (T-AOC), total superoxide dismutase activities (T-SOD), and glutathione peroxidase activities (GPX) as well as intestinal T-SOD, complement 4, and secretory immunoglobulin A contents (Xu et al., 2022). Yang et al. confirmed that protease supplementation significantly improved the hemolymph biochemical indices and decreased the contents of malondialdehyde (MDA) in red swamp crayfish. Similarly, supplementing the diet of Pacific white shrimp with 175 mg/kg of protease significantly improved the non-specific immune system. This was evident through the augmentation of polyphenol oxidase and T-SOD activities, both in the serum and hepatopancreas (Song et al., 2017). Furthermore, a reduction in the accumulation of hematological MDA of the shrimp challenged with Vibrio parahaemolyticus was observed in the study of Song et al. . According to Wu et al. , incorporating protease in diets can enhance serum antioxidant enzyme activities, scavenge free radicals, and regulate the mRNA expression of tnf-α, il-1β, and hsp70, which help protect endothelial cells against stress caused by plant-based diets.Moreover, studies have reported an improvement in antioxidant capacity and immunoreaction in diets composed of protease-treated proteins in fish and crabs. In grass carp fed PSD, Song et al. demonstrated that dietary enzymatically hydrolyzed soy protein can alleviate inflammatory responses by regulating the NF-κB and TOR signaling pathways. In Jian carp C. carpio var, Xiao et al. reported that protease-treated soy protein could decrease MDA and protein carbonyl contents, improve the activities of antioxidant enzymes and glutathione contents, and enhance mRNA expressions of antioxidant enzymes and Nrf2. Similarly, treating the fish with diets including soy protein hydrolysates as a replacement for fishmeal at 30% could significantly increase serum T-SOD and T-AOC activities while decreasing the MDA contents in juvenile starry flounder P. stellatus (Song et al., 2014). In Chinese mitten crab, dietary inclusion of 30 to 60 g/kg of cottonseed meal protein hydrolysate can significantly enhance the antioxidant capacity and immune response by activating immune-related genes such as Tolls and MyD88 (Cheng et al., 2020).Among the above indicators, MDA is a crucial marker for assessing oxidative damage in organisms (Chen et al., 2022a). The results consistently indicated that the addition of protease or the inclusion of protease-treated proteins has a reducing effect on MDA levels. The activity of antioxidant enzymes (such as T-SOD, T-AOC, and GPX) has been identified as a pivotal factor in impeding lipid peroxidation and ameliorating oxidative damage. The above passage partially explained the impact of proteases on oxidative damage and inflammatory responses from a molecular regulatory perspective, but it did not effectively elaborate on the specific mechanisms involved. Recent studies primarily focused on the effects of protease on fish growth and nutrient digestibility, with little attention given to its impact on physiological functions. The immune-enhancing function of proteases appears to be attributed mainly to the hydrolysis of protein. These released small molecule substances are believed to have some bioactive peptides possessing antioxidant and anti-inflammatory properties. These peptides exhibit the potential to function as an effective scavenging machine of reactive oxygen species. Certain peptides that are released show properties of metal ion chelation or the reduction of hydroperoxides to protect fish from oxidative stress (Jia et al., 2021). Therefore, there is a need for further studies to critically investigate this.3.5Disease resistanceMoreover, protease-treated protein inclusion is reported to improve fish survival rates after parasites or bacterial infections (Khosravi et al., 2018). Resende et al. reported that 30 g/kg of swine blood hydrolysates in LFD for European sea bass enhanced the fish's resistance against Tenacibaculum maritimum infection. Furthermore, feeding European sea bass larvae with protein hydrolysate additives significantly improved the fish's resistance to Vibrio anguillarum (Kotzamanis et al., 2007). Another study on the same adult species fed with shrimp protein hydrolysates showed a higher cumulative survival rate when the fish were challenged by an epizootic outbreak of Vibrio Pelagius (Gisbert et al., 2018). Similar results were also reported in the adult red sea bream Pagrus major fed with shrimp protein hydrolysates (Khosravi et al., 2015), and the juvenile fish fed with rill hydrolysate concentrate (Bui et al., 2014) both after being challenged by Edwardsiella tarda. Tuna viscera hydrolysate supplementation in the diets enhanced the resistance to Streptococcus iniae infection in pompano Trachinotus blochii (Pham et al., 2022) and juvenile barramundi Lates calcarifer (Siddik et al., 2018). Furthermore, a reduction in the accumulation of 96 h-cumulative mortality of the Pacific white shrimp challenged with Vibrio parahaemolyticus was observed in the study of Song et al. . Therefore, protein hydrolysate incorporation in fish or shrimp diets could serve as a practical nutritional approach to prevent tenacibaculosis and reduce economic losses in aquaculture. Protease-treated proteins always contain some small peptides with biological activities, possessing potential physiological properties beyond normal and adequate nutrition (Fig. 3) (Hartmann and Meisel, 2007; López-Barrios et al., 2014; Udenigwe and Aluko, 2011). The bioactive peptides contained in proteins subjected to enzymes may be important components that enhance the disease resistance of animals. To date, numerous studies have explored the functional benefits of bioactive peptides derived from protein sources, such as soybean meal (Singh et al., 2014), cottonseed meal (Kumar et al., 2022), rapeseed meal (Ma et al., 2022), and marine or poultry by-product protein (Nirmal et al., 2022; Romero-Garay et al., 2022). In detail, Duan et al. confirmed that rapeseed protein was a good potential source of antimicrobial peptides (AMPs) using in silico approach. An article on multiple meta-analyses has demonstrated the beneficial effects of AMPs added to diets on aquatic animal health and body function (Wang et al., 2023). As gathered above, these bioactive peptides contribute to the health status of farmed animals, which also become the biggest selling point for the promotion of enzymatically hydrolyzed proteins in the feed industry (Singh et al., 2014).Fig. 3The summary of functions related to bioactive peptides released from feed protein sources under protease treatment. Source: Singh et al. .Fig. 33.6OthersThis section provides a primary exposition on the impact of proteases on the gut microbiota, behavioral response, water quality, flesh quality of aquatic animals, and economic effectiveness. Given the scarcity of available literature, the information presented in this section is condensed. The main objective is to incite further scholarly inquiry in this captivating domain.Several studies have reported the positive impact of incorporating dietary protease on the intestinal microflora of various aquatic organisms (Adeoye et al., 2016; Dai et al., 2019; Hassaan et al., 2021; Zhu et al., 2022). For example, a study on Pacific white shrimp reported a significant increase in the abundance of dominant microflora such as Bacteroidetes, Proteobacteria, and Actinobacteria (Zhu et al., 2022). Gut microorganisms have been considered biosensors for the nutritional health of fish, by helping in absorption and improving the immune system (Hassaan et al., 2021). Proteases altered the gut microbiota and subsequently impacted metabolism and immunity, warranting further research.An investigation into the behavioral response of common carp found that fish given dietary papain treatments had higher movement and activity, as well as a more intense feeding response (Tewari et al., 2018). In terms of water quality, Saleh et al. found that including 250 mg/kg of protease in Nile tilapia diets, with a typical protein requirement of 30% CP, contributed to a significant reduction in both ammonia and nitrite concentrations in aquaria water. Likewise, the addition of pineapple waste extract to the diets of tilapia, serving as a source of bromelain, resulted in a considerable reduction in the levels of free ammonia and total nitrogen in the culture water (Yuangsoi et al., 2018). Commercial exogenous enzymes composed of xylanase, alpha-amylase, acidic proteinase, and neutral-proteinase, have also been shown to present ecological benefits by reducing ammonia in aquaculture systems (Hassan et al., 2017). Furthermore, Islam et al. noted a decrease in the levels of phosphate, ammonia, nitrate, and nitrite in the environment of striped catfish (Pangasianodon hypophthalmus) that were fed a diet containing 500 mg/kg pepsin. The enhancement of water quality is largely credited to the improved utilization of nitrogen or phosphorus in feed, which can be linked to the addition of protease. Further evidence supporting this can be found in the previous section. However, Saleh et al. did not observe any significant improvement in water quality when protease was supplemented at 500 mg/kg in groups fed with PSD. A study by Tewari et al. also did not demonstrate a decrease in water NH3 in which common carp fed with a conventional diet supplemented with papain was cultured. Hence, it can be inferred that the treatments of PSD or optimal protein requirement significantly curtail nitrogen emissions, thereby limiting the potential impact of protease on water quality improvement.One study on grass carp reported that protease-treated soy protein supplementation in PSD could significantly improve the flesh tenderness, juiciness, and flavor of grass carp (Song et al., 2019). Good sensory quality always stimulates purchase intentions in consumers, and several studies were conducted to explore feasible strategies to improve flesh sensory quality presently (Tang et al., 2022; Yang et al., 2021). It appears that protease-treated protein supplementation in diets as a nutritional strategy to enhance the flavor of cultured fish is worthy of more research. In food science, food can be processed by enzymatic hydrolysis to improve its freshness and acceptability for human beings (Kang et al., 2019; Selamassakul et al., 2020). Providing fish with high-quality and appealing feed results in a dual advantage of yielding delectable fish fillets while promoting fish optimal growth and development.Multiple studies have investigated the economic effectiveness of protease-enriched diets (Goda et al., 2020; Mo et al., 2020a, 2020b). For instance, one study conducted by Goda et al. explored the application of HPDDG as a replacement for soybean meal in European seabass diets. The finding indicated that supplementing the 50% HPDDG diet with protease resulted in the lowest total cost of feed per kilogram of fish gain. Similarly, Mo et al. (2020b) conducted a study on gold-lined seabream Rhabdosargus sarba, where they investigated two experimental diets with 30% and 60% fishmeal replaced by soybean dregs (SBD). The 30% and 60% fishmeal-replaced diets were supplemented with 0.5% papain (Exp. 1), while both a fishmeal-based diet and the 30% fishmeal-replaced diet were supplemented with 1.3% bromelain (Exp. 2). The results showed that the experimental diet with 60% SBD and papain (Exp. 1) reduced the cost of fish per kilogram by 31.8% compared to the 0% SBD diet. Furthermore, both groups with bromelain (Exp. 2) had a cost reduction of 27.0% and 46.7% compared to fishmeal-based diets. Mo et al. (2020a) stated that supplementing food waste-based diets for grass carp with a combination of papain and bromelain is a viable nutritional strategy.4Conclusions and future perspectiveProtease additives succeed in increasing the digestibility of feed nutrients, boosting fish growth performance, enhancing health status, and improving the aquaculture environment. Moreover, plenty of undesirable protein sources could be hydrolyzed by it to become high-quality protein raw materials with small molecular weight, high digestibility, low ANFs, and more bioactive peptides.As feed additives, some critical bottlenecks may hinder its application in aquatic feed. One of them is their requisite incorporation into the feed components, in a manner that averts denaturation and inactivation during processing, such as extrusion, drying, and subsequent storage, or alternatively, facile solubilization and elimination via a straightforward top-coating process upon administration to the culture pond, particularly when the feed is not quickly consumed (Boyd et al., 2020). Another is fluctuant enzyme activities as the activities of endogenous digestive enzymes in fish are significantly affected by the temperature of the environment compared to homeothermic animals (Hashemi et al., 2018; Ma et al., 2019). It must be emphasized that accurate determination of the protease inclusion level is an important aspect of ensuring the efficiency of exogenous proteases in fish feeding. Low-dose supplementation consistently yielded no discernible effects, whereas high-dose supplementation could potentially exert adverse consequences on both the intestinal health and growth performance of cultivated fish (Liu et al., 2018; Wu et al., 2020). Therefore, more research on estimating the requirement for specific proteases of specific aquaculture species at different water temperatures should be carried out. Additionally, the pH of the digestive tract or stomach of fish is subject to variability among species and changes throughout development, which would also impact the effectiveness of protease additives. Nevertheless, advanced biotechnology holds promise in providing solutions to enhance the stability of proteases (Dotsenko et al., 2021; Navone et al., 2021). For instance, Su et al. successfully applied a combinatorial strategy via bioinformatics analysis and rational design to enhance keratinase thermostability for efficient biodegradation of feathers. Some polymers employed as protein or drug carriers can potentiate the efficiency of exogenous enzymes in medical and feed industries (Liang et al., 2022; Ye and Chi, 2018). One study has suggested that encapsulated shrimp enzymes with alginate–bentonite capsules contributed to a 27% higher enzyme activity when compared with the control diet (Rodriguez et al., 2018). Therefore, further research is needed on techniques that guarantee proper enzyme immobilization (Rodriguez et al., 2018).For protease-treated proteins, pretreatment may negatively impact feed properties, including microbial contamination and final pellet characteristics such as firmness and texture (Liang et al., 2022). Moreover, the processing requirements of these commodities typically involve dehydration, comminution, packaging, and conveyance to feed manufacturers, inevitably leading to a surge in carbon footprint. Hence, optimizing the enzymatic hydrolysis conditions in a targeted manner is necessary. Also, future studies are required to adopt a more environmentally-friendly method, using real-time online enzymatic hydrolysis, so that the enzymatic hydrolysis products can pass directly into the subsequent granulation process. Prior research on phytase, another feed enzyme, showed that its inclusion in the real-time online processing of compound feed was effective in enhancing mineral availability for Atlantic salmon (Salmo salar L.) raised in low-temperature environments (Denstadli et al., 2006, 2007). Our recent work (Xue et al., 2022), published as a patent in China (CN 113005030 B), also confirmed that this method shows high efficiency and low consumption. The online processing operation can be broadly divided into three stages, which are illustrated in Fig. 4.Fig. 4Flow chart of incorporating the protease treatment into the real-time online processing of fish compound feed. The stages include: 1) Mixing proteins with water, protease, and pH regulators, followed by stirring and incubation of the mixture; 2) Direct transfer of the enzymatic hydrolysis products to the next mixer after incubation, along with other powdered and pre-mixed materials; 3) Conveying the mixed material into the flow buffering bin before transferring it to the puffing machine for feed production.Fig. 4Protease preparations utilized in the feed industry are predominantly generated via microbial fermentation. Each stage of the enzymatic production process can substantially influence the safety of the resultant enzymes. As a result, the monitoring of microbial strains is essential to ensure the effectiveness and safety of enzyme products. Additionally, harmful microorganisms and heavy metal pollutants can present significant risks to the safety of enzyme products. Therefore, it is crucial to strengthen the standardization of microbial and enzyme preparations.Author contributionsShiyou Chen: Conceptualization, data curation, investigation, methodology, validation, visualization, and writing-original draft. Sahya Maulu: Writing-original draft, and revising the manuscript critically for important intellectual content. Jie Wang: Review. Xiaoze Xie: Validation, visualization. Xiaofang Liang: Investigation. Hao Wang: Methodology. Junjun Wang: Editing. Min Xue: Conceptualization, supervision, writing-review and editing.Declaration of competing interestWe declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.

PMC

Heliyon

38332863

PMC10851192

12-01-2023

10.1016/j.heliyon.2023.e23189

Erratum to “Facile synthesis and in

Fathy Usama, Abd El Salam Hayam A., Fayed Eman A., Elgamal Abdelbaset M., Gouda Ahmed

In the original published version of this article, the wrong author contribution statement was included. The correct version should be:Usama Fathy: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Resources, Writing – original draft, Writing – review & editing, Visualization, Supervision (Corresponding author).Hayam A. Abd El Salam: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Data curation, Writing – original draft.Eman A. Fayed: Conceptualization, Investigation, Resources, Methodology, Writing – original draft.Abdelbaset M. Elgamal: Methodology, Resources, Formal analysis, Investigation.Ahmed Gouda: Conceptualization, Methodology, Resources, Formal analysis, Writing – original draft, Writing – review & editing.The publisher apologises for the errors. Both the HTML and PDF versions of the article have been updated to correct the errors.Declaration of competing interestThe authors declare no conflict of interest.

PMC

CBE Life Sciences Education

36972334

PMC10228264

Jan-01-2023

10.1187/cbe.20-01-0003

What a Difference in Pressure Makes! A Framework Describing Undergraduate Students’ Reasoning about Bulk Flow Down Pressure Gradients

Doherty Jennifer H., Scott Emily E., Cerchiara Jack A., Jescovitch Lauren N., McFarland Jenny L., Haudek Kevin C., Wenderoth Mary Pat

Pressure gradients serve as the key driving force for the bulk flow of fluids in biology (e.g., blood, air, phloem sap). However, students often struggle to understand the mechanism that causes these fluids to flow. To investigate student reasoning about bulk flow, we collected students’ written responses to assessment items and interviewed students about their bulk flow ideas. From these data, we constructed a bulk flow pressure gradient reasoning framework that describes the different patterns in reasoning that students express about what causes fluids to flow and ordered those patterns into sequential levels from more informal ways of reasoning to more scientific, mechanistic ways of reasoning. We obtained validity evidence for this bulk flow pressure gradient reasoning framework by collecting and analyzing written responses from a national sample of undergraduate biology and allied health majors from 11 courses at five institutions. Instructors can use the bulk flow pressure gradient reasoning framework and assessment items to inform their instruction of this topic and formatively assess their students’ progress toward more scientific, mechanistic ways of reasoning about this important physiological concept.

INTRODUCTIONUsing scientific principles to reason about phenomena is central to scientific thinking (American Association for the Advancement of Science, 2011; National Research Council [NRC], 2012). Oftentimes, students focus on the surface features of a phenomenon to explain how it occurred and thus overlook the underlying principles (Chi et al., 2012). In the field of physiology, Modell identified seven principles he termed “general models” that can be used to reason mechanistically about seemingly different physiological processes that are fundamentally the same. One of these general models, “mass and heat flow,” can be used to describe processes as diverse as oxygen diffusing from the lungs to the blood, ions moving across cell membranes during an action potential, water uptake into plant roots, and chyme moving through the gastrointestinal tract. In each of these examples, the rate of movement of a substance is directly proportional to the magnitude of the driving force (the gradient) and inversely proportional to the magnitude of the factors that impede movement (the resistance); that is, rate of movement of a substance ∝ gradient/resistance (Modell, 2000; Carroll, 2001; Michael and McFarland, 2011). The mass and heat flow general model, which is a law of physics, is also conceptualized as “flow down gradients” in the physiology core concepts work (Michael and McFarland, 2011; Michael et al., 2017).One form of mass and heat flow is “bulk flow,” in which the substances moving are a mixture of molecules in a fluid rather than just one type of molecule via diffusion or osmosis. Common examples of bulk flow are blood flowing through the circulatory system, air moving through the respiratory tree, and sap flowing through the xylem and phloem of plants. In bulk flow, the gradient is a hydrostatic or atmospheric pressure gradient, defined as the difference in pressure between two places, and sources of resistance are tube diameter, tube length, and fluid viscosity (Michael et al., 2017).Applying the mass and heat flow general model to explain bulk flow phenomena is a powerful mechanistic reasoning approach for explaining a multitude of physiological processes. However, postsecondary students seldom use this general model to guide their reasoning and often struggle to apply it appropriately (Michael et al., 2002). One reason students might struggle to understand bulk flow in physiology may arise from the interdisciplinary nature of fluid dynamics, which is grounded in principles of physics and is represented with multiple mathematical relationships (Wang, 2004; Michael, 2007; Breckler et al., 2013). For example, bulk flow is commonly taught in biology courses using the Hagen–Poiseuille equation, often referred to as Poiseuille’s law (Table 1).TABLE 1.Equations related to pressure and courses students might take that typically use the equationName of equationEquationVariablesCourses using this equationHagen–Poiseuille equation Q = fluid flow rate, P = pressure, r = radius of a tube, η = viscosity of the fluid, l = tube lengthPhysiology, fluid dynamicsIdeal gas lawPV = nRTP = pressure, V = volume, n = amount of substance in moles, R = gas constant, T = temperatureChemistryStatic pressureP = F/AP = pressure, F = force, A = areaPhysicsWater potentialΨw = Ψs + ΨpΨw = water potential, Ψs = solute potential, Ψp = pressure potentialBiologyAnother way students struggle with applying the mass and heat flow general model is by misapplying relationships with similar variables they learned in other disciplines. For example, students may use the ideal gas law from chemistry (Table 1) to inappropriately relate the pressure and volume of liquids such as blood. Students may also use the definition of static pressure (Table 1) to incorrectly explain fluid movement along a pressure gradient (i.e., the difference in pressure between two points; Besson, 2004). In plant physiology, students may misapply the water potential equation, which governs water movement in and out of cells via water channels, to the bulk flow movement of sap (Clifford, 2002).Students may also incorrectly relate ideas of pressure, volume, and resistance (Yip, 1998a,b; Carroll, 2001; Michael et al., 2002). In a study exploring students’ ideas about blood flow through the cardiovascular system, Michael et al. found that students thought blood flow determined a vessel’s resistance (e.g., when blood flow increases, vessel resistance either increases or decreases), rather than realizing that the resistance of the vessel determines blood flow and thus blood pressure. This study also noted that students inversely related vessel pressure with blood volume, suggesting that a decrease in venous blood volume would cause an increase in venous pressure (when in fact the opposite would happen, perhaps an example of applying the ideal gas law from chemistry). Similarly, Yip (1998b) found that some students explained that blood could flow from low to high pressure in certain physiological situations. Even in a nonliving context, engineering students studying fluid mechanics struggled to relate pressure and resistance factors to explain fluid flows in pipes (Besson, 2004; Brown et al., 2017; Lutz et al., 2019).Moreover, to reason about how fluids flow down pressure gradients, students must draw on an understanding of energy, such as Bernoulli’s principle, which states that the sum of the pressure energy, potential energy, and kinetic energy of a liquid must be equal between two points, ignoring the loss of energy due to shearing friction between the flowing blood and the vessel walls (i.e., energy must be conserved for flowing fluids). As energy in fluid flow may only be addressed in advanced physiology courses (Badeer and Rietz, 1979), this leaves less advanced physiology students with uncertainty regarding the forces that govern fluid flows down pressure gradients (Besson, 2004; Vitharana, 2015).These challenges may lead to student confusion and interfere with their ability to mechanistically reason about pressure gradients and bulk flow. For example, it may be difficult for students to accurately predict and explain perturbations to physiological systems (e.g., how changes in blood pressure can cause fainting). Faculty are often unaware of these alternative types of student reasoning. A reasoning framework is an evidence-based tool that can help faculty become aware of these alternative types of reasoning. It organizes and characterizes different ways students reason about a topic (e.g., see Scott et al., 2018; Ghalichi et al., 2021). By making explicit the different ways students reason, a reasoning framework can help direct changes or modifications to instruction as well as research into student reasoning (Modell et al., 2015; Lira and Gardner, 2017).An effective way to uncover the different ways students reason about a topic is through thoughtful and timely formative assessments (Chen et al., 2021). To that end, we developed open-ended, formative assessment items to elicit the kinds of reasoning that undergraduate students use to explain the rate of fluid flow through tubes due to a hydrostatic pressure gradient. Phenomena that include osmosis or oncotic pressure, such as fluid flow into and out of vessels at the capillaries (i.e., Starling forces) or into and out of phloem at the source or sink, are beyond the scope of this paper. We identified common conceptual patterns in students’ reasoning about pressure gradients and bulk flow on our assessments and organized them into a bulk flow pressure gradient reasoning framework. This framework describes the different patterns in reasoning that students express about bulk flow and pressure gradients and orders those patterns into sequential levels that instructors can use to understand how their students’ ideas about bulk flow progress toward the mechanistic ideas described in Modell’s general model for mass flow (i.e., using pressure gradients and Poiseuille’s law). Instructors could also use this framework to inform their instructional design. Our two research questions are: RQ1, What patterns and levels of reasoning do undergraduate students use when responding to bulk flow pressure gradient assessment items?; and RQ2, Can we use the bulk flow pressure gradient reasoning framework to evaluate written assessment responses from a national sample of undergraduate biology and allied health majors?Our research is the first to investigate biology students thinking about how pressure gradients are a main determinant of fluid flow and that the size of the gradient is dependent on the difference between those two values.RQ1: WHAT PATTERNS AND LEVELS OF REASONING DO UNDERGRADUATE STUDENTS USE WHEN RESPONDING TO BULK FLOW PRESSURE GRADIENT ITEMS?MethodsThis study is part of a larger project we started in 2014, inspired by Modell’s work on general models , in which we investigated students’ understanding of flow down gradients across multiple physiological contexts (e.g., plants and animals), including bulk flow, ion movement, osmosis, and diffusion. The bulk flow items we developed for this study were modeled after pressure flow illustrations found in the cardiovascular physiology chapter of many undergraduate human physiology textbooks (e.g., Figure 14.3 in Silverthorn, 2013; Figure 12.4 in Widmaeier et al., 2014) and a concept check question in Silverthorn’s Human Physiology (p 470; 2013).To develop a framework that encompasses the full range of undergraduate students’ reasoning about bulk flow pressure gradients, we needed to administer our assessment items to students at different points in their academic careers. Therefore, we administered our items to students at various time points of instruction (e.g., both pre- and postinstruction, introductory and upper-division courses) and from different populations (e.g., students at associate’s-dominant and R1 institutions, biology majors and allied health majors). The ways in which students’ reasoning about bulk flow pressure gradients can be affected and altered by differences in instruction, teaching strategies, and context is not within the scope of this present work. This important question will need to be addressed in the future.We piloted one bulk flow item with 513 students from two institutions (associate’s-dominant and R1 institution) from a range of academic settings (i.e., before and after college physiology course work, biology majors and allied health majors; Table 2) in the 2017–2018 academic year. The item in the pilot study consisted of a simplified diagram with a series of tubes with pressures noted at the beginning and end of the tubes. We asked students to identify which tube had the highest flow rate and to explain their reasoning.TABLE 2.Descriptions of institutions and types of courses providing students’ dataaNumber of students (number of courses)InstitutionCarnegie ClassificationType of coursePilot short answer for RQ1Interview for RQ1Short answer for RQ2Associate’s A4-year, higher part-time, associate's dominantAllied health physiology14 3 Majors introductory biology36 12 Associate’s B2-year, higher part-time, associate's dominantAllied health physiology18 R1 AFull-time, more selective, lower transfer-inAllied health physiology304 8 113 Majors introductory biology159 9 228 Upper-division physiology13 128 R1 BFull-time, more selective, lower transfer-inAllied health physiology242 Upper-division physiology43 R1 CFull-time, more selective, higher transfer-inMajors introductory biology158 R1 DFull-time, more selective, lower transfer-inUpper-division physiology93 aStudents providing written data for RQ2 may not have provided data at both the beginning and end of the course and may have answered both blood and phloem sap items (see Supplemental Table S1). For more information on courses for students who provided interview data, see Supplemental Figure S1.We used the constant comparative method to develop a preliminary bulk flow pressure gradient reasoning framework. The constant comparative method is an inductive data-coding process used for categorizing and comparing qualitative data in which any newly collected data are compared with previous data (Glaser, 1965). Three researchers (J.H.D., E.E.S., J.A.C.) identified qualitatively different types of student reasoning in a subset of 200 written responses in the sample. The researchers then discussed the types of reasoning each identified until reaching consensus on a set of seven distinct reasoning patterns. The researchers then individually recategorized the same 200 written responses using the seven reasoning patterns to test their efficacy. After agreeing on the seven reasoning patterns that captured students’ ideas, we grouped the different reasoning patterns into levels of a preliminary bulk flow pressure gradient reasoning framework. We used this preliminary bulk flow pressure gradient reasoning framework to code the remainder of the pilot data and did not find additional reasoning patterns.To more deeply probe students’ ideas about bulk flow pressure gradients, we collected interview data from students at the same two institutions in the 2018–2019 academic year. This was critical for developing our bulk flow pressure gradient reasoning framework, because it allowed us to differentiate between the reasoning patterns of students who picked the same pressure gradients but offered different rationales for their choices. For example, if a student explained that their choice of pressure gradient showed the “highest pressure,” we were able to probe to see whether “highest pressure” meant “highest starting pressure,” “highest average pressure,” or “least pressure change.” We could also ask students why they did not choose the other pressure gradients. The interviews also allowed us to validate that the preliminary patterns we found from the piloted bulk flow item recurred in additional populations of students.We interviewed 34 biology majors and 11 allied health majors recruited during different points in their academic careers (Table 2 and Supplemental Figure S1). Students were recruited via emails from their instructors asking for volunteers. Given the large enrollment at the R1 institution, we limited interviews to the first five volunteers per course. At the associate’s-dominant institution, we interviewed all volunteers. For some courses, there were fewer than five volunteers. Students were interviewed at one or two time points for a total of 70 interviews (Supplemental Figure S1). Twenty-five of the 45 students were interviewed twice. Students who were interviewed once fell into in one of three categories: students interviewed after their 400-level physiology class (these students were seniors who preferred to be interviewed just once), students interviewed before Introductory Biology I (a course that did not include physiology), and a few students who chose not to schedule a second interview.For the interviews, we developed two assessment items that were based on the structure of our pilot item but were situated in either an animal (i.e., blood flow through the aorta) or plant (i.e., sap flow through phloem) physiological context (Figure 1). When selecting organisms for each item, we chose organisms of a similar size and taxa (e.g., zebra and elk but not a mouse or rabbit). Students were asked to reason about only one of the items in each interview. Items were randomly assigned to students, stratified by course. If students were interviewed twice, they received one item during the first interview and the other item on the subsequent interview. To elicit more student ideas, we followed up the question with prompts related to students’ answers. For each student who explained or implied fluids flowed from high to low pressure, we also followed up by asking why they thought fluids flowed from high to low pressure. Additionally, we asked students’ to describe their ideas about why fluids flow down gradients. This provided us with greater insight into the mechanisms students considered when thinking about bulk flow and how their thinking influenced their responses to our items. These bulk flow items were part of a larger interview protocol that asked students multiple plant and animal physiology questions. Interviews were 45–60 minutes long. Students received a $25 gift card in exchange for their time for each interview.FIGURE 1.Bulk flow pressure gradient assessment items.We used the preliminary bulk flow pressure gradient reasoning framework derived from the pilot written data as a foundation for identifying reasoning patterns and levels in the interview responses to the bulk flow items. Based on our analysis of the student interviews, we revised the bulk flow pressure gradient reasoning framework and created a coding rubric to code all interviews by pattern and levels of reasoning.We tested and calibrated the coding rubric with four researchers (J.H.D., E.E.S., J.A.C., M.P.W.) who each scored eight interview transcripts. After this calibration phase, the four researchers coded the rest of the interviews in pairs. When there were disagreements in coding, the researchers discussed the differences until consensus on a particular code was reached. Consequently, the interrater reliability for interview coding was 100%.ResultsWe developed a three-level reasoning framework that describes the different ways students reason about bulk flow pressure gradients (Table 3). Each level incorporates increasingly more mechanistic ideas that are consistent with Modell’s general model for mass flow in physiology. Specifically, at the lowest level, we identified one pattern of reasoning. At this level, students either used pressure as a measure that indicates how organisms are functioning or had only limited ideas about what the pressure values represented. At the middle level, we identified five patterns of reasoning in which students used a mix of correct and incorrect ideas about how pressure was a driving force that caused fluid flow. At the highest level, we identified one pattern of reasoning. At this level, students consistently reasoned that pressure gradients caused fluids to flow.TABLE 3.Three-level bulk flow pressure gradient reasoning framework describing common conceptual patterns in students’ reasoning about bulk flow of fluid through a tube in an organismaLevelDescriptionLevel 3“Flow down gradients”: The magnitude of the pressure difference is proportional to the rate of fluid flow (i.e., Poiseuille's law).Level 2“Emerging mechanistic reasoning”: A variety of emerging mechanistic ideas about pressure and flow.Sublevel 2.1. “Pressure causes”:Pressures at a single location along the tube, not the pressure gradient, determine fluid flow. 2.1A. High pressure values cause a large force “pushing” on the fluid. 2.1B. Low pressure values at the end of a tube push back less, causing a low resistance to flow.Sublevel 2.2. “Pressures indicate”:The magnitude of pressures are only a result, not the cause, of fluid flow. 2.2A. A small difference between pressure values at the start and end of a tube indicates that flow is maintained, 2.2B. Pressure magnitude indicates the volume of blood that is flowing or has flowed (e.g., high pressures indicates high volumes are flowing, low pressures indicate a high volume of fluid has flowed out of tube). 2.2C. A small difference between pressure values at the start and end of a tube indicates that the tube has a low resistance, thus higher flow.Level 1“Nonmechanistic ideas”: Ideas about characteristics and behaviors of organismsaThough this nomenclature may indicate one sublevel is above the other, in fact we do not ordinate sublevels, as we feel one way of demonstrating emerging mechanistic reasoning is not necessarily “better” than another.In the following sections, we present in greater detail the kinds of ideas students used at each level of the bulk flow pressure gradient reasoning framework. We also present excerpts from our interviews with students enrolled in introductory- to advanced-level biology courses at an associate’s-dominant college and an R1 university as exemplars of the different reasoning patterns we found. These excerpts provide rich insight into students’ thought processes. We use bolding to emphasize ideas critical to, or a hallmark of, a reasoning pattern. Though the scenarios used in the interview question were of blood flowing through blood vessels and sap flowing through phloem in plants, we did not see any indication that the context of the question influenced how students answered. We will further explore the influence of the context of scenario as well as the influence of varying the starting and ending pressures in a future research publication.Level 1: Pressure as a Measure of Organism Function.At the lowest level of the framework, student explanations contained physiology ideas about pressure that were unrelated to pressure gradients and were nonmechanistic in nature. For example, S44 interpreted the difference between the two pressure numbers across different tree species (Figure 1) as indicating the time it takes fluid to travel.S44: The difference between the start and the end [pressure] is smaller so it’s faster for the thing [i.e., sap] going through from the top to the bottom.Interviewer: If we’re looking at the oak again and if this end pressure was also 1.20, so it was the same as the start pressure, would that be even faster or what would that mean? If, instead of this end pressure being 1.10 it was the exact same as the start pressure?S44: It’s super-fast … It just goes straight “boom” … I think it’s going to be really super-fast with the oak tree.S44’s interpretation of the pressure values as representing fluid travel times led them to mistakenly view pressures with the least difference as signifying the fastest flow rate of tree sap, which is contrary to an understanding based on how pressure gradients work.Level 1 explanations also referenced characteristics and behaviors of organisms that were presented in the assessment items rather than referencing principles that govern fluid movement. For example, when asked which of five different animals had the greatest flow rate (liters/minute) of blood (liquid) through their aorta (Figure 1), S38 responded, “So I’m not sure what the normal pressures are for animals … I know that humans have, you know, a regular pressure would be 100 over 70.” This student’s focus on what a “normal pressure” would be for an animal suggested S38 was accessing knowledge about how organisms function to address the task rather than noting changes in pressure that impact fluid movement.Students’ Ideas about Why Fluids Move along Pressure Gradients at Level 1.When asked why fluids move along pressure gradients, explanations at level 1 continued to frame the question around organismal functioning (i.e., meeting demands of daily living that the animal may encounter). For example: S38: I mean I know in the fight or flight response it [blood pressure] will go up. It’s kind of like they stay at certain rates for the body to get what they need at the right times. I guess in an animal it would be at a certain rate so they could get nutrients to run or you know, things like that, so a sloth would probably be pretty slow.Instead of reasoning with fundamental principles of fluid movement, S38 drew on ideas about what blood pressure at “certain rates” enables animals to do, such as having a “fight or flight response,” getting “what they need at the right times,” and having “nutrients to run.” Consequently, S38’s continued framing of the tasks as being about how or why organisms function may have prevented broader reasoning about why fluids move along pressure gradients.Level 2: Emerging Principle-Based Reasoning.Students’ explanations at the second level of the framework demonstrated emerging mechanistic ideas relating pressure and flow. Many of these ideas were linked to scientific relationships that included pressure but were misapplied to the given tasks. Explanations at this level also drew incompletely, or inaccurately, on scientific ideas to explain why fluids move down gradients. We organized students’ explanations at this level into two sublevels, sublevels 2.1 and 2.2. Explanations in sublevel 2.1 reasoned that differences in the magnitudes of pressure at a single location along the tube, not differences in pressure gradient, caused differences in flow rate. Explanations in sublevel 2.2 reasoned that differences in the magnitudes of pressure are a result, not the cause, of fluid flow. Though this nomenclature may indicate one sublevel is above the other, we do not ordinate sublevels, as we currently have no evidence to demonstrate that one type of emerging mechanistic reasoning is “better” than another.Sublevel 2.1 Reasoning Pattern 2.1A: Higher Pressures Cause Higher Bulk Flow Rates.One set of student explanations reasoned that tubes with the highest pressure values would cause greater bulk flow rates, because these tubes had the most “force pushing” on the fluid. For example, S8 suggested the tree with the highest start pressure, the maple tree, would also have the highest sap flow rate: “Because if it has a higher pressure and they all have the same diameter of the [tubes], then it’s probably moving more at one time than the trees with the lower pressure.” We found that students explained which system had the “highest pressure” in different ways; some explanations used the magnitude of the start pressure as the most important value, such as S7 who said: “Well, my instinct is just to say the highest number. So, the zebra at the beginning.” S7 reasoned that: “If you turn a hose on really high, you’re going to get more water out of it than if it’s just lower.” Other explanations calculated the average of the starting and ending pressures and selected the option with the highest value or viewed the two numbers as a ratio and selected the greatest ratio as correct. Some students chose the tube with the highest starting and ending pressure, because a high pressure along the tube meant that the pushing force was maintained along the entire tube. For example, S37 reasoned:The camel has the largest end pressure which means that, I guess, for whatever reason, it’s … the blood is continuing to push equally hard when it reaches the end of the animal as … or very close to when it reaches the end of the animal as when it left.The idea that stronger forces will cause fluids to flow at higher rates is consistent with how physics defines static pressure as equal to the amount of force applied to a particular surface area (P = force/area). Indeed, S19 explained why a high force causes a greater flow rate by reasoning: “It’s force over area and then force is due to acceleration and mass … So, this is why I’m assuming that a greater pressure will mean a greater heart rate.” Using this kind of reasoning may prime students to focus on one pressure value, either measured (i.e., the largest start pressure) or derived (i.e., the highest average pressure), as being most important for determining the driving force behind bulk flow rather than the difference in pressure between the beginning and end of the tube. Thinking about pressure as the force applied to a certain area is productive, in that it helps students conceptualize pressure as a force. However, this definition of pressure alone is unreliable as a reasoning strategy to address fluid flows in tubes; a tube with high pressures at both ends of the tube (e.g., Zebra in Figure 1) will have a lower rate of fluid flow compared with a different tube that has a low beginning pressure but significantly lower ending pressure; that is, a greater pressure gradient (e.g., Sitka deer in Figure 1).Sublevel 2.1 Reasoning Pattern 2.1B: Higher Pressures at the End of the Tube Cause Resistance To Bulk Flow.Another set of student explanations suggested lower pressures at the end of the tube caused higher rates of fluid flows because that lower pressure would provide less resistance or less force to be overcome for fluids to flow. For example, S23 selected the beech tree as having the greatest flow of sap, not because it had the greatest pressure gradient but rather because it had the lowest end pressure (0.30). They used their knowledge of the cardiovascular system to reason:The pressure in the extremities will determine the amount of flow, so resistance can determine blood flow to a certain part … so the beech tree, the end pressure is 0.3 lower than the start pressure. And so therefore … it’s not going to have to overcome as much pressure when the plant glucose is moving down the phloem as opposed to the oak and chestnut, which is only 0.1 difference which is—that’s higher.In these explanations, a large pressure gradient meant less pressure at the end of the tube for the fluid flow to overcome. Thus, pressure at the end of the tube was viewed as an inhibiting force, rather than the difference in pressure being a driving force for fluid movement.Sublevel 2.2 Reasoning Pattern 2.2A: Maintenance of Pressure Indicates Higher Bulk Flow Rates.Another set of student explanations explained that the greatest flow rate occurred when the pressure on the fluid was “consistent” or “maintained” between the two points, no matter whether the pressures were high or low at each end. Students reasoned that this consistent pressure indicated (higher) flow rates were maintained. For example, when S11 was asked why they thought the oak tree (with the smallest pressure gradient) would have the greatest flow rate of sap, they responded: “Well, it’s just the most constant throughout. So, that’s what I would think. Like it, it [pressure] doesn’t really change.” When asked what the flow rate would be in an instance where the start and end pressure were the same, S11 replied:S11: Okay, now I would say that that has the most [flow].Interviewer: Why would you say that?S11: Because it doesn’t change at all. It’s the same throughout.Interviewer: Right. And how does it not changing indicate that it’s the most flow?S11: I don’t, it’s just constant. Like the constant amount of pressure. So it’s the same amount being pushed is what my thinking is. And there’s going to be a lot more at the end if it’s constant compared to a lot more at the beginning and a little bit at the end.Students interpreted the similar pressure values as indicating little change to the system, and therefore little change to fluid flow, because the system was “able to maintain the pressure the whole entire time,” according to S28.Sublevel 2.2 Reasoning Pattern 2.2B: Pressures Indicate the Volume of Blood That Has Flowed.Student explanations using this pattern reasoned that flow rate is a measure of the volume of fluid moving through a tube (accurate) and different volumes of fluid cause different pressures (accurate); therefore, pressure can be used to infer flow rate (in inaccurate ways). Students used this reasoning in several ways.In one way, students explained that organisms with high-pressure values from high blood volumes have the greatest flow rates. S30 explained this by saying:Pressure in the beginning of the aorta probably means that you have some volume of blood being pushed into that area. If you have a larger volume, you could have a larger pressure … I’m going to go with the zebra just because it has the highest starting pressure.This type of reasoning may be based on students’ understanding that increasing the volume of fluid in a compartment will cause an increase in pressure in that compartment. Therefore, these explanations suggest that a high flow rate will cause a high volume of fluid in that space, which in turn causes the high pressure. Given the high pressure, there must have been a high flow rate into that area. Student explanations in this group focus on pressure as a measure of the amount of fluid moving rather than a driving force for fluid movement. It is correct that higher fluid volumes exert more pressure on the walls of a tube; however, there will be only limited fluid flow if the pressure gradient between the beginning and end of the tube is small.In another way, student explanations described lower pressures at the end of the tube, or large pressure differences, as indicating that a greater volume of blood had left the tube. This was exemplified when S29 said:If the starting and ending pressures were pretty similar, that would indicate more of a constant flow of blood and maybe not as much volume of blood flow through. If there was a greater pressure difference, maybe there was a lot of blood that traveled which is why the [end] pressure is so much different than the starting pressure.These explanations interpreted the low pressures as indicating a loss of fluid volume due to the fact that the fluid had already flowed out of the area in question. Consequently, the lower volume of remaining fluid created less pressure on the tube. Similar to the first way of inaccurately connecting pressure, volume, and flow, student explanations using the second way of reasoning described pressure as being directly related to volume with a loss of volume causing the lower pressure.Another set of student explanations discussed pressure and volume as being inversely related. To justify this reasoning, some explanations cited the ideal gas law (PV = nRT), likely because it was a well-known relationship in which “pressure” was a key variable. For example, S35 said: “More pressure is happening when just … less volume. So the volume and the pressure. The formula between both the volume and the pressure, PV[ = ]nRT, if you know it from chemistry.” Although using the indirect relationship between pressure and volume from the ideal gas law frequently led students to select the largest pressure gradient as having the greatest fluid flow, their rationales were not based on pressure gradients as driving forces.Sublevel 2.2 Reasoning Pattern 2.2C: Small Pressure Differences Indicate Low Resistance, which Indicates High Bulk Flow.This set of student explanations inaccurately linked two accurate understandings. The first accurate understanding is that decreased resistance along a path will lead to a smaller pressure drop along that path. The second accurate understanding is that decreased resistance along a path will cause increased flow along that path. By connecting these two understandings, students reasoned that a smaller pressure drop along a path indicates a decreased resistance, which causes a greater flow. For example one student explained, “I wasn’t completely sure but I chose the camel because there is only a small decrease in [pressure] from the ascending aorta to the abdominal aorta so the resistance in the aorta and maybe the rest of the arteries and arterioles are low, which would increase flow by the equation.” However, as the question stated that each tube had the same diameter, length, and blood viscosity (i.e., the same resistance), this sequence of reasoning was inaccurate. While these student explanations correctly noted that resistance moderates fluid flow, they did not attend to the pressure gradient as the driving force for fluid flow.Students’ Ideas about Why Fluids Move along Pressure Gradients.Level 2 explanations generally noted that driving forces caused materials to flow, which represented a shift to a more mechanistic understanding of fluid movement rather than the purpose-driven explanations we found at level 1. However, some explanations revealed that students were struggling to conceptualize these driving forces as energy gradients. Instead, explanations often referenced molecular mechanisms in line with diffusion to explain bulk fluid flows along pressure gradients. For example, explanations contained ideas like materials going to equilibrium (e.g., “If you are going along the concentration gradient, where you’re just trying to equilibrate on both sides, you don’t want to lose any energy so you have to follow the concentration gradient,” S29) or that molecules in liquids move to places that were less “crowded” (i.e., “If you have a bunch of molecules in a really tight space, but they have the chance to escape, then they’re going to want to disperse evenly comparative to their environments,” S39).When explanations did describe energy as playing a role in bulk fluid flows, the ideas were imprecise or vague. For example, some explanations described the challenges associated with moving against gradients (e.g., “You can’t push against a gradient,” S11) or simply mentioned that energy was involved, such as S16, who said: “High to low pressure because just the thermodynamics of it … If there’s a high number here and a low number here, then that’s the path of least resistance for molecules to move.” S37 had a relatively sophisticated understanding about the role of energy in pressure gradients, saying:Because the universe is always trying to decrease potential energy. So, basically, anything at any time is going to go… somewhere that decreases potential energy. So if you have a cliff, and there’s a liquid on it, anything that encourages the water to jump off that cliff is going to be totally fine with water.However, they had previously explained that organisms where high pressure was maintained (and therefore the gradient was small) would exhibit the greatest flow. When confronted with their previous explanation in the face of their energy explanation, they struggled to reconcile the two competing ideas:Interviewer: If we go back to the camel that was the 200 to 200, it’s maintaining its, I think you said, potential energy across that distance?S37: Oh, hm. So I don’t think of it maintaining the potential—well, oh, actually, yes, I do think it would … so if the camel was standing on all fours, I think the blood that was going down to its feet would have less potential energy. But since the pressure is still up, it sounds like the potential energy is being maintained despite that. Yeah.S37 in many ways epitomizes what is characteristic about students who provided responses at level 2; they used scientific ideas that relate to gradients but were uncertain how to apply those ideas to the bulk flow assessment items.Level 3: Principle-Based Reasoning with Pressure Gradients as a Driving Force.At the highest level of the framework, student explanations consistently identified organisms with the largest pressure difference as experiencing the greatest fluid flow. Moreover, the explanations used pressure gradients as driving forces that mediated fluid movement. The following exchange with S4 demonstrates this kind of reasoning, going so far as to explicitly cite the general relationship for bulk flow as part of their reasoning:S4: The deer… I see that, even though the start and end pressures are relatively lower compared with the other animals, there’s a greater difference. And so I’m just looking at the differences between start and end pressure. And you would look to the zebra, and that has a difference of 60, but you also see that the Sitka deer has a difference of 70, which to me indicates a higher flow rate.Interviewer: And why does that indicate a higher flow rate?S4: Hm, that’s a good question. Again, I will think back to my flux model … So we’re assuming that, in all of these animals, you have the same amount of resistance, so that shouldn’t have an impact. So what you’re looking at then are your driving forces, which would come from this start and end pressure. And so if you have a greater difference, then you have a greater numerator, which makes your flux larger.S4 acknowledged that the start and end pressures of the Sitka deer are “relatively lower compared to other animals,” indicating they noticed the different magnitudes of pressures across the animals, but S4 focused on the pressure differences as the most important consideration when making a selection. In the latter part of the response, S4 confirms their selection by drawing on the bulk flow relationship (bulk flow ∝ gradient/resistance), recognizing that when resistance is held constant, the driving forces—as indicated by the pressure gradient—must be driving fluid movement. By using the bulk flow relationship as a reasoning tool, S4 identified the most salient features of the system that would lead to a scientifically correct understanding of the task. Similarly, S31 explicitly used Poiseuille’s law to examine their initial ideas about flow rates:Interviewer: Would any of them [the animals] have a higher flow rate, given that they have the same size aorta, same composition of blood, but these different pressures is the only difference?S31: No, they wouldn’t. Because the flow rate, the flow rate would be Q, I don’t remember the exact formula. But I know it’s, well, it’s change in pressure divided by … 8 pi R to the fourth. And if you’re saying the radius isn’t changing, because they all have the similar diameter … Then, hmm. Flow rate would be proportional to the change in pressure. So if you have a larger change in pressure, you would expect to have a larger flow, right?By using Poiseuille’s law to mechanistically reason about blood flow, S31 realized their initial answer of no differences in blood flow among the different animals was incorrect and that, in fact, the animal with the largest pressure difference would have the most flow.Students Ideas about Why Fluids Move along Pressure Gradients.When students were asked why fluids move down pressure gradients, energy-related explanations were more likely to be associated with level 3 explanations of bulk flow rather than level 2 explanations of bulk flow (i.e., 26% of level 3 interview explanations mentioned energy, whereas only 10% of level 2 interview explanations mentioned energy; the other 74% of explanations were similar to those of level 2, using “equilibrium” and “crowdedness” ideas). However, the energy ideas used were similarly vague or imprecise regardless of the associated bulk flow explanation. Several students mentioned “entropy” as important but were unclear exactly how that played a role in fluid movement. Other students suggested systems moved to lower energy states, like S10 who said: “Things tend towards equilibrium because it’s a lower energy level.” S2 showed one of the more nuanced understandings of the way energy is involved in bulk flow, saying:S2: Because you have a greater force at the high-pressure end. As you go down to a lower pressure, it can’t go back up. Otherwise … you would need energy to go from low to high pressure.Interviewer: Why don’t you need energy to go from high to low pressure?S2: Because there’s a concentrate—or, there’s a pressure gradient existing. So that’s basically your energy.S2 recognized that pressure gradients are also energy gradients, which was uncommon in the students we questioned. Students’ uncertainty with the link between energy and pressure gradients was not prohibitive to their ability to reason productively about bulk flow in a physiology context. However, there may be other contexts where students’ confusion about the link between energy and pressure gradients might be prohibitive.Table 4 presents the frequency of levels observed during our interviews. This information is provided to show the range and frequency in our sample; it is not meant to indicate the prevalence of ideas that might occur in a classroom or show how students’ ideas change over a term, as we only interviewed a small number of students from each course (Supplemental Figure S1).TABLE 4.The number of student interview responses at each level of the bulk flow pressure gradient reasoning framework at the start and end of the term (numbers in parentheses show how total level 2 responses are distributed across different patterns)Start of termEnd of termLevel 1: Nonmechanistic5—Level 2: Emerging mechanistic1610Sublevel 2.1: Pressure causes 2.1A: High pressure pushes 2.1B: Low pressure is less resistanceSublevel 2.2: Pressures indicate 2.2A: Pressure maintained 2.2B: Pressure indicates volume 2.2C: Pressure indicates resistance—Level 3: Flow down gradients1623RQ2: CAN WE USE THE BULK FLOW PRESSURE GRADIENT REASONING FRAMEWORK TO EVALUATE WRITTEN ASSESSMENT responses FROM A NATIONAL SAMPLE OF UNDERGRADUATE BIOLOGY AND ALLIED HEALTH MAJORS?MethodsTo further validate the bulk flow pressure gradient reasoning framework, we recruited students from 11 courses at five institutions across the United States taking a course that included instruction on bulk flow. These students took our assessment items as low-stakes, formative assessments. This allowed us to obtain responses to our assessments from an additional, larger group of students. We did this in order to investigate whether the framework captured the diversity of student reasoning in this larger group of responses and collect validity evidence based on response processes (American Educational Research Association et al., 2014). It also provided us with a snapshot of reasoning levels pre- and postinstruction in different student groups. Four of the five institutions were public R1s, very high, research-active, “more selective” institutions (Carnegie Classification of Institutions of Higher Education, n.d.; Table 2).In each course, students were given an online assessment of six short-answer physiology items. One of the six items was a bulk flow pressure gradient item similar to our interview items (Figure 1) but with values for three rather than five tubes in a plant or animal scenario (Supplemental Figure S2). The instructors selected the most appropriate scenario(s) to give to their students. The other five items were part of a different study.We collected students’ written responses at the beginning and/or end of a term for three student groups: students in introductory physiology courses for allied health majors, students in introductory biology courses for majors, and students in upper-division physiology courses (Table 2). We collected 1050 responses from 935 students in 11 courses at the beginning of the term and 882 responses from 752 students in 11 courses at the end of the term (see Supplemental Table S1 for sample sizes by item, time point, and course). While physiological topics dealing with the concept of bulk flow were presented in each of these classes, the data collected are meant to serve as validity evidence to evaluate whether or not the bulk flow pressure gradient reasoning framework can capture the breadth of reasoning used in a diverse sample and are not intended to assess specific instructional practices.We used the coding rubric created for analyzing the interview data for RQ1 to identify the patterns and levels of reasoning in students’ short-answer responses. We calibrated coding on students’ responses by having two researchers (J.A.C. and a research assistant) use the coding rubric to code 114 responses into one of the seven patterns. If two independent coders coded the students’ responses to the same reasoning pattern, this was considered a match. Interrater reliability for this calibration phase was greater than 90% agreement. After this calibration phase, one researcher (research assistant) coded the rest of the responses, with a second researcher (J.A.C.) coding 10% of those data. Final agreement was greater than 90%.ResultsWe collected data from 11 courses across five institutions to confirm that our reasoning framework could be used to categorize reasoning offered by a larger and more varied group of students. We found examples of all the reasoning patterns described in our bulk flow reasoning framework in all three student groups at both the beginning and the end of term (Table 5 and Figure 2). Additionally, we were able to use the bulk flow pressure gradient reasoning framework to code all student responses collected. We noticed that fewer than 10% of all students reasoned at level 1 at the beginning or the end of the term, while more than 50% of students enrolled in majors introductory biology or upper-division physiology courses began the term using level 3 reasoning. We also found that, regardless of when the items were given (i.e., the beginning or end of a term), a greater proportion of students in allied health physiology courses reasoned at level 2 compared with students enrolled in majors introductory biology or upper-division biology courses. Within these level 2 responses, we found most students used reasoning pattern 2.1A (higher magnitude pressures cause higher bulk flow rates) with the second largest proportion of responses coded as 2.2A (maintenance of pressure indicates higher bulk flow rates). By the end of the term, students in allied health physiology courses reasoned at roughly equal proportions for levels 2 (44%) and 3 (49%), whereas students enrolled in majors introductory biology or upper-division biology courses reasoned predominantly at level 3 (60% and 70%, respectively).TABLE 5.Example student responses from each pattern and level from the national validation sample for RQ2Level and patternBlood flow item: zebra, 106–102; camel, 93–91; elk, 83–75Phloem sap flow item: American beech, 0.60–0.35; white oak, 1.20–1.00; American chestnut, 0.90–0.801: NonmechanisticElk: “I compared it to what I know about humans. People with a high blood pressure usually have a heart that beats faster. The heart, although it beats faster, pumps less blood which is probably why it beats faster, to compensate for the difference.”American beech: “It has the greatest number of vessels.”2.1A: High pressure pushesZebra: “The zebra has the greatest flow rate because there is more pressure which pushes blood through the aorta faster.”White oak: “There is highest pressure both at the beginning and the end so the sap in the phloem will be pushed more to move faster.”2.1B: Low pressure is less resistanceElk: “It has the greatest flow rate since it has the least pressure in the aorta so it has the least resistance to the blood flow.”American beech. “Lowest pressures mean the least resistance. Therefore, the sap would be flowing the fastest because the size of the ‘tubes’ are generally the same size in all of the trees.”2.2A: Pressure maintainedCamel: “The camel was able to maintain almost the same blood pressure meaning that the blood pressure remained high.”American chestnut: “I think the American chestnut has the greatest flow because it has the smallest difference in start and end pressure.”2.2B: Pressure indicates volumeZebra: “Since the pressure is the greatest, I assume that it means that there is more blood in the area, meaning that it has the greatest flow rate.”American beech: “Because it had the greatest loss in pressure over the same amount of time as the other trees, meaning that more sap flowed out flowed through the vessel over the given time, and thus faster than the others.”2.2C: Pressure indicates resistanceCamel: “The pressure from the beginning of the vessel to the end of the vessel decreased the least, meaning resistance is the least in this animal, which means that decreased resistance will increase flux.”American chestnut: “The rate of flow would be greatest at the tree with the least resistance. Since the starting and ending pressure in the American chestnut is similar, the resistance must have been low.”3: Flow down gradientsElk: “Elk has the greatest flow rate due to having the largest difference in start pressure and end pressure.”American beech: “There is a greater pressure gradient in the American beech. So the sap will flow down its pressure gradient faster.”FIGURE 2.Pattern and level of reasoning in written responses from the national validation sample described in Table 2. Responses are grouped by time point (beginning and end of the term) and course type (introductory physiology for allied health majors, introductory biology for majors, and upper-division physiology for majors). Data from blood and phloem sap items were combined.DISCUSSIONWe developed the first reasoning framework that describes how undergraduate students reason about the role of pressure gradients in determining the rate of fluid flow through tubes. The bulk flow pressure gradient reasoning framework has three levels and is based on 70 interviews and 2445 responses to short-answer assessment items from biology and allied health majors in introductory to upper-division courses. Our framework focuses on a simple bulk flow system and targets students’ understanding of how the magnitude of the pressure gradient determines the flow of fluids through tubular structures of similar length and diameter. Despite this constrained focus, we found that student explanations displayed a diversity of ideas concerning the concept of pressure and how pressure gradients influence fluid movement.Students providing bulk flow explanations at level 1 in the framework used nonmechanistic ideas about pressure, often relying on ideas about characteristics and behaviors of organisms. As with many science concepts (e.g., energy, evolution), pressure is both a scientific term and a term used commonly in everyday language (Pramling, 2009; Jin and Anderson, 2012; Slominski et al., 2020). Pressure is used colloquially (e.g., people are under pressure to meet a deadline), in medical situations (e.g., systolic and diastolic blood pressure), and in relation to water flow in homes (e.g., lack of water pressure causing low shower output). Students providing explanations at level 1 draw on these surface understandings. Therefore, instructors can be aware that students may be interpreting their words through a different lens than intended. Providing assignments to compare and contrast the colloquial and scientific use of the word “pressure,” in addition to teaching Poiseuille’s law may be helpful.We found that, when explaining blood flow through vessels or sap flow through phloem, students using level 1 reasoning often relied on teleological reasoning. In these cases, students reasoned the blood had to flow because the animal needed to deliver the blood with all its nutrients and oxygen to the tissues to keep the animal alive. Students used similar logic for the delivery of sap with its water and nutrients to the various parts of a plant. We were not surprised by this teleological thinking, as we see it in the students in our classes and it has been well documented in a robust body of literature from the fields of biology and physiology education research (Richardson, 1990; Tamir and Zohar, 1991; Michael, 1998; Mohan et al., 2009; Slominski et al., 2020).Students using any of the five patterns of reasoning seen in level 2 demonstrate emerging ideas about how pressure relates to flow. Many of these ideas were linked to scientific relationships that included pressure but were misapplied to the given tasks.Students using reasoning pattern 2.1A explained that tubes with the highest pressure values, be that the highest starting pressure, highest average pressure, or highest ratio of pressures, would cause greater bulk flow rates, because these tubes had the most force “pushing” on the fluid. This pattern is similar to one that Brown and colleagues found in some engineering university students; even after completing a course on fluid mechanics, students can reason that a high pressure at one point is a pushing force causing fluid flow. Students using reasoning pattern 2.1B explained that lower pressure at the end of the tube causes higher rates of fluid flows, because there is less resistance to overcome. These students see the ending pressure as a source of resistance to flow, yet pressure and resistance are independent variables in the bulk flow equation. This connection between a change in pressure causing a change in resistance was also found by Michael et al. . Students using reasoning patterns 2.1A and 2.1B might understand the pressure vocabulary used in class (e.g., high pressure, low pressure, mm Hg) and interpret pressure as a force that impacts fluid flow, but may not be cueing into the instructors’ emphasis on pressure gradients.Students using reasoning pattern 2.2A explained that little to no pressure difference between the start and the end of the tube indicated that the flow was maintained (i.e., at a high level). These students are using pressure in the tube as a measure of flow rather than as a cause for it. This use of pressure as indicators and not causes of flow was similar to what we observed in students using reasoning pattern 2.2B. Some students explained that a high flow rate will cause a high volume of fluid to accumulate in a space, which in turn causes the high pressure, so given the high pressure at the end of the tube, there must have been a high flow rate into that area. Other students explained that lower pressures at the end of the tube, or large pressure differences, indicated that a greater volume of blood had left the tube. A third group of students reasoned that pressure and volume are inversely related, as in the ideal gas law. This incorrect use of an inverse relationship between pressure and volume for liquids was also found by Michael et al. . All three groups of students using 2.2B reasoned that flow rate is a measure of the volume of fluid moving through a tube per unit time, and because different volumes of fluid cause different pressures, pressure can therefore be used to infer flow rate. These students are using pressure as an indicator of volume and fluid flow rather than focusing on the pressure gradient as a driving force for fluid flow.Students using reasoning pattern 2.2C reasoned that a smaller pressure drop along the tube indicates a decreased resistance and decreased resistance will cause a greater flow. However, in this case, the students failed to note that the question specifically stated that the tubes were identical (i.e., have the same resistance) and that any difference in flow would be due to the difference in the stated pressure gradients. Like students who used reasoning pattern 2.1B, these students focused on their accurate understanding of the inverse relationship between resistance and flow rate, but failed to incorporate the direct relationship between pressure gradients and flow rate.Students providing level 2 explanations are calling on multiple resources about pressure, volume, and resistance that they have accumulated through their academic or everyday life. As many students in biology and physiology classes have taken physics or chemistry either in high school or college, they may have encountered many of the principles associated with the concept of pressure (e.g., Poiseuille’s law, ideal gas law, static pressure; Table 1). Instructors should therefore be very clear on what principles are appropriate for hydrostatic fluid flow in organisms and provide students an opportunity to practice applying these principles in situations with pressure gradients of different magnitudes to confirm that the students are using the proper principle. It may also be beneficial for biology and physiology instructors to build collaborations with their colleagues who are teaching introductory physics courses to coordinate how principles such as Poiseuille’s law are taught. Such interdisciplinary collaborations have been shown to be quite beneficial to both the instructors and the students who take these courses (Redish and Cooke, 2013).The results from our national validation sample indicate that only 10% of our sample (predominantly from R1 institutions) reasoned at level 1 at the start of term. This suggests that, by the time students reach the undergraduate level at R1 institutions, most have an emergent mechanistic understanding of pressure rather than an indicator of an organism’s functioning. We also found that, as students experience more biology and physiology courses, many gain an understanding that a pressure gradient rather than just pressure is the driving force for fluid flow. These are the 70% of upper-division students who provided level 3 reasoning when explaining fluid flow. However, this development is not inevitable, as indicated by the 30% of upper-division students who continued to demonstrate uncertainty about how pressure gradients work (i.e., reasoned using levels 1 or 2) even at the end of the term.LIMITATIONSWe acknowledge that our national sample for validation is composed almost exclusively of public R1 institutions. To get a more comprehensive data set, we need to expand our sample to include more associate’s-dominant institutions as well as regional public institutions and private schools.Although we did not observe a difference in the pattern or type of student reasoning between the blood flow and phloem sap flow items, we realize that context may influence the resources students call on to answer a question (Nehm and Ha, 2011; Slominski et al., 2020). That is, students may draw from different patterns or levels of reasoning as they reason through problems in different contexts (Lira and Gardner, 2020). To that end, we are currently analyzing a new set of data from items that not only vary the context of the organism and the magnitude of the pressure gradient but also investigate changing or keeping constant starting pressures. Additionally, the ways in which students’ reasoning about bulk flow pressure gradients can be affected and altered by differences in instruction and teaching strategies is not within the scope of this present work. This important question will need to be addressed in the future.Conclusions and Implications for Teaching and ResearchPedagogical content knowledge (PCK) is “information about typical difficulties students encounter as they attempt to learn about a set of topics; typical paths students must traverse in order to achieve understanding; and sets of potential strategies for helping students overcome the difficulties that they encounter” (NRC, 2000). There are several different types of knowledge that make up PCK: knowledge of students, assessment knowledge, content knowledge, curricular knowledge, and pedagogical knowledge (Carlson et al., 2019). Of particular relevance for this study, “knowledge of students” includes instructors’ ability to anticipate how students are likely to reason and what students will find confusing or challenging about a topic (Ball et al., 2008). Assessment knowledge includes knowing how to design formative assessments and make changes to instruction based on responses to these assessments (Chan and Hume, 2019). We propose that instructors can use our bulk flow pressure gradient assessment items to make student reasoning visible and provide instructors with greater knowledge of their students’ current understanding of the topic. Our bulk flow pressure gradient framework can guide the instructors’ interpretation of the reasoning students offer when solving bulk flow problems, which in turn can inform instructors’ design of future formative assessments and course activity (i.e., assessment knowledge; Auerbach et al., 2018; Chan and Yung, 2018).We realize it is often challenging to untangle students’ explanations of physiological phenomena. However, by taking the time to dissect their reasoning, instructors can gain valuable insight into student thinking about the relation of variables to one another, as well as students’ misinterpretations of what is a cause and what is an effect of a change in a physiological variable. Our bulk flow pressure gradient reasoning framework provides some guidance as to the multitude of ways students use pressure, volume, flow, and pressure gradients. We suggest instructors can use our formative assessments and reasoning framework to enhance their teaching of bulk flow down pressure gradients in three ways: 1) Use our bulk flow pressure gradient reasoning framework to anticipate the kinds of ideas students will bring to the classroom or laboratory and plan instruction accordingly. 2) Have students take a bulk flow pressure gradient item as a low-stakes formative assessment near the beginning of the unit to reveal students’ incoming ideas about bulk flow along pressure gradients. Instructors can then modify planned instruction to meet the learning needs of their students. 3) Have students take a bulk flow pressure gradient item again at the end of the unit or term and use the responses to reflect on the impact of the teaching methods used. By uncovering and summarizing patterns in students’ explanations, our bulk flow pressure gradient reasoning framework provides faculty with the opportunity to more effectively design their course topics about pressure and pressure gradients and thus enhance their PCK and thus their students’ mechanistic reasoning on this challenging topic (Ergönenç et al., 2014).In the beginning of the Discussion and in the paragraphs above, we have endeavored to provide suggestions for how faculty might respond if they uncover their students using the different types of reasoning in our bulk flow pressure gradient reasoning framework. At this point, these instructional strategies are mostly our own personal suggestions based on our years of teaching. We suggest that biology education researchers could use our bulk flow pressure gradient reasoning framework as a tool to assess the distribution of patterns and levels of reasoning in different populations of students, including investigating the effectiveness of new teaching strategies focused on bulk flow pressure gradient reasoning.Supplementary MaterialClick here for additional data file.

PMC

Heliyon

PMC10245010

5-24-2023

10.1016/j.heliyon.2023.e16498

A concise summary of powder processing methodologies for flow enhancement

Shah Devanshi S., Moravkar Kailas K., Jha Durgesh K., Lonkar Vijay, Amin Purnima D., Chalikwar Shailesh S.

The knowledge of powder properties has been highlighted since the 19th century since most formulations focus on solid dosage forms, and powder flow is essential for various manufacturing operations. A poor powder flow may generate problems in the manufacturing processes and cause the plant's malfunction. Hence these problems should be studied and rectified beforehand by various powder flow techniques to improve and enhance powder flowability. The powder's physical properties can be determined using compendial and non-compendial methods. The non-compendial practices generally describe the powder response under the stress and shear experienced during their processing. The primary interest of the current report is to summarize the flow problems and enlist the techniques to eliminate the issues associated with the powder's flow properties, thereby increasing plant output and minimizing the production process inconvenience with excellent efficiency. In this review, we discuss powder flow and its measurement techniques and mainly focus on various approaches to improve the cohesive powder flow property. Graphical abstractImage 1 Highlights•Elaborated factors affecting powder flow.•Types of flow include mass flow and core flow.•Different types of powder flow testers are described and compared.•Several techniques can be applied to improve flow of powders.•All the techniques reduce the intermolecular interactions.

1IntroductionAs a solid state, powders have diverse applications in different manufacturing fields, such as dyes, ceramics, petroleum, cosmetics, pharmaceuticals, and food. On average, 75% of manufacturing processes in chemical industries involve particulate solids or powders at least once or twice throughout the cycle. In the pharmaceutical industry, where most of the products are solid dosage forms, transport and handling of powdered solids are especially important . Free-flowing powders are desirable to enable robust powder processing operations such as bin filling, hopper discharge, and capsule and die filling. In a pharmaceutical plant where the powders undergo different processes, there is significant variation in the physical particle properties, which affect the powder flow. Thus, it is essential to understand the processes and critical parameters to ensure product quality and efficient manufacturing performance. Being such an integral part of all the manufacturing processes, it is essential to understand the phenomenon of powder flow, which finally dictates the process efficiencies of fluidization, blending, crushing, granulation, tableting, and flow from the storage tanks. Powder flow properties are critical, particularly in the pharmaceutical, food, cosmetic and ceramic industries. Really for the development, conveyance, packaging, and transport of goods, powder flow activity is essential . Powder flow can be of diverse types, i.e., “static” and “dynamic”; measurement of the former involves shear cell and wall friction analyses of a consolidated state of the powder, and the latter requires flow studies using a rotating helical blade in a less consolidated state of the powder sample. The static flow determinations, such as shear cells, highlight the dominant friction and mechanical interlocking in some process operations, such as wall friction . The behavior of the powder flow is measured by various material properties, mainly pertaining to the physical and chemical properties of the substance . As a simple observation, poor flow gives poor product uniformity and causes undesirable process breakdown, affecting the production line's required speed and thereby providing substantial financial losses.Flowability is a derived powder property, unlike other physical properties, which are not inherent but dependent on a range of fundamental powder properties and other material and environmental parameters. The complex phenomena of powder flow are known to be affected by factors such as particle size distribution [, , ], particle geometry [8,9], moisture content, inter-particle forces which eventually reflect the particle surface energies [10,11]. Most of these properties affect the interparticle interactions within the bulk of the powder. Various interparticle forces, such as van der Waals, capillary forces, electrostatic forces, mechanical interlocking, and other frictional and gravitational forces, exert their effects and determine the powder flow . For fine dry particles, the van der Waals force is the most influential type of force responsible for the cohesiveness of powder . The decreased particle size causes gravitational forces to be less dominant. At specific particle sizes (66When the angle of repose exceeds 50°, the flow is rarely acceptable for manufacturing purposes. The angle of repose is determined by measuring the height of the cone of the powder and calculating the angle of repose, α, from the following Eq. :tan(α)=height0.5×base1.2.1.2Compressibility index and Hausner ratioIn recent years the compressibility index and the closely related Hausner ratio have become simple, fast, and popular methods of predicting powder flow characteristics. The compressibility index has been proposed as an indirect measure of bulk density, size and shape, surface area, moisture content, and materials cohesiveness because these can influence the observed compressibility index. The compressibility index and the Hausner ratio are determined by measuring both the powder's bulk volume and tapped volume. The basic procedure is to measure the apparent unsettled volume, V0, and the final tapped volume, Vf, of the powder after tapping the material until no further volume changes occur. The compressibility index and the Hausner ratio are calculated by the following Eqs. , :CompressibilityIndex=100×V0−VfV0Hausnerratio=V0VfAlternatively, these parameters can be calculated by replacing the volumes with their respective density values. For the compressibility index and the Hausner ratio, the generally accepted scale of flowability is given in Table 3 .Table 3The acceptable scale of flowability for compressibility and Hausner ratio.Table 3CompressibilityFlow characterHausner ratio1–10Excellent1.0–1.1111–15Good1.12–1.1816–20Fair1.19–1.2521–25Passable1.26–1.3426–31Poor1.35–1.4532–37Very poor1.46–1.59>38Very, very poor>1.60Like the angle of repose, the two properties are not intrinsic and dependent on the methodology used. Thus, there have to be experimental considerations for evaluating these, including the diameter of the cylinder used, the number of times the powder is tapped to achieve the tapped density, the mass of material used in the test, and the rotation of the sample during tapping.1.2.1.3Flow through an orificeSeveral particle-related and process-related factors affect the powder flow rate, which can significantly measure powder flow ability. However, determining the flow rate through an orifice is useful only with free-flowing materials. The flow rate through an orifice is generally measured as the mass per time flowing from any container. The three important experimental variables include the type of container used to contain the powder, the size and shape of the orifice used, and the method of measuring powder flow rate. Either mass flow rate or volume flow rate can be determined. Mass flow rate is a more accessible alternative, but it biases the results to favor high-density materials. Since die fill is volumetric, determining volume flow rate may be preferable. No general scale is available because the flow rate is critically dependent on the method used to measure it. The flow rate through an orifice is not an intrinsic property of the powder and is, therefore, dependent upon the methodology used.1.2.1.4Shear cell methodsVarious powder shear testers and methods that permit a more thorough and precisely defined assessment of powder flow properties have been developed to correlate the powder flow with the hopper design. These methods enable the scientist to obtain several parameters such as yield loci, the angle of internal friction, the unconfined yield strength, the tensile strength, and a variety of derived parameters such as the flow factor and other flowability indices. These methods have been successfully used to determine critical hopper and bin parameters. A cylindrical shear cell forms a shear plane between the lower stationary base and the upper movable portion of the shear ring. Annular shear rings require less material; however, the design does not allow a uniform shear distribution inside and outside the annulus. The plate type of shear cell consists of a thin sandwich of the powder between a lower stationary rough surface and a rough upper surface that is moveable. A significant advantage of shear cell methodology is greater experimental control. The methodology generally is rather time-consuming and requires substantial amounts of material and a well-trained operator .All these methods, though simple and conventional, hold a great significance in formulation development in the pharmaceutical industry. These basic methods are simple to perform and give a fair idea about the powder flow behavior. Over years now, the pharmaceutical industry has been completely dependent on the Pharmacopeial methods for powder flow evaluation to compare powders and predict their behaviors during large scale processing. However, with growing trends, research and developments of novel APIs and excipients for complex processing in the pharmaceutical industry, the nature of the newer materials differs greatly and therefore, advanced characterization tools for powder flow evaluation become inevitable.1.2.2Modern methods for evaluating powder flow propertiesDespite the simplicity of these methods in terms of execution and result interpretation, reproducibility, predictability, and sensitivity are the general issues. It is also challenging to establish correlations between the derived values and the actual flow behavior of the powders. Also, these compendial powder flow characterization methods are semi-quantitative and do not indicate the powder flow behavior under shear when subjected to downstream processing such as tableting . A proper characterization technique should mimic the stress and powder compaction state of the powder process condition. Such limitations have paved the way for developing advanced characterization techniques . With technical developments, newer methods such as powder flow categorization techniques to determine cohesivity, determination of avalanches, dielectric imaging, microscopy of atomic force, and penetrometers are coming into practice. Such strategies suffer from multiple factors that contain reproducibility, conditions of efficiency, and predictability. E.g., measurement of the angle of repose and avalanching has limitations for a cohesive powder that does not flow through the funnel, and vibrating the funnel creates inherent complexity and variability in measurement. When the cohesive powder is subjected to vibrations, it reduces interparticle friction and locks the particles into certain positions, which causes inherent variability and may obstruct the flow . Although the smaller avalanches may get suppressed due to the vibration, the vibration consolidates small amounts of powder on the slope of the equipment until sufficient powder is built up to cause a larger avalanche . Another way of calculating powder flow requires using a photovoltaic array with a proprietary technology called “AeroFlow” . But this method includes a narrow disk which imposes a significant frictional barrier and doesn't accurately mimic the mixing or filling process. Another method to evaluate the flow properties of a powder under shear is a shear cell method which is most commonly used and is convenient . In recent years, image analysis techniques based on high-speed imaging and particle image velocimetry (PIV) are being used to measure flow patterns and agglomeration behavior of fluidized powder using luminous intensity and powder movement inside a cuvette. This direct optical evaluation of powder is suitable for testing a wide range of pharmaceutical powders, even with a small sample size [12,38].These standard techniques cannot measure the entire range of powder flowability/cohesion. Flow meters require a funnel to flow through the powder or granular material and are thus ineffective for cohesive powders. The method known as miniaturized powder flow through an orifice introduced state-of-the-art image analysis for powder flow analysis. It recognized that measuring large powder volumes has prevented a detailed description of the cohesive phenomena that govern many industrial processes. Since the sample size was drastically reduced, which introduced powder flow measurements into pharmaceutical pre-formulation stages, this method can be applied to process operations such as die and capsule filling, mixing, powder coating and conveying, and to applications such as dry powder inhalers, in which particle-level behavior dominates over bulk behavior. Blanco, D. et al. have successfully predicted die-filling efficiency for the first time in pharmaceutical literature .For highly cohesive powders, tap density testers are often insufficient because the tapping pressure is insufficient to resolve the tight inter particular cohesive bonds. When tapped, the powder bed will not consolidate. Another issue with this research approach when dealing with granular materials would be that the heavier or denser particles are far more likely to stack easily under their weight, resulting in excessively low efficiency. For large granular compounds, commercially available approaches are not commonly available due to particle size limitations. There is no precise method for calculating powder flow throughout all four flow regimes (plastic, inertial, fluidized, and entrained gas) .In the modern innovative powder flow method techniques, different instruments can measure powder characteristics, such as powder flow tester, FT4 powder rheometer, ring shear tester, and gravitational displacement rheometer . The advanced powder flow analyzers, mainly shear cell testers, are trends in the industry to determine good flow behaviors of powder due to the strong theoretical and science-based processing and design. The Powder Flow Tester evaluates powder flow properties in industrial processing equipment rapidly and efficiently [40,41]. Flow Function, Wall Friction, Bulk Density and Time Consolidation Test with Flow Function, Arching diameter, Rat-hole Dimension, Hopper Half Angle, etc. be determined and calculated with the help of simple function in powder flow tester [24,42]. The FT4 Powder Rheometer was initially developed to explain powder rheology or powder flow properties. This range of measuring capabilities makes the FT4 a universally functional powder tester and perhaps the most reliable instrument to test and evaluate powder behavior. A powder rheometer has been discovered to provide a reproducible and straightforward calculation of the powder response to different conditions .The Gravitational Displacement Rheometer (GDR), an experimental apparatus used to characterize the cohesiveness and flowability of powders based on their avalanching behavior, quantifies the flow properties of the mixture under unconfined conditions . A flow index is calculated from GDR measurements. It is directly associated with the flow via hoppers, providing a hopper design predictive approach and simple experimentation for evaluating materials and assessing their suitability for a particular hopper system . Jenike was the pioneer of the shear cell tester. Shear cell measurements are distinguished by Schwedes, 2003 in two variants-direct (major principal stress rotates during the test) and indirect (major principal stress is fixed during the test) tester . Historically, the shear cell process and design were more complex and time-consuming. It required one and half days to collect test measurement, chart recorder output, and produce yield locus, for example, Walker annular ring shear cell (the 1980s). The Jenike shear tester was even more complex because it required manual pre-consolidation stress and multiple sample preparation to define a single yield locus. It could be done by only a trained, experienced and skilled person. Another type of shear tester is the constant -volume shear tester with a lower movable shear mechanism, as reported by Y. Shimada et al., 2018 . The proposed method of evaluation allowed to obtain powder yield locus, consolidation yield locus, critical state line, shear cohesion, powder bed void fraction, and stress transmission ratio by with a single shear test . In order to overcome these difficulties variety of automated and computer-controlled shear cell techniques have been proposed which include uniaxial, biaxial and triaxial compression testers, rotational ring shear cells like FT4 rheometer, Schulze, and most recently powder flow tester (Brookfield PFT).Uniaxial, biaxial, and triaxial compression testers are indirect types and generally not commercially available; therefore, little research is found in the literature relating to pharmaceutical and other industry-relevant powders. Table 4 summarizes the principle, pros, and cons of these non-commercial testers collected from several reviews on testers [44,47,48].Table 4Comparison criteria for indirect uni, bi, and triaxial testers.Table 4Indirect testersPrincipleProsConsUniaxialBulk density and unconfined yield strength can be obtained, sample filled into a cylinder and consolidated using normal consolidating stress to get the bulk densitySample loaded again after removal of the cylinder using normal stress, which leads to unconfined yield strengthQuick and easyTime consolidation of the coarse particle can be measured.Can be used for testing cohesive powder only (stable after removing cylinder)Low accuracyDetermination in the low-stress region is not possible for cohesive powderConsolidation by vertical force cannot guarantee a steady state flowBiaxialFour steel plates can realize steady-state flow and uniaxial compression on the lateral X and Y axis.It avoids vertical deformation and friction between the plates using rigid plates and thin silicon rubber membranes.Stress and strain can be controlled on the X and Y axis and measured in all X, Y, and Z directions.Able to determine time-dependent stress-strain behaviorsTime-consumingNot recommended for silo design and quality control purposeTriaxialPrincipal stress can be applied and measured in all three directions.Normal triaxial tester – stress increases in a vertical direction until failure by two movable stampsTrue triaxial tester – deformation in all X, Y, and Z directions can be possible with six wall boundaries of the sample.Both the tester used in soil mechanicsA true triaxial tester can measure all three principal strains and stresses.The advantage of a true triaxial tester is the complete determination of the state of stress and the state of strain, because all three principal stresses and strains are measured.Over consolidated samples cannot be tested after unconfined failure strengthThe availability of several advanced powder flow testers makes their selection and understanding a prerequisite for evaluating powder flow behaviors. Therefore, a comparative study of these powder flow testers would better assist in selection . Table 5 is one such comparative summary of the different powder flow testers.Table 5Comparison criteria for FT4, Ring Shear Tester, and PFT shear cell testers.Table 5Commercial instrumentCharacterized features and differencesFT4 powder rheometer•A blade, piston, and shear head are applied to rotate a compact and incremental load that interacts and allows particles to flow, resist (axial and rotational force), and measure the bulk flow of the powder sample.•Initial consolidation stress is not included in the data collection•Measures applied normal force (torque) at the bottom of the powder bed and displayed force, torque, and height data•The range of volume of vessel available is 10–400 ml, providing the capability to test variable sample range•The instrument can control and maintain the stress on highly dilating powder•The initial compaction load range for FT4 powder rheometer is 3–15 kPaRing shear tester (RST)•The bottom ring (annular) of the solid specimen's shear cell is rotated while the lid is fixed by two tie rods and connected to the load beam. The extorted force on the lid is transferred to the solid specimen to measure the flow behavior at different shear stress.•Initial consolidation stress is not included in the data collection•The ring (annulus) is used to load the sample•Measure the applied normal force at the top of the powder bed (force), and the torque is displayed•High spillage of sample due to scrapping of overfilled powder•At low normal stress (below 2 pKa), the tester does not control/respond to force created by rapidly dilating (particles roll over each other) powderBrookfield Powder flow tester (PFT)•The lid applies the vertical compression downward into the sample contained in the shear cell (annular trough). A calibrated beam controls the compaction stress on the rotating sample trough. Torque experienced on rotating sample trough against the stationary lid is measured to determine the flowability of the powder.•Initial consolidation stresses included in the data collection•The ring (annulus) area used to load the sample•The instrument can measure the low consolidation stresses and determine a flow function from one test with five data points•Initial compaction load can be set in the range of 0.3–5 (4.82) kPa•High spillage of sample due to scrapping of overfilled powder and need separate balance to weigh•Normal stresses during pre-shear and shear steps cannot be set.•The longest shear displacement (measurement of rotational speed and time elapsed to start rotation) is required to attain a steady state.Compared to conventional techniques, these novel techniques are successful. These new techniques offer automatic handling with no errors and fast results. On the other hand, the inter particulate forces, which also affect the powder flow, can be determined by vibration, centrifugation, or impact separation. Such methods allow the determination of the adhesion forces by measuring the number of particles detaching from a surface at a given force .2Approaches to enhance powder flow properties2.1Addition of glidantsAlthough the addition of glidants must have resolved several failures in pharmaceutical manufacturing operations, this category of excipients usually does not gain the required attention during product development. As a well-known fact, adding glidants affects the powder mixture's tablet properties and flow behavior in a specific concentration range. The small glidant particles bind to the powder and granule surface, enhancing the separation between the two particles and thereby reducing the attracting forces between the particles. Glidants on the powder or granulate particles form a monoparticulate coating that allows movement over one other. Thus, the rough surface is softened completely, reducing the friction and adhesive forces among materials . Though not clearly known, there are two mechanisms by which glidants improve the flow properties of the powders. Since the interparticle force hinders the powder flow, reducing this friction is one of the most important techniques to promote powder flow. This reduction can be achieved by modifying the surfaces of the powders, increasing the distance between two particles, which in turn leads to a reduction of the attraction forces between them. The second mechanism is the theory of ball bearing effect, wherein the tiny glidant particles form a monolayer on the surface of the powder or granule particles, making them roll over each other and giving an enhanced flow. This corroborates with a sandwiched contact system of the small particle between two larger particles. The improved flow behavior greatly depends on the degree and uniformity of the coverage of glidant particles over the host powder particles. Such a covering mechanism of the glidant has been proven by Jonat et al., by scanning electron microscopic studies. The study reports colloidal silicon dioxide particles adhering to the surface of the Microcrystalline cellulose particles, hence improving the latter's flow. The addition of colloidal silicone dioxide increased the bulk and tapped density and reduced the angle of repose of the MCC powder. In another study, the work done by the mixing blade to mix lubricated and unlubricated granules was compared, and it was found that lubrication done to improve the powder flow can reduce the work done by 75% .The efficiency of the glidant to improve the flow properties also depends on the material attributes and the properties of the glidants. Understanding these factors, which affect the formulation, is therefore essential. A paramount concern in this approach is the particle and the consequent surface energy of the host particles and glidant, which eventually affect the inter-particle forces and the flow properties. Some researchers in their study have shown that the material attributes in terms of the surface energy affect the coating efficiency of the glidant over the host material in dry powder systems [13,21]. Though these are also affected by the mixing process, Jallo et al., concluded that a large difference in the surface energies (i.e., high interaction potential) between the two entities is also required for optimal coating. However, the evaluation of this surface energy has not been validated to predict the glidant effectiveness for pharmaceutical powders . As stated earlier, the mixing process also affects the glidant's effectiveness. The energy type and intensity of the mixing process are crucial for the glidant to disperse uniformly on the host particles. Glidants within themselves do not usually have good flow and exhibit tendencies to agglomerate owing to their small particle size. Therefore, low energy or low shear mixing fails to fully disperse the glidant uniformly on the surface of the host particles giving suboptimal flow improvement. The nature of the glidant, in terms of its hydrophobicity and triboelectric behavior, may also affect the flow properties enhancement because of the direct effect on the inter-particle force . It has been reported that the combined use of a glidant and a lubricant in the tablet formulation usually has a better impact on the powder flow .One of the commonly used glidants, silica (silicon dioxide), owing to its small particle size and low density, has been identified as the most potent glidant . Generally, an approach of adding glidants separately into two phases, i.e., API-glidant mixture and pharmaceutical excipient-glidant mixture, is used to optimize the glidant distribution. Such a two-step approach enables the various glidant forms to adjust their right concentrations for each particle characteristic at each glidant mixing point . Abe et al. In their study, used a two-step glidants mixing method with an API and lactose, microcrystalline cellulose, sodium starch glycolate, and magnesium stearate as the pharmaceutical excipients. Two types of silicon dioxide were tested as glidants; non-porous silica and porous silica, both of which were mixed in high-shear blender. Their study results show that, with their optimal concentrations, the required glidants combinations improved the flowability of the two-step process relative to the one-step operation. The two-step operation removes the key to powder flow bottleneck and makes it more beneficial to apply direct compression process for improvement trials.While using glidants for improving the flow of powders, it is essential to know that the order of mixing of glidants in the formulation is a crucial factor that affects the effect of glidants . A specific order of mixing of glidants plays a vital role, as described by Kalyana et al. in their study on two glidants, i.e., Cab-O-Sil (CS) and Magnesium Stearate (MgSt). In their study, the authors reported that the mixing of glidants in the tableting blends leads to the formation of microlayers, affecting the product properties. These microlayers formed on the surface of particles are the reason the mixing order is significant. Three mixing orders were evaluated to obtain blends containing Avicel PH200, Pharmatose, and micronized acetaminophen (mixing order-1: CS added first; mixing order-2: MgSt added first; mixing order-3: CS and MgSt added together). It was found that the mixing order has a significant impact on hydrophobicity and flowability. Both these properties increased when CS mixed into the blend before the MgSt. These observations were mainly attributed to the onion-like ordered mixtures, explicitly formed due to the microlayers on the particulate surface, giving a distinct microstructure. Another critical factor affecting the improved flow properties of the addition of glidants is the nature of the glidant. As reported by the previous authors, the hydrophobicity and hydrophilicity of the glidant affect the flow properties of the blend. Ahamad et al. determined the glidants' effects of hydrophobic and hydrophilic silica on a poorly flowing API using various methods. Ibuprofen was mixed with hydrophobic and hydrophilic silica and evaluated using conventional flow properties like the angle of repose, bulk density, and the advanced technique, i.e., powder flow tester (PFT). The results show that, according to the multiple flow measurements. However, both the silica increased the flowability of ibuprofen to a significant degree, hydrophobic silica performs better than hydrophilic silica in terms of a reduction in angle of repose, cars index values, and hydrophilic silica is considered a good flow enhancement for the flow factor . This observation is due to the simple reason that increased hydrophobicity decreases cohesion and hence improves flow .Similarly, a study by Jonat et al., also revealed that hydrophobic glidants are most effective for hydrophilic drugs . The difference in the glidant geometry structure on the flow properties of powder mixture has also been evaluated. It was noted that porous flow aids improve flow properties more than nonporous agents because porosity contributes to the reduction of adhesion force between the individual host particles .2.2Thin coating to the powder with polymersCoating fine particles on the surface with a polymeric solution to improve the flow properties of the core particles in a controlled manner is a well-known approach . The flow improvement by such an approach is due to a ball-bearing effect, as stated earlier, which means that the modification of the surface properties helps improve the powder flowability . The formed inter-particulate barrier will decrease attractive forces such as electrostatic and molecular interactions, resulting in improved powder flow and packing properties. This approach to coating particulate systems can also be applicable for taste masking and controlled release properties.Natalja et al. studied the effect of polymer coating on Ibuprofen's cohesiveness, mainly due to its varying particle size and distribution . The powder was then coated with hydroxypropyl methylcellulose (HPMC) to improve the flow with the help of an ultrasonic nebulizer. The coated powder showed improved physical properties since polymer treatment affected the particle size. Morphology evaluation revealed a decrease in the API's cohesiveness and an improved particle surface homogeneity. The enhancement in flow properties increased as the uniformity of the HPMC layer increased. Because the glidants affect tablet parameters such as hardness, friability, dissolution, etc., the polymer coating can be a preferred method for pharmaceutical industries. Genina et al. worked on improving the flow properties of Ibuprofen powder by HPMC coating in a top-spray fluid bed granulator. An increased flow of the powder was noted due to the trace amounts of hydroxypropyl methylcellulose deposited onto the particle surfaces .Li et al., in their work, utilized nano-coating by electrostatic deposition to improve the flow properties hindered due to crystal growth. The coating significantly improved physical stability, wetting by aqueous media, dissolution rate, powder flow, and tabletability. Thus, it can be seen that coating cohesive powders with polymeric solution has an essential application in improving the flow of powders. The process can be carried out by several techniques, as discussed above .2.3Surface coating of the powder with silicaSurface modification of particulate solids by dry coating to reduce the cohesion between particles can be done by dispersing guest nano-particles over the surface of host particles of cohesive powders. Coating with nano silica provides nanoscale roughness, which eventually causes a reduction in the contact surface between the cohesive particles and hence reduces the interparticle cohesion force, which is responsible for the reduction in the agglomerate formation . Such a technique for surface modification reduces the cohesiveness of the powders by forming a film or thin coat over the host particles . Van der Waals forces are responsible for keeping the host and the guest particles in contact with each other. The guest remains attached to the host when the force of attraction between the two is more than the guest's weight .Different types of silica produce different results when used to improve a powder's flowability. Microcrystalline cellulose (MCC) coated with hydrophobic colloidal silicone dioxide types showed better flow properties in terms of angle of repose as compared to those coated with hydrophilic silicone dioxide types. Moreover, hydrophobic silica coatings are independent of the mixing conditions, unlike hydrophilic silica, which shows improved flow under forced mixing conditions. The SEM data showed that the coverage by the silica particles on the MCC particles dictates the improvement of the flow properties. The nature of silica particles also determines the extent and uniformity of this coverage. The silica particles are linked to stable MCC aggregates, forming larger aggregates owing to the hydrogen bonds. Hydrophobic silica has a comparatively lesser ability to form hydrogen bonds giving friable aggregates compared to those with hydrophilic silica. Agglomerates of hydrophobic silica are easily breakable, whereas those with hydrophilic silica require higher energies . Generally, the flow of MCC or starch-like particles improves when hydrophilic colloidal silicon dioxide is used with higher mixing time and energy. On the other hand, hydrophobic silica tends to spread evenly on the hydrophobic surface. Higher surface coverage can be achieved by mixing hydrophobic silica with host particles leading to flow improvisation .Qi tony et al., in their study, treated cohesive lactose powder with magnesium stearate and fumed silica using tumbling blending or intensive mechanical dry coating. The results show that a cohesive powder of lactose improves flow properties after treatment with magnesium stearate and fume silica due to reducing inter-particular forces . El-Say et al. improved the rheological properties of cohesive lactose powder by treating the powder with different kinds of acrylic resins . A magnetically assisted impaction coater and a hybridizer have also been to coat cohesive cornstarch powder with silica particles to improve the powder flow. The Hosokawa powder flow tester results showed that nanosized silica provided the best flowability enhancement . Performed a similar study of coating a cohesive API with silica particles in a vibratory mixer. Their study showed that dry coating improves a cohesive powder's bulk density and flow function coefficients. Another method that can be used from such dry coating is comilling, as reported by several researchers [5,68]. The above observation and results indicate that a dry coating is a simple and easy technique to improve the flow of cohesive materials. In addition, the problems faced in a polymeric coating involving liquid solution spray, that can be devoid in a dry coating include lumps formation, degradation of drugs from moisture, heat, etc. The dry coating is helpful for light and moisture-sensitive drug. Fig. 2 illustrates the mechanism of flow enhancement by silica coating.Fig. 2Schematic representation of the mechanism of flow improvement by silica coating.Fig. 2Silica coating helps to improve the flow properties by counteracting the different forces which hinder the powder flow, such as van der Waals forces, liquid bridging, electrostatic charge, mechanical locking, and frictional and gravitational forces. The silica particles, at first, adhere to the material's surface, filling the voids and irregularities on it. Such adherence causes a reduction in mechanical and interlocking friction. The increased distance between the direct particle surfaces reduces van der Waals forces and electrostatic interactions. Thus, the silica particles act as barriers and lubricant to enhance the powder flow. Such a mechanism is mostly observed for hydrophobic silica. On the other hand, the hydrophilic silica retains moisture, preventing liquid bridging [12,69].Surface modification should be used in conjugation with micronization to together obtain benefits of enhance dissolution properties as well as good flow. Micronization is another widely used technique to improve poorly soluble drugs' surface area and solubility – dissolution rate . However, this process usually leads to highly cohesive fines forming agglomerates due to electrostatic interactions between the particles. Though micronization may lead to enhanced dissolution characteristics, it hampers the flow properties and density of the powders. Simultaneous micronization and surface modification can be achieved by milling the particles with nano silica, which provides fine surface-modified particles with a lower tendency to agglomerate. A water-soluble polymer can be an alternative to such a process which would further assist water wettability. Along similar lines, X. Han et al. have used a water-soluble Polyvinyl pyrrolidone for co-milling the drug particles along with nano-silica to give a combined advantage of water wettability and nano-silica to improve the flow in a lower mechanical stress fluid energy mill. A reduced electrostatic charging tendency on the surface of the micronized powder is observed due to the coating of nano-silica, which prevents agglomeration of the powder and enhances flowability. In such a process, the mill operating conditions and amount of silica used significantly affect its flowability and compressibility . With increasing grinding air pressure, smaller median particle size was obtained due to more energy input, leading to more intense and faster particle-particle and particle–wall collisions. A higher feeding rate results in larger ground particles owing to the shorter residence time of the particle in the grinding chamber and lower kinetic energy available for grinding. The amount of silica increases the flow properties, but beyond a certain value, no significant improvement is obtained. Such a coating and co-grinding process also increases the powders' compressibility compared to pure or pure micronized powder. As also dispersibility of the powder drug. Thus, such surface modification improves the flow properties and the powdered drug's dissolution characteristics.2.4Addition of anticaking agents or flow conditionersOne of the significant issues in handling solid hygroscopic powders is agglomeration or caking to form lumps, mainly due to partial dissolution of particles and subsequent recrystallization due to environmental moisture content . Deliquescence is the phenomenon responsible for the chemical and physical instabilities of powdered materials, which is nothing but a first phase temperature-induced, moisture-dependent phase change from solid to liquid which appears for crystalline deliquescent ingredients at a particular relative humidity . The absorbed moisture forms a stable bridge causing what is called caking—during manufacturing, processing, storage, and consumer use, caking and lumping cause severe problems in powdered products . The anticaking substances, also called the flow conditioners, overcome flow issues inside the manufacturing units (Barbosa-muriet Canovas et al., 2005). Anticaking agents are usually crystal growth inhibitors . A flow conditioner is sub-sieve particle size and is typically very fine powder. The added flow conditioners compete for moisture uptake with the host molecules and form a protective moisture barrier on the surface of the particles or physical barriers between ingredient particles . This physical barrier eliminates surface friction and further inhibits crystal growth. For optimal agent effectiveness, interactions between the anti-caking agent and the host molecules are necessary .Various forms of fine silicon oxide, sodium aluminum silicates, tricalcium phosphate, and calcium stearate powder are widely used as anti-caking agents. In a study by Nurhadi and Roos, calcium silicate and calcium stearate were added to honey powder as flow conditioners. A comparative analysis was performed to evaluate the agents' performance by influencing water sorption and flowability. Calcium silicate did not seem to affect the water sorption properties of honey powder, whereas calcium stearate showed an inhibitory effect on recrystallization. The addition of flow conditioners improved the flow of honey powder from easily flowing to free-flowing powder. Calcium stearate was a better aid since it reduced interparticle friction. The flow conditioners also improved honey powder's bulk density and flow factor index . Rebeca et al. also studied the impact of anticaking agents on the action of delicate ingredients and blends on moisture sorption, flowability, and caking characteristics. Out of the anti-caking agents evaluated, Calcium stearate showed a delay in the onset of deliquescence, reduction of moisture sorption, and the preservation of flowability at different humidities over time whereas the addition of calcium silicate decreased the overall moisture sorption event .Silicon dioxide makes a barrier between the host particles, which can be decreased powder stickiness and slow down the deliquescence . One another research done by M. Peleg et involves the study of anticaking agents like calcium stearate with aluminum silicate for improving the flow of sugar powder. Using a Jenike Flow Factor Tester, the evaluation showed decreased cohesion and compressibility upon the addition of anticaking agents. The incorporation of calcium stearate caused a reduced angle of internal friction . Another work done by Christopher et al. on egg powder reported particle surface alteration by flow conditioner such as silica and sodium silica-aluminate, which eventually increased the flowability of egg powder. Without a conditioner, it was found that whole egg powder could absorb more water and the same powder with flow conditioners yet retain strong flow properties . In another work, the scientist Konstance et al. researched Sucrose, lactose, and flour; these three powders as encapsulated materials for butteroil, and Sylox is used as an anticaking flow agent for the same. They reported that encapsulated powders are less flowable, but flow characteristics are improved by the addition of the anticaking/flow agent .2.5By changing the particle shape and sizeResearchers have attempted to improve this powder property to enhance flow by considering particle size and shape as critical factors in powder flow properties. Particle morphology plays a vital role for powders that must be directly compressed to form tablets. Consequently, it is possible to improve the flow as well as compaction properties of the powders by modifying the particle shape of the particles. One example is Ascorbic acid; wherein spherical agglomerates were prepared using a spherical crystallization process by a binder. The process involved precipitation of the crystals in the form of spherical agglomerates by a bridging liquid which eventually enhances the micrometric properties, flowability, packability, and compactibility required for direct tableting. The improved compactibility of the spherical agglomerates was attributed to enhanced fragmentation, increased contact points during compression, and improved plasticity of the particles .It is known and studied that lowering the particle size minimizes the flowability. Detergent powder with particle sizes of 125–180 μm and 180–250 μm gave more significant bulk powder flow properties because their circular shape makes them roll or move over each other when the shear stress is applied to them. However, the particles (<125 μm) having a high surface area to volume ratio showed flow suppression of powders and particle interaction when pressure was applied . The powder flowability range of these particles (125–355 μm and 710–1000 μm) is quick to flow, and other powder size ranges indicated poor flowability .Preparation of granules of powders to modify the particle size and morphology is yet another approach to improve powder flow. Granules improve flow, handling, and compression properties . Depending on the method used to enable powder particle agglomeration, the granulation method can be divided into two types: dry granulation and wet granulation. Wet granulation means increasing the size using a liquid binder; fine particles are agglomerated into bigger ones into permanent structures. The granulation method has unique benefits, enabling powders' mechanical processing without loss of blend consistency. The flow properties of a powder mixture are enhanced by growing the size and surface area of the particles—the uniformity of powder density increases and the strength. The process minimizes air trapping between granules and reduces particle and cross-contamination levels .In a study reported by El-Say et al., the physical and technological properties of the poorly-flowing and remarkably coherent drug Bezafibrate were addressed. Bezafibrate's flow property and compression properties were enhanced by wet granulation. The result showed that the flow property improves in terms of enhanced micromeritic parameters, including acceptable mean size, narrow size distribution, low lump percentage, low fines percentage, and no sticking to the wall surface . Another group of researchers also prepared granules by adding water as a binding agent, using two pre-processed excipients, such as lactose monohydrate and cellulose, and evaluated their flowability. Using the wet granulation process, all these excipients can be directly compressed. The result shows that the wet granulation method enhanced the flow and improved permeability properties and compression, which is not observed in dried powder . Granulation can be done using different techniques and various instruments; hence, Limin et al. prepared granules in a high-shear granulator and scientifically assessed the effects of massing on flowability. This study evaluated microcrystalline cellulose as an excipient, and the granulating agent was distilled water. The process enhanced flowability due to dramatic changes in granule porosity, morphology, and specific surface area . Hence agglomeration is one process implemented to enhance the powders' functionalities involving flow properties . Agglomeration improves the flow property of fine powders since it results in a powder with higher bulk density and lots of reduction in its wall friction and the cohesive strength . From all the above studies, it can be concluded that granulation is the most widely used technique to improve powder flow and minimize production process problems in different industries.2.6Sprouting the crude drugSprouting is another technique that has been found to improve powder flow, as reported by a few studies, mainly for food powders. Sprouting of food seeds and nuts significantly affects the nutritional value since the bioavailability, and improved digestion of the nutrient leads to better nutrient utilization. The sprouting process leads to the release of metabolic enzymes, such as proteinases, that increase the amino acid content of the food products . Apart from improved nutrition, few researchers studied the effect of sprouting on the flow properties of the sprouted material compared to their counterparts. The raw and sprouted onion powders were examined for their individual flow properties, and the flow indicators such as bulk density, Hausner's ratio, Carr's index, angle of repose, coefficient of friction cohesion index, and caking index indicated the improvement in the flowability of the powder. Powder flow analysis also showed the more coherent nature of the powders obtained from sprouting, which is the free-flowing nature. Also, the caking tendency of the sprouted onion powder can be reduced, which provides reduced segregation potential during transportation and storage . Sprouting leads to increased density of the powders. The improved flow properties of the sprouted powder can be the plausible reason for enhanced flow properties. Sprouting process parameters like sprouting days affect the flowability of the powder. Likewise, the increased number of sprouting days increases the flowability of powder . Thus, it can be concluded that sprouting improves the functional potential of the food powders and the powder rheology and flowability. By considering the above results of different scientists, we could conclude that sprouting is a novel technique to enhance powder rheological characteristics.2.7Crystallization techniqueCrystallization is another study that has proven to improve the flow properties of poorly flowable materials. The technique allows researchers to modify the crystal habit of various materials which affects their powder flow properties. Garekani et al. showed different sizes and shapes of ibuprofen particles crystallized from multiple solvents showed varied flow properties. Crystals obtained from methanol and ethanol were lath/plate-shaped and exhibited better flow properties than the needle-shaped crystals from hexane . Crystallization modifies the particle size and shape, eventually modifying the powder flow properties . C. Sun studied poorly flowing citric acid anhydrate by exposing it to relative humidity to prepare pure monohydrate with almost equal particle size and morphology but varying surface properties than citric acid anhydrate. Results by ring shear cells indicated that hydration could significantly increase the anhydrous citric acid flowability . Kawashima et al. carried out another method of crystallization to improve ascorbic acid flow, i.e., spherical agglomeration technique by emulsion solvent diffusion. In this study, ascorbic acid crystal agglomerations with the mechanism of emulsion solvent diffusion (ESD) and spherical agglomeration (SA) were precipitated by a solvent to alter the system. In contrast with the initial pharmaceutical crystals, the resulting angle for agglomerated crystals decreased significantly. These results showed that agglomerated crystals' flowability and packability were ideally enhanced for direct tableting . Information from the above research work reveled that the crystallization techniques is effective for poorly flowable anhydrous drugs for improving the drug flow properties. If the anhydrous drug can be converted into hydrous form, it improves their compaction, flow and tableting performance. One more research performed by Kaerger et al., in 2004 reported the influence of the size and shape of paracetamol particles blends on the flow and compression behavior. The engineered particles by SAXS (Solution Atomization and Crystallization by Sonication) technique exhibited improved properties in bulk, tapped density, and overall flow compared to the blend with micronized particles .To summarize, there are several proposed and implemented methods for enhancing flow properties of pharmaceutical powders. Based on the material properties and suitability of the technique, either of these can be explored for an application. Literature reports a vast array of examples which employ the above-described methods and the results are promising for all. A summary of a few of these examples is tabulated for reference in Table 6, indicating the approach, instruments, observations and inferences reported for those.Table 6Summary of some reported studies for improvement of powder flow with different techniques.Table 6Sr. No.MethodsTechniques/materials usedInstruments used for checking powder flowObservationsReference1Two steps glidants mixing processSilicon dioxide mixed with the help of high shear mixer (HSM)Powder tester (PT-R, Hosokawa Micron Corporation, Japan) and critical orifice diameter testerDecreased repose (AOR) angle, required minimum hopper outlet diameter, and improved powder flow.2Thin coating to powderUsed ultrasound-assisted mist of HPMC (Hydroxy propyl methyl cellulose)In-house designed flowability testing methodSuperficial changes occur3Used HPMC solution by using top spray fluidized bed granulatorIn-house designed flowability testing methodSmall improvements in particles4Deposition of chitosan by Electro-statistic deposition over powdersCompendial methodsInhibit crystallization and reduce AOR.5Dry coating of silicaFT4 powder testerEnhance flow function coefficients (FFC) and bulk density (BD)6Mixed hydrophilic nono silica via continuous fluid energy millFT4 powder tester and conventional methodsIncrease BD and FFC and reduce electrostatic forces (ESF)7Intensive mechanical dry coating of silicaFT4 powder rheometer and conventional methodsReduce inter particular forces, cohesiveness, AOR, compressibility, and increase in pour and tapped density.8Used acrylic resin via phase separation techniquesConventional methodsReduce AOR, Hausner ratio (HR), and increase in density9The coating is done by using Magnetically assisted impaction coater (MAIC) and the hybridizer (HB)Hosokawa powder flow testerReduction in AOR due to uniformed coating to powder10Adding anticaking agentsCalcium silicates and calcium stearate are used as anticaking agents by using manual mixingPowder flow tester (PFT) Brookfield's engineeringInhibit recrystallization, collapse structure of amorphous honey powder, reduce internal friction and enhance BD and flowability index.11Calcium silicates, calcium stearate, and silicon dioxide are used as anticaking agents via manual mixingRevolution powder analyzerReduce moisture sorption, delaying the onset of deliquescence and preventing the caking12Silica and sodium silico-aluminate are used and mixed by using a hand wire mixerCompendial methodsEnhance Surface smoothness, fill inter particular voids, absorb powder and bulk density moisture, and reduce inter particular forces.13Sylox is used as an anticaking agent and mixed by manual mixingConventional or compendial methodsReduction in cohesion in powder hence enhances powder flow14Mixing order of glidants and lubricantsCab-o-sil and magnesium stearate are used as glidants, mixing done via a v-shape blenderGravitational displacement rheometer (GDR)An increase in hydrophobicity and mixing order also affects powder flow properties15By altering particles' shape and sizeThree different shapes and sizes of lactose powder useShear cellSpherical size particles, similar size distribution, large size particles, less specific energy, and low compressibility are all parameter that increases powder flowability.16Black soybean has different particles sizes, powder usePFTIncreased high sensitivity circularity (HSC) reduces internal friction and requires minimum critical hopper opening diameter as it reduces HSC and particle size decreases the powder flow.17The detergent powder having different sizes was usedSchulze ring shear cell testerThe spherical shape particles have a higher flow than less spherical shape particles. The particles having small sizes having higher surface area increase particle interaction and reduce flow.18Paracetamol and microcrystalline are prepared by using novel engineered Solution Atomization and crystallization by Sonication (SAXS)Compendial methodsImprove powder flow because of increased spherical morphology, BD, and tapped density (TD).19Addition of hydrophilic and hydrophobic glidantsHydrophobic and hydrophilic silica is used as glidants mixing done in a v-shape blenderPFT and compendial methodsThe reduction in AOR, carr index (CI), and enhanced flow factor indicated enhanced powder flow.20Hydrophobic and hydrophilic colloidal silicon dioxide utilize similar to a glidant mixing done via different mixers (free fall mixer, high-speed mixer, pin mill)Conveyer belt and compendial methodsUniformed distribution of glidants, as well as protection from moisture, improves powder flow.21By prepare granulesPrepare granules of Bezafibrate by using wet granulation techniques (high shear granulator).Compendial methodsAfter granulation, the narrow size distribution minimizes lumps formation, and fine powder percentage minimizes sticking property, required low ejection force indicated improved powder flow.22Prepare granules by using wet granulation techniques (Cuisinart mixer)FT4 rheometer and compendial methodsIncreasing particle size reducing size distribution and increasing density indicated that it improves powder flow.23Granules prepare by using wet granulation techniques (high shear granulator)Ring shear cell testerIncreasing circularity, granules size, and reduced specific surface area and porosity indicated that enhanced powder flow24By sprouting the crude drugSprouted (Freeze-dried) onion powder is utilizedPowder flow analyzer and compendial methodsAn increase in particle size and a decrease in the CI, cohesive index, and caking strength indicated improved powder flow properties.25Sprouted sorghum grains powder is utilizedCompendial methodsA decrease in CI indicated that enhanced flow of powder26Crystallization techniques to improve powder flowabilityCrystal hydration of citric acid is done with anhydrous citric acidRing shear cell testerIt reduces crystal surface interaction and increases density, enhancing powder flow.27Ascorbic acid is used by utilization of emulsion solvent diffusion (ESD) techniquesCompendial methodsIncrease plastic deformation, lower elastic recovery and AOR indicate that enhance powder flow.3ConclusionPowder flow is a crucial parameter necessary for the smooth functioning of a pharmaceutical production line. Freely flowable powder improves feed parameters' reproducibility, resulting in efficient processing. From a pharmaceutical point of view, it gives reliable tablet hardness, friability, dissolution rates, and ease in manufacturing. There is an increase in plant output when the powder is smoothly flowing. Poor or uneven powder flow can result in excessive trapped air inside powders, thereby facilitating capping or laminating or even creating lubrication issues in high-speed tableting operations. This problem can be solved to improve powder flow properties. It will diminish air-pocket forming by a smooth downward flow of the material. The dosage cavity is filled very precisely, which increases the average weight and eliminates the deviation in the average weight coefficient, and induces force during compression, decreasing stress on system components. In a nutshell, the powder flow evaluation is crucial for understanding the suitability of a manufacturing process and for giving efficient drug development strategies. With the help of the techniques mentioned above, novel ways and means of powder flow enhancement can be used to provide overall benefits.Author contribution statementAll authors listed have significantly contributed to the development and the writing of this article.Data availability statementData will be made available on request.Declaration of competing interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Indian Journal of Dermatology

PMC10718247

Jan-01-2023

10.4103/ijd.ijd_649_21

Unilateral Syndactyly, Hemihypertrophy, and Hyperpigmentation with Mosaic 2q35 Deletion

Ali Akhtar, Kumar Ajeet, Dubey Pawan K, Pandey Vivek, Singh Ankur

Pigmentary mosaicism (PM) is a clinical condition of dyspigmentation with chromosomal abnormality. PM presents with both cutaneous and extracutaneous manifestation. Hypomelanosis of Ito and linear and whorled nevoid hypermelanosis are syndromic disorders in which PM is one of the manifestations. We present a case of a 1-year-old child with a unique constellation of symptoms of unilateral syndactyly, hemihypertrophy, and skin hyperpigmentation. Karyotype from peripheral blood was normal. We found genetic aberration (mosaic 2q35 deletion) in the present case from fibroblast cultured from the affected area. This unique constellation of symptoms was previously reported once but genetic study was not done from the affected tissue. This case highlights the need of considering fibroblast culture-based genetic study rather than doing simple karyotype from peripheral blood. Genetic study also established the molecular basis of symptoms in the above case.

Case ReportA 1-year-old male child presented to genetic clinic with complaints of overgrowth of a few parts in the right side of the body and syndactyly of the third and fourth fingers of the right hand. The child was born as a full-term, normal vaginal delivery, in a public hospital. He cried immediately after birth. Child development was normal. Three-generation pedigree analysis showed no family member was affected with the same type of skin abnormality. He had one brother who had died on day 4 of life at home. The cause of death could not be ascertained. Examination revealed hemihypertrophy of the right side of the body [Figure 1]. There was a fusion of the third and fourth fingers of the right hand [Figure 2]. Facial dysmorphism was present in the form of depressed nasal bridge and everted nostrils. There was a sandal gap in the right foot. There was skin hyperpigmentation on the right side of the neck, throughout the upper arm, axilla, abdomen, thigh, leg, and foot along the lines of Blaschko [Figure 3]. There was no involvement of hairs, nails, teeth, and mucosa. Ocular examination was normal. X-ray examination of hands revealed nonosseous syndactyly of third and fourth fingers in the right hand [Figure 4]. The skeletal survey was normal. Neuroimaging, ultrasonography of the abdomen, and echocardiography were done to look for other malformations. All were normal. Punch skin tissue was obtained from the affected area. Fibroblast culture was done as per guidelines. Cells were grown and arrested at metaphase for visualizing the defect. A total of 90 metaphase plates were captured under the microscope and karyotyping was done with 450 G-banding resolution using automated karyotyping workstation having Metasystem's software (Ikaros®, Carl Zeiss® Microscopy GmbH, Göttingen, Germany). Chromosomes were analyzed following guidelines provided by the International System for Human Cytogenetic Nomenclature (ISCN 2016). A deletion on the long arm of chromosome 2 (2q35) was observed in 10 out of 90 metaphase plates, analyzed for karyotyping and cytogenetic anomalies [Figures 5-7]. The karyotype of the patient in the present case was 46, XY, del q35. This abnormality was not observed in any of the 50 metaphase chromosome plates prepared from the peripheral blood of the same patient. The size of this deletion was about 4.8 Mb. This region contains IHH, NHEJ1, SLC2343, E2BP3, FAM134A, ZFAND28, ABCB6, ATG9A, MTABC, ANKZF1, UNQ229, GLB1L, STK16, MPSK1, TUBA4BDNAJB2, PTPRN, RESP18, DNPEP, DES, SPEG, GMPPA, ACCN4, CHPF, OBSL1, INHA, LIP1, STK11IPSLC4A3 genes.Figure 1Hemihypertrophy of the right half of the bodyFigure 2Nonosseous syndactyly of third and fourth fingers of the right handFigure 3Pigmentary mosaicism of right half of the bodyFigure 4X-ray of Right hand showing non-osseous syndactylyFigure 5Metaphase plates showing mosaic 46, XY, del q35 in the patientFigure 6G banding human karyogram 46, XY, del q35Figure 7Slide showing metaphase plate 6 to 10 with normal karyotyping (without del 2q35)Patient was managed conservatively. Plastic surgery referral was done for nonosseous syndactyly which they planned to correct it. There are other systemic features present in the child. There is no report of mosaic 2q35 deletion associated hemihypertrophy in literature; guarded prognosis was given to parents. The risk of reoccurrence could not be ascertained as parents refused to provide their samples for further testing.DiscussionPigmentary mosaicism (PM) is characterized by dyspigmentation (hyperpigmentation and hypopigmentation) of body parts along the lines of Blaschko. Two types of genetically different cell populations are derived from a single zygote. PM can further be classified along lines of Blaschko as narrow bands, broad bands, checkerboard, phylloid pattern, and patchy pattern. PM can be associated with two common syndromes: hypomelanosis of Ito and linear and whorled nevoid hypermelanosis. PM presents with both cutaneous and extracutaneous features. The PM is mainly somatic with postzygotic mutation and has been explained by various hypotheses earlier. Some are including co-migration of genetically different cell populations, functional Xchromosome mosaicism, spreading of X inactivation to autosomes in balanced X autosomal translocations, partial activation or silencing of pigmentary genes by transposons, genetic imprinting, and phenotypic reversion. Taibjee et al. have put forward the hypothesis that chromosomal abnormalities reported in PM disrupt the expression or function of pigmentary genes. Chromosomal abnormalities have been previously reported in the largest case series of PM (N-651 patients); 263 patients underwent cytogenetic analysis (40%). Forty-two percent (n = 111) of the patients showed abnormal cytogenetic results. Ninety-three patients (n = 93) showed a mosaic state and 18 patients showed a nonmosaic abnormality. Common chromosomes involved in above case series were 2,4,5,7,8,9,10,12,13,14,15,16,17,18,20,22, and sex chromosomes. Trisomy 2 mosaicism had been previously reported by two authors.Differential diagnoses of above condition are as follows: Klippel–Trenaunay syndrome (KTS), CLOVES Syndrome, and PIK3CA-related overgrowth syndrome (PROS). Clinical features of KTS include soft tissue overgrowth, congenital vascular malformation manifesting as port wine stain, varicose veins. The affected limb may be larger or longer. CLOVES Syndrome is an acronym that stands for congenital lipomatous overgrowth, vascular malformations, epidermal nevi, and scoliosis/skeletal/spinal anomalies. PROS is a group of disorders of overgrowth of body parts due to genetic defect in the PIK3CA gene. Specific disorders under this condition are CLOVES syndrome, Megalencephaly-capillary malformation syndrome (MCAP), hemihyperplasia-multiple lipomatosis syndrome (MCAP syndrome), facial infiltrating lipomatosis. Pigmentary defect along the lines of Blaschko, hemihypertrophy, and syndactyly prompted to do cytogenetic analysis from the affected part. Molecular defect mosaic 2q25 deletion was found to be a basic defect in the present case.PM is not an uncommon condition for pediatricians in their daily outpatient clinic. Hemihypertrophy or asymmetric overgrowth was reported in many case series. The reported frequency of asymmetric overgrowth was ranging from 7% to 100% in various reported case series. Genetic abnormalities associated with PM are variable. In a series of 76 pediatric patients, the frequency of chromosomal abnormality was 31%. The most common chromosomes involved are 12 and 22 in 19 patients having a mosaic chromosomal abnormality. Nonmosaic alternations were found in three cases and polyploidy in one case.In the present case, the chromosomal investigation showed a 46, XY, del q35. This karyotype abnormality was not found in peripheral blood. This chromosomal aberration was found in fibroblasts cultured from skin tissue. This was found in only 11% of cells examined. The size of this deletion lies between 4.5 and 5.5 MB. One of the important genes found in this deleted area is IHH (Indian Hedgehog Homolog). IHH gene-coded proteins play a role in bone growth and differentiation. Mutations in this gene are the cause of Brachydactyly type A1 which is characterized by malformations of toes and fingers. Chromosomal abnormality in the mosaic state is a commonly observed genetic aberration in the largest case series report on PM by Kromann et al. Similar case of hemihypertrophy, syndactyly, and PM was reported in a 6-year-old male child having a normal karyotype performed on peripheral blood sample, although no karyotype from fibroblast culture was mentioned (Srinivas et al. 2015). The present case and the one reported by Sriniwas et al. showed extra-cutaneous manifestation of asymmetry of limbs and nonosseous syndactyly of third and fourth fingers of the right hand. Extracutaneous manifestations are common in PM. These involve mainly neurological deficits, learning disability, epilepsy, hypotonia, spasticity, asymmetry of limbs, kyphoscoliosis, and syndactyly.The present case highlights the importance of analyzing karyotype from the affected areas. This way, there is a high chance of detecting chromosomal aberration in such cases. The 2q35 deletion in the mosaic state has not been previously reported. The mosaic 2q35 deletion from the affected areas significantly correlates with the clinical manifestations in the present case.Photo consentPhoto consent was taken from parents to publish it, following all due procedures.Declaration of patient consentThe authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.Ethical approvalEthical approval was taken.Financial support and sponsorshipNil.Conflicts of interestThere are no conflicts of interest.

PMC

Health Promotion Journal of Australia

36054639

PMC10091986

1-01-2023

10.1002/hpja.656

A pragmatic strength and conditioning intervention for firefighters: Feasibility of the Tactical Athlete Resilience Program (TARP)

Sharp Paul, Caperchione Cristina M., Brown Georgia A., Stadnyk Antony, Marin Elizabeth, Hulin Billy, Wade Jarrod, Mott Brendan, Gabriel Mark, Impellizzeri Franco, Fullagar Hugh H. K.

AbstractIssue AddressedFirefighting is physically and mentally taxing and recruits are expected to have optimal health and fitness. However, physical fitness tends to decline following initial training, placing firefighters at an increased risk for stress and injury. Efforts are needed to engage and support firefighters in maintaining adequate health and fitness to withstand the rigorous demands of their occupation. This study examined the feasibility of TARP, a pragmatic strength and conditioning intervention for metropolitan‐based firefighters, delivered in collaboration with a professional National Rugby League club.MethodsA mixed‐methods approach was utilised to examine program implementation, recruitment and sample characteristics, intervention satisfaction and acceptability, and participants' response to the intervention. Evaluation measures included field notes taken during steering committee meetings, participant flow data, baseline and follow‐up outcome measures, self‐report questionnaires, and telephone interviews with a sample of participants.ResultsParticipants (N = 113) were predominantly men (82%) with a mean age of 43 ± 9.3 years and BMI of 26.6 ± 2.9 kg/m2. Program satisfaction was high (95% very satisfied or somewhat satisfied) among program completers (42% retention). Key strengths of the program included delivery through the professional sports club, quality of facilities and equipment, and scheduling flexibility. Future programs should consider incorporating education or training to support behaviour change maintenance and strategies to retain participants at follow‐up.ConclusionsResults provide valuable insights into the design and delivery of interventions for firefighters and demonstrate the importance of strong partnerships between community stakeholders.

1INTRODUCTIONAustralian firefighters enter the workforce through a recruitment process bound by physical, tactical, and psychological parameters (eg, psychometric assessments, fitness and medical tests, as well as other merit‐based processes). Due to the physicality of entry‐level testing, firefighters typically begin employment with an adequate level of strength and endurance to meet the inherent requirements of firefighting. It is expected that they maintain good physical fitness in order to work in the dynamic and unpredictable environments that are commonly associated with the firefighter role. 1 , 2 However, research indicates that in the years following entry, the physical fitness of firefighters declines, impacting their abilities to respond to the high physical demands of firefighting and leaving them more susceptible to short and long term physical and psychological stress and injury. 3 , 4 , 5 Changes over time that may impact a firefighters' performance have been observed including decreased aerobic capacity, muscle strength, and endurance, all of which have been associated with increased cardiovascular disease risk and decreased job performance. 6 , 7 , 8 In addition, continual exposure to hazardous environments that cause psychological trauma can have long‐term detrimental effects on firefighters' mental health, emotional fatigue, employment burnout and post‐traumatic distress. 9 , 10 As such, there has been a call from researchers and fire agencies alike for increased action surrounding the potential benefits of health interventions and training programs which could encourage firefighters to maintain their health and fitness. 2 , 11 , 12 Moreover, it is important that these programs consider how to best empower individuals to increase their control over, and improve, their physical and mental well‐being.Finding innovative ways to promote, engage, and support the health and fitness of firefighters can be difficult for fire agencies alone, especially given the dynamic and unpredictable nature of the profession. Borrowing from health promotion research and practice, creating partnerships with relevant community stakeholders can improve engagement in health promotion programs and has the potential to positively impact physical and mental health outcomes. 13 , 14 , 15 For example, The Healthy Eating Activity and Lifestyle (HEAL 16 ) program is an Australian lifestyle modification program aimed at improving lifelong healthy eating and physical activity. It was designed and implemented by a collaboration of stakeholders from local government health districts, national health and exercise accreditation organisations, local government councils, and researchers from academic institutions. Process evaluation and feasibility results of the HEAL program indicated that partnerships were vital to the success of local implementation and are necessary to reach and engage community members in preventative health promotion programs. 13 Similarly, the Canadian Harmonization Program 17 was a collaborative effort amongst health professionals, cancer care professionals, academics and community members to advance cancer prevention approaches through smoking cessation, healthy eating and physical activity in rural communities. Like HEAL, they highlighted that these partnerships were integral to engaging and reaching community members. Moreover, they also reported the interventions utilised as part of the Harmonisation program had positive impacts on smoking and physical activity behaviours. 18 , 19 , 20 Although these programs are not specifically centred on firefighters, the learnings reported are highly transferable to programs with similar objectives (eg, improve program uptake and fitness related outcomes), and with a focus on stakeholder collaborations in program design, delivery, and evaluation.Guided by the success of previous stakeholder partnerships, Fire and Rescue New South Wales (FRNSW; Australia's busiest, and one of the world's largest, state‐level urban fire and rescue services), iCare (the workplace insurance and care services agency for New South Wales Government agencies), and the South Sydney Football Club (SSFC; a professional National Rugby League club in Sydney, Australia), collaborated to co‐design and develop a health and fitness program for firefighters. Similar stakeholder partnerships (ie, health agencies, community organisations, academic institutions, sporting teams, etc) aimed at developing and delivering health promotion programs have been highlighted in general populations 21 , 22 ; however utilising this approach with firefighters is a new endeavour.The Tactical Athlete Resilience Program (TARP) was developed as a pragmatic strength and conditioning intervention for metropolitan‐based firefighters aimed at improving physical and mental health. Delivered in collaboration with the SSFC, the 10‐session program was delivered on site in the club's training facilities and led by professional performance staff (eg, strength and training coaches, physiotherapists), utilising the high‐performance sport context as a draw to program participation. To accommodate firefighters varied schedules, participants were given the flexibility to choose from several available options each week and self‐nominate the frequency that they attended sessions (eg, weekly, bi‐weekly, varied). Up to six participants could register for each session, providing an opportunity for social interaction with other firefighters, often from other stations across the city. Each session lasted approximately 1.5 to 2 hours and included a warm‐up (ie, foam rolling, stretching, movement patterns), gym‐based exercises (eg, weights, stationary bike), and mobility training (indoors). SSFC coaches utilised participants' baseline measures to create an individually‐tailored experience and participants were encouraged to engage in additional, unsupervised sessions outside of the program. While previous initiatives for fire and rescue services have concentrated on physical health and fitness outcomes, 2 , 6 few studies have explored program feasibility. Understanding the processes of program implementation is necessary to inform future research and support program sustainability and dissemination. 23 Thus, the aim of this study was to determine the feasibility of the TARP related to program implementation, recruitment and sample characteristics, intervention satisfaction and acceptability, and participants' response to intervention.2METHODSProgram development, delivery, and evaluation were guided by the TARP steering committee, which included organisational stakeholders from FRNSW, SSFC, and iCare, and researchers from the University of Technology Sydney (UTS). The focus of the committee was to define the scope and objectives of the program, to monitor program progress, and provide a forum for feedback and joint decision making across stakeholders. All ethical procedures were approved by the institutional Human Research Ethics Committee (ETH19‐3632) and registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12619001116112).2.1Study designTARP was evaluated using a pre‐post quasi‐experimental design. Program feasibility was based on evaluation objectives identified by the TARP steering committee and guiding questions for health promotion programs using mixed methods. 24 In line with Orsmond & Cohn , feasibility objectives were operationalised as an evaluation of recruitment capability and sample characteristics, acceptability and suitability of intervention and study procedures, and preliminary evaluation of participant responses to intervention. Data were collected from participants at baseline and post intervention (ie, completion of 10 TARP sessions) including self‐reported questionnaires of health‐related behaviours and psychosocial outcomes, objective measures of health and fitness, and semi‐structured telephone interviews. Process data regarding recruitment, intervention fidelity, and attendance records were obtained from program facilitators.2.2Participants and recruitmentParticipants were firefighters (18‐65 years) from FRNSW platoons with no history of illness or injury that would contraindicate participation in a strength and conditioning program. Recruitment occurred between August 2019 to March 2020 and September 2020 to April 2021 and was facilitated by FRNSW using a combination of induction events at SSFC and informational emails to targeted stations and crews. Interested individuals were encouraged to contact a member from SSFC and the research team to determine eligibility, register participation, and schedule baseline measurements. To maintain participant confidentiality, FRNSW did not have access to recruitment records or data. All participants provided written informed consent and completed an exercise pre‐screening tool (PAR‐Q) prior to data collection.2.3Measurements2.3.1Self‐report questionnairesValid and reliable self‐report questionnaires were completed online by participants and emailed to research staff prior to baseline assessments. Psychological well‐being was assessed using the single item Cantril ladder. 25 Self‐esteem was assessed using the 10‐item Rosenberg self‐esteem questionnaire. 26 Psychological resilience was assessed using the abbreviated version (2‐item) of the Connors‐Davidson Resilience Scale. 27 Health related quality of life was assessed using the EuroQoL questionnaire. 28 Overall health was assessed using the EuroQol visual analogue scale. 28 Physical activity was assessed using two previously validated questions relating to the number of days participants engaged in physical activity over the past week and over the past month. 29 Post intervention, questionnaires were completed again at the commencement of the follow‐up measurement session and included additional Likert‐scale questions to assess program satisfaction and acceptability (eg, How likely are you to tell other firefighters about the TARP program?)2.3.2Health and fitness measuresParticipants completed a battery of health and fitness measures at baseline and post intervention to determine the feasibility of the assessment protocol, determine effectiveness of recruitment for engaging individuals that would benefit from participating, and to provide an indication of intervention effect. Body weight and height were measured without shoes or heavy clothing using an electronic flat scale to 2 decimal places. Resting systolic and diastolic blood pressure was measured on participant's nondominant arm with a blood pressure monitor (Omron 705‐CPII) after sitting at rest for 5 minutes. Grip strength was assessed by maximal isometric hand grip strength using a Harpenden Handheld Dynamometer. Participants completed three alternating attempts per hand and peak values were retained for analysis. The Knee‐to‐Wall (KTW) test was used to assess ankle mobility and the range of ankle dorsiflexion. 30 The Apley Test was used to assess shoulder mobility, particularly around the glenohumeral joint. 31 The Sit‐and‐Reach Test (SRT) was used to assess hamstring and lower back flexibility, 32 balance was assessed using the Y‐balance Test (YBT 33 ), whilst lower body power was assessed using the Standing Broad Jump (ie, farthest of 3 jumps recorded in centimetres). Upper body strength was assessed using the 3RM (3‐repetition maximum) bench press (kg). Aerobic capacity was assessed using the 3‐Minute Mean Maximal Power cycling test on a Wattbike Pro (Wattbike, Nottingham, UK) and average power output (Watts) was retained for analysis. All measures were collected on site at SSFC by trained performance staff and research assistants using standardised procedures, including a period of familiarisation prior to assessment. Further detail about the health and fitness measures and procedures are available with the TARP evaluation report. 34 2.3.3Follow‐up interviewsPost intervention, a sub‐sample of participants (50%; n = 24) were invited to participate in a one‐on‐one telephone interview to gain a deeper understanding of program satisfaction and acceptability. Participants invited to complete an interview were randomly selected following completion of the program. A semi‐structured interview guide was developed to explore participants' perceptions and experiences with program implementation, delivery, content, and recommendations for improvement (Appendix A). Interviews were audio recorded and lasted approximately 10 to 20 minutes.2.3.4Field notes and process dataField notes were taken during TARP steering committee meetings from a member of the research team and any deviations or interruptions in the intervention protocol were recorded. Intervention dose was collected from registration and attendance records kept by SSFC staff.2.4Data analysisQuantitative data analysis (ie, self‐reported questionnaires, health and fitness measures) was performed using IBM SPSS version 25. Descriptive data are reported as mean (SD) and/or percentages where appropriate. Independent t tests were conducted to identify potential differences between baseline measures of completers and non‐completers. Differences in outcomes from baseline to post intervention were tested using repeated measures ANOVAs and reported with 95% confidence intervals (CIs).Qualitative data (ie, telephone interviews, field notes) were transcribed and managed using Nvivo 12. For the purposes of feasibility testing, transcribed data were content analysed by one of the researchers (PS) and a trained research assistant (GB). A coding framework was developed inductively using low inference codes to enable a descriptive summary of the data. 35 All transcripts were independently coded by both researchers and regular checks on consistency of coding were conducted. Codes were reviewed for similarities and grouped under the following categories: recruitment and sample characteristics; program implementation and engagement; acceptability and satisfaction; and, participants' responses to intervention. According to best practices, 24 results from the quantitative analysis were integrated with the qualitative findings to provide a rich report of program feasibility.3RESULTSResults are presented under four topics as they relate to the feasibility of TARP: program implementation; recruitment and sample characteristics; intervention satisfaction and acceptability; and, participants' responses to the intervention.3.1Program implementationSignificant external factors that impacted program delivery included the 2019 to 2020 Australian bushfires, the COVID‐19 pandemic, and the 2020 flood events. Notably, public health orders implemented to control the spread of COVID‐19 caused a pause in program delivery between April and August 2020. Significant “in‐program” learnings and pivots were made, driven primarily through stakeholder collaboration across 5 steering committee meetings. Deviations in the registered trial protocol as a result of these disruptions include an extended recruitment and study period and modified recruitment target from 300 to 100 firefighters. All stakeholders (iCare, SSFC, UTS, FRNSW) agreed there was a clear commitment to adapt and see the program through, particularly through the work of key operational staff in all organisations, and that recruitment and delivery challenges were negotiated through strong community stakeholder collaborations.3.2Recruitment and sample characteristicsRecruitment efforts focused on firefighters within the Metro East and South Commands, 36 encompassing approximately 1449 firefighters within the target catchment area. A total of 129 participants registered for TARP, and 113 completed baseline assessments. Telephone interviews revealed that the most common method of recruitment was by word‐of‐mouth, and that knowing others that were participating in TARP, and/or hearing positive experiences from past participants, encouraged participants to join. Participants' motivations for program participation varied, yet most reported an interest in getting more active, increasing fitness, and/or improving exercise technique. Potential deterrents to program participation included concerns about the confidentiality of physical measurements and their potential impact on employment, as well as a general lack of confidence, interest, or experience with structured exercise programs.Participants (N = 113) were predominantly men (82%) and had a mean age of 43 ± 9.3 years, and BMI of 26.6 ± 2.9 kg/m2 (Table 1). Post intervention, 48 participants completed follow‐up measures (43% retention). Differences in baseline characteristics between program completers and non‐completers (ie, lost to follow‐up) were examined (Table 1). Non‐completers were younger (45.7 ± 9.2 vs. 39.5 ± 8.3 years; P = .002) and reported a lower initial overall health rating (80.9 ± 8.8 vs. 75.6 ± 12.8; P = .016). There were no other differences in participant characteristics or program outcomes at baseline between completers and non‐completers.TABLE 1Baseline characteristics of study participantsAll (N = 113)Completers (n = 48)Non‐completers (n = 65) P valueAge (years)43 (9.3)45.7 (9.2)39.5 (8.3) .002* Sex (M/F)93/2039/955/11—Height (cm)176.6 (7.6)175.1 (7.6)177.7 (7.3).072Body weight (kg)82.6 (12.3)80.9 (12.6)84.1 (12.0).234BMI (kg/m2)26.6 (2.9)26.5 (3.0)26.7 (2.8).693SBP (mm Hg)140 140 139 .720DBP (mm Hg)85 87 84 .178Grip strength R (kg)49 49 50 .649Grip strength L (kg)47 47 47 .946Perceived wellbeing6.0 (0.9)6.1 (0.9)5.9 (0.9).302Self‐esteem32.8 (4.2)33.1 (4.2)32.6 (4.3).557Psychological resilience6.5 (1.7)6.8 (1.5)6.3 (1.8).131Quality of life0.89 (0.12)0.90 (0.11)0.87 (.012).177Health rating77.7 (11.6)80.9 (8.8)75.6 (12.8) .016* Physical activity (days/wk)4.2 (2.0)4.6 (1.6)4.0 (2.2).199Physical activity (days/mo)17.2 (8.2)18.6 (6.9)16.2 (8.8).119Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; L, left; R, right; SBP, systolic blood pressure.*P < .05.3.3Intervention satisfaction and acceptabilitySatisfaction and acceptability were examined using process data (ie, attendance records), a self‐report questionnaire (n = 39), and telephone interviews (n = 24) with a subsample of participants. Throughout the 20‐month program period, registered participants collectively completed a total of 1,037 supervised sessions, with only 59 recorded failures to attend (5.4%). Participants completed an average of 7 ± 3 training sessions, including 71% who completed at least 5 sessions, 53% who completed at least 8 sessions, and 43% who completed all 10 sessions. Among program completers, 95% of participants reported performing additional offsite sessions throughout the program, exercising an average of 3.2 ± 1.6 sessions per week in addition to their participation in TARP. Median time to complete the 10 TARP sessions was 12.6 weeks (range = 7‐20 weeks). No adverse events were reported as a result of the intervention or testing procedures.Participants' self‐report questionnaire responses indicated a high level of satisfaction with TARP, with 82% reporting that they were very satisfied, 13% somewhat satisfied, and 5% neutral. None of the participants reported that they were dissatisfied with the program. Table 2 reports participants' likelihood to tell others about TARP and engage in future workshops or programs.TABLE 2Program satisfaction and training and lifestyle effects (n = 39)Very likely (%)Somewhat likely (%)Neutral (%)Somewhat unlikely (%)Very unlikely (%)Tell other firefighters about TARP878005If offered the opportunity, engage in future TARP workshops or programs878005Engage in exercise on a regular basis8510033Engage in exercise more often than prior to completing TARP41262383Engage in exercise with other firefighters or other people46318133Continually make conscious changes to your behaviour to promote a healthy lifestyle6426335Through the telephone interviews, participants described the program as a straightforward fitness program targeted at strength, cardiovascular endurance, and flexibility. Participants believed that the exercises were relevant to their work and could be tailored to various levels of fitness. Working together in small groups with other firefighters was considered a strength of the program. This social support was perceived to provide accountability, motivation, and comradery. The flexible delivery and number of available sessions were also considered to be a strength of the program. Additionally, most participants identified the benefit of living near to the training facilities and noted that it may be more challenging for colleagues who lived farther away to attend session. Measurement was important as participants appreciated tracking their progress (eg, weight lifted) and seeing changes over time. Finally, the fitness trainers were perceived to be highly knowledgeable, approachable, motivating, and engaging. Participants appreciated their reputable experience and qualifications working for SSFC. Commonly identified strengths and recommendations for program improvements are summarised in Table 3 with representative quotes.TABLE 3Strengths and recommendations for program improvementsStrengthsExample quote(s)Free to join The fact that it was free, a lot more people would do it. We were very lucky to get it for free. A very good initiative by the uni, souths, and the fire brigade. – Participant 4Knowledgeable/experienced trainers To have the NRL training staff, and to have access to all of their knowledge is unbelievable… getting more education about specific training was the key to staying motivated and seeing results. – Participant 10Partnership with SSFC The fact that it's a bit of a reputable program and high‐performance trainers and all that sort of stuff, so people like the idea of training with them. Separate from the employer and confidential. – Participant 13Easy to schedule training sessions They provided multiple times on multiple days and you could just fit it in around your rotating roster and your lifestyle. I thought that worked really well. – Participant 7Flexible delivery It was good that it was flexible. It wasn't that you have to attend these particular sessions. They offered sessions that you can slot into. – Participant 15Working together with other firefighters Meeting different people and training with different people from all different ranks, when you're sweating in the gym it doesn't matter if you're a commissioned officer or a firefighter. It was actually a really good social experience as well. – Participant 22Small group size I think the smaller classes worked well because all those people that hadn't been regularly training. Having the trainer there with less people to look at got more specific guidance where needed. Where you might lose that in the larger class size. – Participant 18Facilities and equipment It was quite interesting to see what they do there and how they apply their type of training techniques and to see the actual facilities, which were amazing. – Participant 4 Recommendations More personalisation and individualised feedback No one sat me down and told me “oh wow you have improved or you really need more work here…your deficiencies lie in this area and this is how it's going to affect you in the long run, so here are some tips on what to do.” – Participant 8Transition plan post‐program Admittedly I wasn't too familiar with the nutrition component but I know there's a lot of programs that we can access in terms of exercise and building towards harder training. If there's nutrition options too I'll certainly look into it. – Participant 1Follow‐up assessments Maybe a follow‐up assessment or something down the track just to see if you have maintained your levels…if you had an annual or six‐monthly test, it would give you inspiration to get back to those figures. – Participant 4Continuation of specialised programs There'd be a great opportunity to enhance the program as it is sort of an introduction and then look at having possibly a strength and conditioning program, cardiovascular program, even a mindfulness program so meditation or something like that. – Participant 173.4Participants' responses to the interventionData collected from questionnaires, telephone interviews, and health and fitness measures suggested that the intervention had potential to improve a range of study outcomes. During the telephone interviews, participants reported experiencing improvement in strength and flexibility as well as increased exercise frequency and confidence in performing movements properly. Participants also reported an increased knowledge of exercise technique and programming; however, some participants reported a lack of skills, knowledge or uncertainty in how to continue making lifestyle changes post‐program. Some participants reported that they had incorporated the techniques and exercises they had learned into their existing training regime and/or increased the amount of exercise they were engaged in. Although many of the participants reported that they regularly engaged in exercise prior to joining TARP, their participation refreshed or expanded their understanding of exercise programming and movements. This was further supported by questionnaire data, which indicated most participants were confident that they could continue to engage in physical activity and make healthy lifestyle changes (Table 2). Finally, the intervention showed potential to improve several health and fitness outcomes, with the greatest observed changes in self‐esteem (1.8, 95% CI = 0.8‐2.7, P < .001, d = 0.57), flexibility (2.3 cm, 95% CI = 1.3‐3.3, P = .001, d = 0.68), Y balance R (7.2%, 95% CI = 5‐9.5, P < .001, d = 0.99), Y balance L (7.9%, 95% CI = 5.6‐10.2, P < .001, d = 1.06), broad jump (9.3 cm, 95% CI = 5.9‐12.7, P < .001, d = 0.80), bench press (7.2 kg, 95% CI = 5.4‐9.1, P < .001, d = 1.17), and mean maximal power (25.6 W, 95% CI = 18.6‐32.7, P < .001, d = 1.06). Changes in health and fitness measures of program completers are reported in Table 4.TABLE 4Changes in health and fitness measures for program completers (n = 48)BaselinePost scoreDifferenceMean (SD)Mean (SD)Mean (95% CI) P value (ES)BMI (kg/m2)26.5 (3.0)26.5 (3.1)0 (−0.2, 0.1).892 (0.02)Body weight (kg)80.9 (12.6)80.8 (12.5)−0.1 (−0.5, 0.4).825 (0.04)Perceived wellbeing6.1 (0.9)6.5 (1.1)0.3 (0, 0.8) .029 (0.34)* Self‐esteem33.1 (4.2)34.8 (4.4)1.8 (0.8, 2.7) <.001 (0.57)* Psychological resilience6.8 (1.5)7.1 (1.1)0.1 (−0.2, 0.3).486 (0.10)Quality of life0.90 (0.11)0.91 (.10)0 (0, 0).999 (0.00)Health rating80.9 (8.8)83.9 (9.3)2.9 (1, 5.7) .041 (0.31)* Physical activity (d/wk)4.6 (1.6)4.6 (1.5).1 (−0.3, 0.5).614 (−0.08)Physical activity (d/mo)18.6 (6.9)19.3 (7.2)0.9 (−1.1, 2.8).368 (0.14)SBP (mm Hg)140 135 −5.4 (−10.0, −0.8) .022 (0.35)* DBP (mm Hg)87 84 −2.6 (−5.5, 0.3).080 (0.26)Grip strength R (kg)49 50 1.5 (−0.4, 3.3).116 (0.24)Grip strength L (kg)47 47 0 (−1.8, 1.8).995 (0.00)KTW R (cm)12.2 (3.2)12.8 (3.1).6 (0.1, 1.1) .020 (0.36)* KTW L (cm)12.2 (2.9)12.5 (2.9).3 (−0.1, 0.7).133 (0.23)Apley test R (cm)13.1 (7.7)12.2 (7.0)−.9 (−2.3, 0.4).167 (0.20)Apley test L (cm)16.4 (8.2)15.4 (9.1)−1.0 (−2.6, 0.5).184 (0.20)Sit and reach (cm)7.8 (12.3)10.1 (12.2)2.3, (1.3, 3.3) .001 (0.68)* Y balance R (%)84 91 7.2 (5, 9.5) <.001 (0.99)* Y balance L (%)84 92 7.9 (5.6, 10.2) <.001 (1.06)* Broad jump (cm)189 198 9.3 (5.9, 12.7) <.001 (0.80)* Bench press (kg)70 77 7.2 (5.4, 9.1) <.001 (1.17)* 3‐minute MMP cycling (W)254 280 25.6 (18.6, 32.7) <.001 (1.06)* Abbreviations: BMI, body mass index; CI, confidence intervals; DBP, diastolic blood pressure; KTW, knee‐to‐wall test; L, left; MMP, mean maximal power; R, right; SBP, systolic blood pressure. Note: Significantly different from baseline (*P < .05).4DISCUSSIONFirefighting is physically and mentally taxing and requires optimal health and fitness for safe and effective work – factors that tend to decline after recruitment. 37 Previous studies have demonstrated the potential for exercise interventions to improve outcomes of health and fitness among firefighters 2 , 6 ; however, less is known about the feasibility of implementing these interventions in the real world. Feasibility studies can provide valuable insight into the process of development and implementation of interventions and are needed to better address challenges related to engaging firefighters in health promotion programs (eg, shift system, varied schedules). The present study examined the feasibility of TARP, a pragmatic strength and conditioning intervention for metropolitan‐based firefighters delivered in collaboration with the SSFC. Our findings provide direction for program refinement as well as consideration for the design and delivery of interventions aimed at increasing the health and well‐being of firefighters.It is important for programs to engage participants that would benefit most from making healthy lifestyle changes, as poor health and fitness may impact firefighters' ability to respond to the high physical demands, leaving them more susceptible to physical injury. 3 In this regard, the characteristics of participants that underwent baseline testing suggests that recruitment was effective at engaging with firefighters that varied in age (23‐66 years) and weight status (20.3‐34.3 BMI kg/m2). The relatively high proportion of men (82%) is in line with that of the overall FRNSW firefighting staff (89% 38 ). Participants reported several strengths of the program that may have supported engagement, notably the high‐performance training environment and staff, schedule flexibility, and small peer‐group setting. Community collaborations in particular, such as the partnership with SSFC, have been shown to improve engagement with exercise interventions 39 and are valuable in recruiting and retaining participants. These qualitative assessments support the feasibility of the intervention and have been previously utilised in effective interventions for firefighters 6 as well as healthy adult populations. 40 For example, social support provided in group training from trainers and other exercise participants may increase program adherence, quality of life, and social interaction. 41 Program adherence (43%) was found to be lower than previous studies involving firefighters (50%‐83%). 6 Reasons for low participant retention may be related to program design and delivery and/or external factors such as program interruptions due to COVID‐19. In particular, the program was interrupted by two major environmental events (bushfires and floods) and the COVID‐19 pandemic. Specifically, the 2019 to 2020 Australian bushfires resulted in the largest number of deployed firefighters in Australian history, with over 5700 firefighters in New South Wales deployed between October 2019 and February 2020. 42 These program interruptions presented serval logistical challenges including cancelled training sessions and delays in individual program completions. Further, during the COVID‐19 pandemic, the program was paused for 6 months, which likely impacted participant recruitment, retention, and satisfaction. Given the high satisfaction rating (95% satisfied; 5% neutral) by participants that completed follow‐up measures, it is possible that adherence rates without these disruptions would have approached levels more in line with those reported in previous interventions. However, it can be reasonably anticipated that similar environmental or global events will continue to occur in the future, highlighting the importance of flexibility, resiliency, and creativity from researchers, stakeholders, and participants alike. In the present study, strong cross‐sectoral collaboration and community partnerships likely contributed to the continued delivery of the program following these disruptions. 43 However, additional strategies (eg, incentivisation) may be required to encourage more participants to complete follow‐up measures. Future research is needed to better understand the factors that contribute to low adherence and dropout.As a real‐world trial, the intervention delivery was adapted by facilitators and focused primarily on the delivering of a gym‐based exercise program. Although originally more behaviour change strategies and personalisation were planned, the delivery did not include this aspect. While this was not identified by the Steering Committee, this may be due to program interruptions, a lack of resourcing, or non‐engagement from participants. Introducing participants to a “toolbox” of behaviour change techniques (eg, setting and reviewing goals, action planning, self‐monitoring, and information about health and emotional consequences of change) may encourage internalised and self‐relevant motivation for the maintenance of health behaviours 44 and support program adherence and retention. Behaviour change theories (eg, Social Cognitive Theory 45 ) have been utilised previously in interventions targeted at firefighters 2 and may help to inform the implementation of these techniques. Given participants reported a lack of skills/knowledge or uncertainty in how to continue making lifestyle changes post‐program, these techniques would likely be beneficial in future programs. To support performance staff, additional training or guidance may also be needed to effectively incorporate these aspects of the program, as well as appropriate resourcing to effectively deliver it.While it was not an outcome of this feasibility study nor is it appropriate to assume intervention effectiveness, it is relevant to consider if the intervention shows promise of being successful with the intended population. For feasibility studies, it has been suggested that researchers use a combination of quantitative and qualitative methods to assess if an intervention shows promise of being successful with the intended population. 24 Results from participants' interviews, survey questions, and physical outcome measures suggest the potential for several desirable improvements in modifiable risk factors, performance, and self‐perception. Among program completers, participants demonstrated changes in blood pressure, unilateral leg balance, and hamstring/lower back and right ankle flexibility. Changes in upper‐body strength, lower‐body power, and maximal‐effort work capacity were also observed, aligning with previous exercise interventions in firefighters (see Andrews, Gallagher 6 for review). Furthermore, participants reported higher perceived wellbeing and self‐esteem, which highlights the potential for psychosocial benefits of training interventions. These improvements, if reproducible with an adequately‐powered effectiveness study, may translate to a reduced risk of cardiovascular disease, 7 , 12 , 46 musculoskeletal injury, 47 improved overall work performance, 48 and resilience to physiological and psychological stressors and fatigue encountered during firefighting duties. 47 Limitations of this research must be acknowledged. First, the large loss to follow‐up and lack of associated data (eg, motivators/barriers to participation, reasons for dropout) limit our ability to identify potential issues with the program's design and delivery and confidently determine feasibility and acceptability. While this may have introduced a source of attrition bias, post‐hoc analysis showed few differences in baseline measures between program completers and those lost to follow‐up. Regardless, future research may need to account for this loss by implementing strategies that encourage participants to complete follow‐up measures or allow greater flexibility in measurement schedules. Second, findings from the present study are drawn from a single urban location amid major national and international crises and may not be generalisable to other settings or contexts. These promising findings point to a need to further explore the potential for this type of program to improve firefighters' health and well‐being. Future research is needed to examine the effectiveness of TARP through a fully powered randomised control trial.5CONCLUSIONUnderstanding program feasibility is necessary to support the sustainability and dissemination of initiatives. The present study provides valuable insights into the design and delivery of interventions for firefighters. A strength of this intervention was the pragmatic delivery of a flexible training program to an active‐duty workforce through the collaborative partnership of community stakeholders. In particular, delivering interventions for firefighters in collaboration with a professional sports club offers unique opportunities for recruitment and allowed firefighters to participate in an independently delivered program with the support of their employer. The sustainability and scalability of TARP, as with similar interventions, is dependent on several factors including the availability of strategic partnerships as well as the extent to which stakeholders see value (eg, reducing injury claims) in maintaining these partnerships. Strong collaborations between stakeholders such as these are critical to the development of robust and sustainable interventions.FUNDING INFORMATIONFunding for this research was provided by FRNSW.CONFLICT OF INTERESTBilly Hulin and Jarrod Wade were employed by the South Sydney Rabbitohs Rugby League Football Club. Brendan Mott and Mark Gabriel were employed by Fire and Rescue NSW. Data collection, analysis, and interpretation were exclusively conducted by researchers at the University of Technology Sydney and these authors did not have access to participant data. All other authors declare no conflict of interest.ETHICS STATEMENTEthical approval for this research was provided by the University of Technology Sydney Human Research Ethics Committee (ETH19‐3632).PATIENT CONSENTAll participates agreed to the publication of research findings in scientific journals.Supporting information Appendix S1 Supporting informationClick here for additional data file.

PMC

Journal of Stroke

37813674

PMC10574304

9-01-2023

10.5853/jos.2023.01753

Association Between Slow Ventricular Response and Severe Stroke in Atrial Fibrillation-Related Cardioembolic Stroke

Ha Sang Hee, Jeong Soo, Park Jae Young, Yang So Young, Cha Myung-Jin, Cho Min-soo, Chang Jun Young, Kang Dong-Wha, Kwon Sun U., Kim Bum Joon

Dear Sir:Atrial fibrillation (AF)-related strokes usually have a higher initial stroke severity and frequently lead to severe disability and mortality [1,2]. Stroke recurrence was not found to be linked to heart rate (HR); however, there was an association between HR and mortality in patients with AF-related stroke . While AF typically presents with tachycardia, a slow ventricular response (SVR) is also observed, albeit less frequently.Hemodynamic changes associated with AF have been studied, and AF with SVR may lead to intracardiac hemodynamic alterations, thrombus formation, and hypoperfusion in the ischemic area . However, the association between SVR and initial stroke severity, early neurological deterioration, and functional outcome in patients with AF-related stroke is not known.We retrospectively reviewed the data of patients who had acute AF-related stroke (within 7 days of stroke onset) and were admitted to Asan Medical Center between January 2017 and March 2022. We included patients who fulfilled the following criteria: cardioembolic stroke, known or newly diagnosed AF, and relevant acute ischemic lesions on diffusion-weighted imaging (DWI). We excluded patients who had AF with rapid ventricular rate in the initial twelve-lead electrocardiogram (ECG), poor initial magnetic resonance imaging (MRI) quality, incomplete clinical data, presence of stroke mechanisms other than cardioembolism, and presence of low temperature or electrolyte abnormalities that may systematically reduce the HR. The study protocol was approved by the Institutional Review Board Committee of Asan Medical Center (IRB number: 2022-1178) and informed consent was waived because of the retrospective nature of the study. Demographic data and risk factors were obtained by reviewing the medical records. Neurological deficits at admission were evaluated using the National Institutes of Health Stroke Scale (NIHSS) score, and severe stroke was defined as patients whose NIHSS score at admission was >15 points . All patients underwent neurovascular MRI with a 3.0T Philips scanner (Philips Healthcare, Eindhoven, The Netherlands) within 24 hours of admission. We also used the Olea Sphere® imaging system (Olea Medical SAS, La Ciotat, France) for automatic post-processing and measurement of the DWI lesion volumes.ECGs were obtained from the emergency department after >5 minutes of rest in the supine position. Paroxysmal AF was defined as the spontaneous restoration of normal sinus rhythm within 7 days, and persistent AF was defined as AF lasting >7 days. In terms of ventricular response, patients with an HR <60 beats per minute on an initial ECG were considered to have SVR . Diagnosis of AF and transthoracic echocardiography were performed during admission by an experienced cardiologist and ejection fraction (EF) and left atrium (LA) diameters were measured.The baseline characteristics were compared according to the presence of SVR. The chi-square test, Fisher’s exact test, Student’s t-test, or Mann–Whitney U test were used as indicated. Univariate and multivariate analyses were performed to identify the factors associated with severe stroke. According to the results of the univariate analyses, age, male sex, and variables yielding P values <0.10 were included in the multivariate analysis. IBM SPSS Statistics for Windows, version 21.0 (IBM Corp., Armonk, NY, USA) was used for all analyses, and P<0.05 was considered statistically significant.A total of 496 patients (mean age, 73±11 years; male, 53.7%) were included in this study; 31 (6.2%) had SVR, and 109 (22.0%) had severe stroke. There were no significant differences in the demographics or vascular risk factors according to the presence of SVR. However, patients with SVR had higher initial NIHSS scores and larger DWI lesion volumes than those without SVR. Patients with SVR had larger LA diameters and were more likely to have persistent AF and severe stroke than those without SVR (Table 1).Univariate analysis showed that older age, diabetes mellitus, lack of prestroke antiplatelet use, newly diagnosed AF, and SVR were associated with severe stroke. In the multivariate analysis, older age (adjusted odds ratio [aOR]=1.03, 95% confidence interval [CI] 1.01–1.05; P=0.008), diabetes mellitus (aOR=1.66, 95% CI 1.04–2.64; P=0.034), no prestroke antiplatelet use (reference= none; aOR=0.50, 95% CI 0.28–0.90; P=0.020), and SVR (aOR=2.30, 95% CI 1.05–5.04; P=0.038) were independently associated with severe stroke (Table 2).In this study, 6.2% of patients with AF-related cardioembolic stroke had SVR at presentation. We found that SVR was associated with severe stroke. The mechanisms underlying our findings remain unclear. One possible mechanism is that SVR leads to prolonged left ventricular diastolic filling, elevated LA pressure, and deterioration of left atrial appendage contractility, which, in turn, leads to blood stasis and large thrombus formation . We also found that LA size was significantly larger in patients with SVR than in those without SVR. An enlarged LA is an independent risk factor for thromboembolism in patients with AF . Furthermore, most patients with SVR have persistent AF, which can lead to decreased flow velocity in the LA through structural remodeling and endocardial fibroelastosis, thereby increasing the risk of large thrombus development .In patients with AF-related cardioembolic stroke, both low and high HRs during the acute stage were associated with an increased risk of mortality. The potential reason for this association is decreased heart function, which could result from either a low or high HR [3,9]. However, our results showed that the EF was similar between those with and without SVR. Instead, the high proportion of patients with severe stroke may at least partially explain the association between high mortality and low HR in patients with AF-related stroke. Nevertheless, data on the optimal HR for patients with acute AF-related stroke are insufficient. This study focused on the association between SVR and severe stroke. Thus, future studies focusing on the effect of rate control on stroke severity are needed.This study had several limitations. First, this study was performed at a single center and included a small number of patients. Second, due to the retrospective study design, we were unable to obtain information on the sustained presence of SVR prior to stroke onset. Despite these limitations, we found that SVR was strongly associated with initial stroke severity in patients with AF-related cardioembolic stroke.

PMC

Molecular Ecology Resources

35980602

PMC10087395

1-01-2023

10.1111/1755-0998.13702

Genomic investigation of the Chinese alligator reveals wild‐extinct genetic diversity and genomic consequences of their continuous decline

Yang Shangchen, Lan Tianming, Zhang Yi, Wang Qing, Li Haimeng, Dussex Nicolas, Sahu Sunil Kumar, Shi Minhui, Hu Mengyuan, Zhu Yixin, Cao Jun, Liu Lirong, Lin Jianqing, Wan Qiu‐Hong, Liu Huan, Fang Sheng‐Guo

AbstractCritically endangered species are usually restricted to small and isolated populations. High inbreeding without gene flow among populations further aggravates their threatened condition and reduces the likelihood of their long‐term survival. Chinese alligator (Alligator sinensis) is one of the most endangered crocodiles in the world and has experienced a continuous decline over the past c. 1 million years. In order to identify the genetic status of the remaining populations and aid conservation efforts, we assembled the first high‐quality chromosome‐level genome of Chinese alligator and explored the genomic characteristics of three extant breeding populations. Our analyses revealed the existence of at least three genetically distinct populations, comprising two breeding populations in China (Changxing and Xuancheng) and one breeding population in an American wildlife refuge. The American population does not belong to the last two populations of its native range (Xuancheng and Changxing), thus representing genetic diversity extinct in the wild and provides future opportunities for genetic rescue. Moreover, the effective population size of these three populations has been continuously declining over the past 20 ka. Consistent with this decline, the species shows extremely low genetic diversity, a large proportion of long runs of homozygous fragments, and mutational load across the genome. Finally, to provide genomic insights for future breeding management and conservation, we assessed the feasibility of mixing extant populations based on the likelihood of introducing new deleterious alleles and signatures of local adaptation. Overall, this study provides a valuable genomic resource and important genomic insights into the ecology, evolution, and conservation of critically endangered alligators.

1INTRODUCTIONA large proportion of earth biodiversity is severely impacted by human activities and endangered species are forced to survive in small and isolated populations (Haddad et al., 2015). Indian tiger and Scandinavian wolf (Kardos et al., 2018) are representative examples threatened with local extinction. These populations usually suffer from reduced genetic diversity and severe inbreeding, leading to a reduced potential to adapt to environmental changes (Kardos et al., 2018; Khan et al., 2021). Moreover, inbreeding will lead to the exposure of deleterious alleles in homozygous state, thus reducing the survival of individuals via inbreeding depression. Assisted gene flow via translocations from other populations is a promising way to maintain and/or recover small populations and thus induce a genetic rescue effect (Foote et al., 2019). For example, translocations of the puma subspecies (Puma concolor) successfully improved the survival and fitness of the receiving population (Saremi et al., 2019).It is worth noting that assisted gene flow poses a potential risk of outbreeding depression when mixing long‐term isolated populations. Because isolated populations may have been exposed to different evolutionary pressure and have fixed different adaptive alleles, the combination of distinct haplotypes may interfere with the interaction between genes (Frankham, 2005; Kelle & Waller, 2002). Moreover, there is a likelihood of introducing new deleterious alleles that would increase the mutational load of the receiving population (Kyriazis et al., 2020; Robinson et al., 2019). Scientific management and conservation require a full understanding of the genetic status of all populations of a target species (Hedrick & Garcia‐Dorado, 2016).The Chinese alligator is one of the species on the brink of extinction and is currently listed as “critically endangered” on the IUCN Red List (Jiang & Wu, 2018). It belongs to an ancient reptilian lineage that has survived since the Mesozoic, and once inhabited a large area of wetlands, marshes and ponds of the lower Yangtze River (Thorbjarnarson et al., 2002). However, this species was restricted to the border area of Anhui, Jiangsu and Zhejiang Provinces by the 1900s. At present, no more than 100 mature Chinese alligators remain in the wild, with a fragmented distribution in Xuancheng, Jingxian, Guangde, Nanling, and Longxi, five narrow regions in Anhui Province (Jiang & Wu, 2018). According to a census data, their age structure is unbalanced and egg laying performance has declined in the wild populations (Ding et al., 2001).Conservation and breeding projects were carried out at the Anhui Research Centre of Chinese Alligator Reproduction (hereafter Anhui Centre, acronym XC) and Changxing Yinjiabian Chinese Alligator Nature Reserve (hereafter Changxing Centre, acronym CX in 1979, based on 212 and 11 wild founders, respectively (Wu et al., 2002). In addition, breeding programmes were also implemented at the St. Augustine Alligator Farm, Bronx Zoo, and the Rockefeller Refuge in America (acronym ACA) (Ross et al., 1998). Although the overall population has been increasing, their genetic diversity was reported to be very low (Wan et al., 2013; Wu et al., 2002; Zhai et al., 2017) and inbreeding within each population seemed unavoidable (Wu et al., 2006). Indications of inbreeding depression have both emerged in Anhui and Changxing Centres, characterized by reduced reproductive ability and physical deformities in offspring (Wu et al., 1999, 2006). Translocation programs have been carried out to help increase the fitness of the small CX population. To weigh the pros and cons, it is essential to examine the genomic background of extant populations, which would aid in managing measures for the conservation of Chinese Alligators.Here, we assembled and annotated the first chromosome‐scale genome for Chinese alligator, and sequenced 23 samples from three isolated populations. To clarify the genetic relationship and survival potential of different populations, we investigated population structure, demographic history, genetic diversity, inbreeding status, mutational load and local adaptation. This study will provide a valuable genomic resource and important genomic insights into the ecology, evolution, and conservation of this critically endangered species.2MATERIALS AND METHODS2.1Samples collection and ethics statementThe blood sample was collected from an adult Chinese alligator reared at Changxing Centre, China. The blood sample was divided into four tubes with 2.5 ml for each tube for PacBio long‐read sequencing, Illumina short‐read sequencing, RNA‐seq sequencing, and Hi‐C sequencing. We also collected blood or umbilical cord samples (2 ml for each individual) from 23 wild or semi‐wild individuals for resequencing, including eight individuals from CX (including a wild founder “CX1”), nine individuals from XC and six individuals from ACA (Figure 2a, Table S1). Research and sample collection were both approved by the Animal Ethics Committee of Zhejiang University (ZJU20210267) and the Institutional Review Board of BGI (BGI‐IRB E22002).2.2 DNA and RNA extraction, library construction and genome sequencingHigh molecular weight genomic DNA was extracted from blood samples using the DNeasy Blood and Tissue kit (Qiagen) for PacBio sequencing, with an average insert size of 20 kb. SMRTbell libraries were constructed using SMRTbell Template Pre‐Kits (Pacific Biosciences), according to the manufacturer's instructions. Genomic DNA used for resequencing and genome analyses was extracted using a phenol‐chloroform protocol together with ethanol precipitation (Sambrook et al., 1989). DNA libraries with short insert sizes were constructed according to the manufacturer's instructions of the Illumina sequencing platform. For Hi‐C sequencing, we first performed cross‐link process with formaldehyde for the blood sample, and then the Hi‐C library was constructed according to the procedures of Lieberman‐Aiden et al. . Total RNA was isolated using TRlzol reagent (Invitrogen), and Agilent 2100 Bioanalyser system (Agilent) and Qubit 3.0 (Life Technologies) were used for RNA quantity, integrity, and purity evaluation. DNA libraries of RNA‐seq, whole‐genome resequencing and genome analyses were all sequenced on the Illumina HiSeq X 10 system (Illumina) (Tables S1 and S2).2.3De novo assembly and assessmentGenome size and heterozygosity of the Chinese alligator were estimated by k‐mer frequency method (Lander & Waterman, 1988). We first assembled an initial genome with error‐corrected PacBio long reads based on the Overlap‐Layout‐Consensus algorithm (Li et al., 2011). Daligner in Falcon (version 0.5) software (Chin et al., 2016) was used to map all PacBio reads to the longest single‐pass reads and then LASort, LAMerge and pbdacgon were used to generate consensus of mapped reads. Contigs were polished using Quiver (version 2.3.1) consensus‐calling algorithm (Chin et al., 2013) with PacBio long reads. Contigs were further corrected by the Pilon (version 1.18) (Walker et al., 2014) software with 276.83 Gb Illumina short reads. Hi‐C reads were first filtered using the program “filter_data_parallel” in the SOAPdenovo2 package (r240) (Luo et al., 2012) before genome mapping. We then mapped Hi‐C clean reads to the draft assembly using Burrows‐Wheeler aligner mem (BWA, version 0.7.17) (Li & Durbin, 2010) with default parameters. Finally, 3d‐DNA pipeline (version 180,922) (Durand et al., 2016) was used to concatenate the contigs to the chromosome‐level genome. All Illumina short reads were remapped to the final assembly for error‐correcting of the misassembled bases. The completeness of the genome was evaluated by BUSCO analysis using the vertebrata_odb10 database. We also mapped the Illumina short reads, RNA‐seq data and Hi‐C data to our assembled genome by BWA software with default parameters.2.4Genome annotationDe novo prediction and homology‐based method were both used for repetitive elements annotation in our Chinese alligator genome. De novo prediction was performed using RepeatModeler2 (version 1.0.9) (Flynn et al., 2020) with default parameters. De novo predicted repetitive elements were then added into the RepBase as known repeats. RepeatMasker (version 4.1.1) (Tarailo‐Graovac & Chen, 2009) was finally carried out by searching in RepBase library (Jurka et al., 2005) for identifying and classifying transposable elements. Tandem Repeats Finder (TRF version 4.09) (Benson, 1999) was also used to identify tandem repeats.Gene annotation was performed based on repeat‐masked genome. Gene prediction was carried out following the procedure described here (Wang et al., 2017). Briefly, de novo gene prediction, RNA‐seq method and homologous proteins alignment were used to annotate protein‐coding genes for our genome with Maker (version 2.31.11) (Campbell et al., 2014). We used SNAP (version 1.0) (Korf, 2004), Genescan (version 1.0) (Burge & Karlin, 1997), glimmerHMM (version 3.0.3) (Majoros et al., 2004) and AUGUSTUS (version 2.5.5) (Keller et al., 2011) for de novo gene prediction to identify protein‐coding genes in our assembled genome. We collected protein sequences from Anolis carolinensis, Alligator mississippiensis, A. sinensis (GenBank ID: GCF_000455745.1), Crocodylus porosus, Gavialis gangeticus, Gallus gallus, Homo sapiens, Meleagris gallopavo, Taeniopygia guttata and Xenopus tropicalis in the NCBI database for homology‐based predictions. GeneWise (version 2.2.0) (Birney et al., 2004) was used for gene model prediction. Transcripts after filtering and assembling by Trimmomatic (version 0.27) (Bolger et al., 2014) and Trinity (version 2.9.0) (Haas et al., 2013) were aligned to our genome by program to assemble spliced alignments (PASA) (version 2.2.0) (Haas et al., 2008) to predict gene structures. The final consensus gene set was obtained by combining the above‐mentioned three gene sets by Maker (version 2.31.11) (Campbell et al., 2014). All protein‐coding genes were aligned to databases of InterPro (Apweiler et al., 2001), KEGG (Kanehisa & Goto, 2000), Swiss‐Prot and GO for the functional annotation.2.5Syntenic analysisTo examine the synteny between the GCF_000455745.1 genome and our assembly, we firstly performed the whole‐genome alignment using LAST (version 973) (Kielbasa et al., 2011) software with the following parameters: lastdb ‐uNEAR ‐cR11; lastal ‐P16 ‐m100 ‐E0.05; last‐split ‐m1. Synteny blocks from the GCF_000455745.1, which were aligned to the same chromosome in our assembly, were firstly sorted together and then visualized using Circos (version 0.69–9) (Krzywinski et al., 2009) software.2.6Divergence time estimationWe first performed protein alignment of 15 species (A. sinensis, A. carolinensis, A. mississippiensis, C. porosus, G. gangeticus, G. gallus, H. sapiens, M. gallopavo, T. guttata, X. tropicalis, Ophiophagus hannah (GCA_000516915.1), Gekko japonicus (GCA_001447785.1), Pelodiscus sinensis (GCA_000230535.1), Chrysemys picta (GCA_000241765.2) and Chelonia mydas (GCA_000344595.1)) by blastp in BLASTtools (version 2.2.26) (Altschul et al., 1990) with default parameters and clustered by Orthomcl (version 1.4) (Li et al., 2003) with the inflation parameter of 1.5. A total of 2595 single‐copy genes shared by all species were identified and then used to build a phylogenetic tree by IQTREE (version 1.6.12) (Lam‐Tung et al., 2015) with the maximum‐likelihood method. We used the MCMCTREE (version 4.5) in the PAML software (Yang, 2007) to estimate the divergence time among species with the parameter “burnin = 1000, sample‐number = 1000,000, sample‐frequency = 2”. Multiple fossil time points were used for time calibrations from Timetree ( (Table S3).2.7Read mapping and variant callingRaw sequencing reads from the 23 Chinese alligator individuals were filtered with Trimmomatic (version 0.27) (Bolger et al., 2014). Low‐quality reads, reads with more than 10% “Ns” and reads with adaptor sequences were filtered out. Clean reads were mapped to our improved assembly with BWA mem with default parameters and one bam file was generated for each individual. SAMtools (version 1.3) was then used for sorting, indexing, and removing duplicates from bam files. For variant calling, we first used Samtools to generate a raw variant call format (vcf) file with the strict “mpileup ‐q 1 ‐C 50 ‐t SP ‐t DP ‐m 2 ‐F 0.002” (Li et al., 2009) to conduct BQSR using the vcf file as reference variants. Next, the genome variant call format (gvcf) file for each individual was generated by using the Genome Analysis Toolkit (GATK version 4.0.3.0) (Depristo et al., 2011) with the function of HaplotypeCaller. Joint calling was then performed to generate the combined VCF file. Hard filtering was applied to get the single‐nucleotide polymorphism (SNP) sites set with “QUAL 60.0 || SOR > 3.0 || HaplotypeScore > 13.0 || MQRankSum < −12.5 || ReadPosRankSum < −8.0”. We also filtered out biallelic SNPs with the highest and lowest 0.25% depth. In the population structure analysis, we only maintained the loci with a missing ratio less than 10% and minor allele frequency (MAF) larger than 0.05 and finally avoided the bias caused by linkage disequilibrium (LD) by Plink (version 1.9) (Chang et al., 2015) with the parameter “‐‐indep‐pairwise 10 kb 1 0.5”.2.8Population structure analysisPairwise Weir and Cockerham's F ST (Weir & Cockerham, 1984) for the three populations were calculated using VCFtools (version 0.1.16) (Danecek et al., 2011). VCFtools was used to convert VCF files to plink format files to conduct principal component analysis (PCA) with Plink (version 1.9). We used the program ADMIXTURE (version 1.3.0) (Alexander et al., 2009) to infer genetic clusters representing distinct ancestral components. Values of 1–5 were run with “–cv” flag to compute the cross‐validation error and to infer the most likely value of K. To analyse the phylogenetic relationship, we first aligned A. mississippiensis genome against our assembly using the LAST (version 973) software with following parameters: “lastdb ‐uNEAR ‐cR11; lastal ‐P16 ‐m100 ‐E0.05; last‐split ‐m1” to identify conserved regions between the two alligator genomes. Then the alleles of A. mississippiensis were added to the vcf file of Chinese alligators and finally kept 312,525 SNPs in these conserved regions. A phylogenetic tree was then constructed with A. mississippiensis serving as the outgroup using IQTREE (version 1.6.12) with 1000 bootstraps. The tree layout was generated using the online tool iTOL ( We then performed F3 statistics using qp3Pop implemented in ADMIXTOOLS (version 5.1) (Patterson et al., 2012) to test if one population was an admixed population of the other two. LD was calculated on SNP pairs within a 1000‐kb window using PopLDdecay (version 3.40) (Zhang, Dong, et al., 2018).2.9Demographic inference and population divergenceWe combined multiple sequentially Markovian coalescent (MSMC) and approximate Bayesian computation (ABC) methods to track fluctuations in effective population size (N e) from 10 ka BP to the present day for the three populations, considering the different limitations and resolutions of each approach. Firstly, SNPs were phased by BEAGLE (version 5.0) (Browning et al., 2018) with default parameters. We randomly selected four individuals from each population and masked uncovered regions with bamCaller.py for them. MSMC (Schiffels & Durbin, 2014) was then run with following parameters: ‐R ‐i 20 ‐t 6 ‐p ‘10*1 + 15*2’. The final result was visualized with a generation time of 20 years and the mutation rate of 7.9*10−9 substitutions per site per generation (Green et al., 2014). To further infer the most recent population history of Chinese alligator, we only used SNPs with a MAF > 0.2 in the VCF file to run PopSizeABC (version 2.1) (Boitard et al., 2016) with following parameters: mac (minor allele count threshold for AFS and IBS statistics computation) = 0; mac_ld (minor allele count threshold for LD statistics computation) equals 2,3,4 respectively; L (size of each segment, in bp) = 4,000,000; nb_rep (number of simulated data sets) = 500; nb_seg (number of independent segments in each data set) = 30.Population divergence was inferred using four randomly selected samples from each population by MSMC2 (version 2.1.2) (Schiffels & Durbin, 2014) with following parameters: ‐‐skipAmbiguous ‐I 0–8,0‐9,0‐10,0‐11,0‐12,0‐13,0‐14,0‐15,1‐8,1‐9,1‐10,1‐11,1‐12,1‐13,1‐14,1‐15,2‐8,2‐9,2‐10,2‐11,2‐12,2‐13,2‐14,2‐15,3‐8,3‐9,3‐10,3‐11,3‐12,3‐13,3‐14,3‐15,4‐8,4‐9,4‐10,4‐11,4‐12,4‐13,4‐14,4‐15,5‐8,5‐9,5‐10,5‐11,5‐12,5‐13,5‐14,5‐15,6‐8,6‐9,6‐10,6‐11,6‐12,6‐13,6‐14,6‐15,7‐8,7‐9,7‐10,7‐11,7‐12,7‐13,7‐14,7–15 ‐i 20 ‐t 6 ‐p ‘28*1 + 1*2’. When the relative cross‐coalescent rate (RCCR) dropped to 0.5, the split between pairwise populations was estimated to occur at corresponding time point.2.10Gene flowWe performed a D‐statistics test (A, B; X, Y) by using qpDstat in ADMIXTOOLS, where we set A. mississippiensis as Y, XC as X, CX and ACA as A and B, respectively. TreeMix (version 1.13) (Pickrell & Pritchard, 2012) was then used to infer models of population split and migration between different populations by setting A. mississippiensis as an outgroup with the parameter “‐m 1‐5 ‐k 1000 ‐root AM”. Identity by descent (IBD) analysis was performed in refined‐ibd software (16May19.ad5.jar) (Browning & Browning, 2013) with default parameters. Gene flow within and among populations was then estimated by the pairwise IBD fragments from different generations (g). We inferred generations with the equation l = 100/(2 g), where l was the length of IBD in cM (Thompson, 2013). The estimated recombination rate (1 Mb = 0.89 cM) was calculated by dividing the overall recombination rate map length by whole genome size (Stapley et al., 2017) from a genetic linkage map of a female C. porosus based on microsatellite markers (Miles et al., 2009). We assumed that the recombination rate was conserved between Chinese alligator and C. porosus, considering that crocodilian families had similar karyotype, genome size and evolutionary rate.2.11Genetic diversityWe quantified genetic diversity by estimating genome‐wide heterozygous rate (H) and nucleotide diversity (π). Whole‐genome H of each sample was by calculated dividing the total number of heterozygous SNPs by successfully assembled autosomal genome size (Cho et al., 2013). H of genic, intron, exon, CDS and UTR regions were also calculated. Comparison of different populations and genomic regions were conducted using two‐sided pairwise t‐test in R (version 4.1.2) (R Development Core Team, 2012). π was estimated in 5‐Mbp windows (Feng et al., 2019) across all autosomes by VCFtools (version 0.1.16) (Danecek et al., 2011). GO enrichment analyses were performed on the diversity hotspot regions with top 20 π values in R by using the package “clusterProfiler” (Wu et al., 2021; Yu et al., 2012). Each significantly enriched category included at least two genes, and the hypergeometric test was used to estimate significance (p 150, the mutation can be designated as deleterious (Li et al., 1984). The ratio of homozygous to homozygous and heterozygous derived alleles and the number of these variants were then compared using two‐sided pairwise t‐test in R (version 4.1.2) (R Development Core Team, 2012). The contribution of inbreeding in the accumulation of homozygous missense mutations (Rnhom) was calculated by dividing the ratio of missense to synonymous counts of homozygous derived alleles inside ROH by the corresponding ratio outside ROH (Wang et al., 2021).We predicted the risk of assisted gene flow (i.e., assuming successful post‐translocation mating) by counting newly introduced deleterious alleles as in rhinoceros populations (von Seth et al., 2021). We showed the number of unique homozygous LOF and dnsSNP in each individual selected for translocation, while the mutations were absent in all individuals of the receiving population. We also performed a statistic on shared and unique nonsynonymous mutations for all pairwise individuals within and between populations.2.14Evidence for recent positive selectionWe identified all candidate SNPs under recently positive selection in each population by applying the integrated haplotype score (iHS, version 1.3) (Voight et al., 2006) with the major allele in three populations as the ancestral state. The iHS values were normalized by subtracting the genome‐wide mean iHS and dividing by the standard deviation (whole‐genome homozygosity analysis and mapping machina [WHAMM], Sites with an iHS score above or below the threshold (top or bottom 1%) were considered as candidate ancestral or derived mutations under positive selection. We used four approaches to identify genes in these candidate regions: sliding 100‐kb windows by 50‐kb step on the whole genome and summing up iHS scores of candidate SNPs in each window. Genes intersecting with these windows were sorted by the iHS score; Genes were selected in a 5‐kb flanking region around each candidate mutation; Nonoverlapping 50‐SNP windows were used to select genes; Each gene with candidate mutations was used to count iHS score. Finally, genes detected by all four methods were considered under recently positive selection.The population branch statistic (PBS) was then performed to investigate the recent selective effect (Yi et al., 2010) in each population. We estimated F ST for each gene between pairwise populations in VCFtools and used 0.999999 to replace 1 to avoid infinite PBS value. The divergence specific to the branch for each gene was calculated by the following formulas: t12=−log1−FST12 t13=−log1−FST13 t23=−log1−FST23 pbs=t12+t13−t23/2 All genes with a PBS value larger than the 99.8th quartile of the distribution of the PBS values were reported.3RESULTS3.1Improved de novo assembly of the Chinese alligatorWe assembled a chromosome‐scale genome of the Chinese alligator with high quality, contiguity, and accuracy by using a combination of Illumina short reads (135.65 Gb, 120‐fold), PacBio long reads (230.03 Gb, 100‐fold) and Hi‐C reads (309.80 Gb, 135‐fold) (Table S2). Genome size was estimated to be 2.42 Gb with a heterozygosity rate of 0.07% by calculating the frequency of 17‐mer using 135.65 Gb Illumina short reads (Figure S1 and Table S4). The total length of our assembly was 2.30 Gb, accounting for 95% of the estimated genome size. The contig N50 and scaffold N50 were 22.53 Mb and 219.05 Mb, respectively (Tables 1 and S5). The number of chromosomes of Chinese alligator is reported to be 2n = 32 (Zeng et al., 2011). Here, scaffolds totaling 2.26 Gb (98.26% of our assembly) were anchored into 16 chromosomes (length: 32 to 307 Mb) (Figure 1a) consistent with the karyotype study, indicating the assemble accuracy of our genome on the chromosome‐scale. BUSCO analysis showed that 96.6% of 2586 BUSCO genes (database: vertebrata_odb10) were identified, with 95.9% single and 0.7% duplicated copy. The remaining 2.4% and 1.0% were fragmented and missing. The GC content of this genome was 44.93% (Figure S2), which is very close to three other crocodiles (A. mississippiensis: 44.3%, GenBank ID: GCA_000281125.4; C. porosus: 43.85%, GenBank ID: GCA_000768395.2; G. gangeticus: 44.95%, GenBank ID: GCA_001723915.1) (Green et al., 2014). At last, 99.24, 99.47 and 91.70% of the Illumina short reads, Hi‐C reads and RNA‐seq data were successfully mapped onto our assembled genome, respectively.TABLE 1Comparison of assembly statistics between the assembly in this study and the previous version (GenBank ID: GCF_000455745.1)ParametersCurrent assemblyGCF_000455745.1Assembly approachWGS, PacBio and Hi‐CWGSSequence depth (X)355109Contig N50 (Mb)22.530.02Scaffold N50 (Mb)219.052.2Genome size (Mb)2296.172274.86Predicted genes (n)21,86222,200FIGURE 1Landscape of Chinese alligator genome. (a) Distribution of genome landscape: population‐scale π‐values across 16 chromosomes; density of SNP; density of indels; gene count; read depth mapped to the genome; GC content density. The statistics were calculated using a 500‐kbp window. (b) Divergence time of fifteen species generated by MCMCtree using the maximum likelihood method. The numbers in brackets show 95% confidence intervals of estimated divergence time between lineages.Further, our assembly showed extremely high collinearity with the scaffold‐level genome released previously (GenBank ID: GCF_000455745.1, Figure S3) (Wan et al., 2013), and with an increase of 21.31 Mb of the assembled size and 1.8% of BUSCO score (Table S6). The statistics on contiguity showed an improved scaffold N50 of 99‐fold and contig N50 of 979‐fold when compared to the new assembly with the GCF_000455745.1 genome. In addition, our genome covered 99.59% of the short‐read‐based genome, and filled 114.85 Mb of gaps in it. Using comparative genomic analysis, our genome supported that the Chinese alligator and A. mississippiensis are sister groups that separated from each other c. 32 million years (My) BP (Figure 1b). Moreover, we confirm that the divergence between the common ancestor of birds and the crocodilian lineage occurred c. 240 My BP (Wan et al., 2013).3.2Improved genome annotationWe identified 814.74 Mb repetitive elements in our assembled Chinese alligator genome, representing 35.48% of the total genome (Table S7), which was comparable with three other crocodiles (Green et al., 2014). The most abundant repeat category was LTRs (20.10%), followed by LINEs (12.93%), DNA elements (5.74%) and SINEs (0.04%) (Figure S4 and Table S8). There also existed 8.27 Mb (0.36%) unknown repeat elements. We masked all these repeat sequences for genome annotation.By combining evidence from de novo prediction, transcript mapping and homology‐based alignment, we predicted 21,862 high confident protein‐coding genes (Figure S5 and Table S9), which is generally consistent with gene numbers in the previous prediction (22,200 genes) (Wan et al., 2013). The average gene length, intron length and exon length were 36.25 kb, 4.47 kb and 173.79 bp (8.77 exons per gene), respectively (Figure S6 and Table S10). An obvious peak around 1000 bp was found in the gene length distribution of the GCF_000455745.1 and A. mississippiensis assemblies, but not in our chromosome‐scale assembly. Finally, 19,962 (91.31%) protein‐coding genes were functionally annotated in at least one of the four databases we used (see Section 2) (Figure S7 and Table S11), which was significantly more than the gene set count of GCF_000455745.1 (17,615 genes) (Wan et al., 2013). In addition, 2681 miRNA, 544 rRNA, 1545 tRNA and 1077 snRNA were predicted in our assembly (Table S12).3.3Population structure analysisThe average whole‐genome sequencing coverage and depth for the 23 individuals were 98.89% and 10.50‐fold, respectively (Table S13). We finally obtained 1,129,456 SNPs after filtering (see Section 2) for downstream analysis. PCA, admixture and phylogenetic tree analysis all supported that the CX, XC and ACA populations were assigned to three distinct clusters, which is consistent with geographical distribution (Figure 2a–d). The F3 statistics also supported a lack of admixture among the three Chinese alligator populations (Figure 2e). Pairwise F ST further revealed that they were significantly distinct from each other (F ST‐ACA‐CX = 0.23; F ST‐CX‐XC = 0.20; F ST‐ACA‐XC = 0.16). According to the rooted phylogenetic tree of the three populations with A. mississippiensis (Figure S8), XC is more likely the ancestor population, and the LD decay curve showed the fastest decrease of the r 2 value to its 50% in XC (Figure S9).FIGURE 2Distribution and genetic population structure of Chinese alligator populations. (a) Distribution of Chinese alligators in China and translocated individuals in America and sampling locations in this study. n represents the number of samples. Pictures above and below show original and external distribution, respectively. (b) Principal component analysis of 23 individuals showing the first and second principal components. Pairwise F ST is added near the bidirectional arrow. Asterisk represents the founder of CX population. (c) Inferred population genetic structure of the 23 individuals using the maximum likelihood method with a model with two to four ancestral components. (d) Unrooted tree constructed using the neighbor‐joining method from biallelic SNPs among 23 Chinese alligators and A. mississippiensis (acronym AM). The p‐distance is indicated by the scale bar. (e) F3 statistics for all three populations to detect the potentially admixed relationships.3.4Population demographic historyAncient demographic trajectories of three populations were highly similar with a continuous decline from 20 ka BP to the present day (Figures 3a,b and S10). This decline could be divided into three phases, including the first decline around 20–4 ka BP, a relatively stable state c. 4.0–1.5 ka BP, and the second obvious decline after 1.5 ka BP with an extremely low N e. We further inferred that the divergence among the three populations started c. 4.2 ka BP with a total separation within the past 1000 years (Figure 3b). The ACA and CX split c.1.0 ka BP, CX and XC c. 0.8 to 1.1 ka BP, ACA and XC c. 0.5 ka BP, according to the RCCR curves.FIGURE 3Estimated demographic history and divergence time of the three Chinese alligator populations. (a) Thick coloured lines depict temporal fluctuations in effective size (N e) over the past 10–0.5 ka. The x‐axis corresponds to the time before present in years on a log scale, assuming a substitution rate (μ) of 0.79 × 10−8 substitutions/site/generation and a generation time (g) of 20 years (Green et al., 2014). The coloured rectangles depict several extreme climate events including the last glacial maximum (LGM), the Younger Dryas cold period (YD), MWP‐1A, 1B and the 8.2 ka BP cooling event (Lambeck et al., 2014) as well as 4.2 ka BP aridification event (Zhang, Cheng, et al., 2018). Thin coloured lines indicate the mean annual temperature (TANN) for the lower of the Yangtze region in the Holocene (Li et al., 2017). (b) Recent effective population size inferred by PopSizeABC. Dotted lines indicate a 90% confidence interval and grey rectangles depict the little ice age (LIA) (Jiang & Zhang, 2004). (c) Split times between pairwise populations calculated by MSMC2. The grey rectangle depicts the time of Song Dynasty coinciding with serious damage to the habitats of Chinese alligators (Barker, 2012).3.5Gene flow among the three Chinese alligator populationsGene flow is essential to understand the degree of connectivity among populations, and to guide translocation and genetic rescue efforts for endangered species. We first performed the ABBA‐BABA test (A, B; X, Y), by setting A. mississippiensis as the outgroup. For most of the combinations, no significant deviations were found in the shared derived alleles between XC‐ACA and XC‐CX (D [CX,ACA; XC,AM] = 0.024, Z = 1.359), except for two individuals in XC (XC1, XC3), with significantly more shared derived alleles with ACA and CX, respectively (Figure 4a). TreeMix analysis detected gene flow between ACA and XC, which was somewhat consistent with the result of ABBA‐BABA test (Figure 4b). A detailed scanning of IBD fragments found that very long IBD fragments (within 5 generations) were absent among populations, although these can be found within CX (Figure 4c). However, shared IBD fragments increased from five generations (100 years) before present within and between populations (Figures 4d,e and S11).FIGURE 4Gene flow between the three Chinese alligator populations. (a) Estimates of D (CX, ACA; XC, AM), in which only one positive and negative statistics, respectively, were considered statistically significant following correction for multiple testing, based on Z‐score > 3 and <−3. (b) Maximum‐likelihood tree with A. mississippiensis serving as an outgroup calculated by TreeMix. (c–e) Lengths of IBD (cM) shared between pairwise individuals within and between populations that were generated from 0–5 (c), 5–10 (d) and 10–15 (e) generations before the present.3.6Genetic diversityGenome‐wide H in all samples showed extreme depletion with an average H = 1.20 * 10−4 ± 1.16 * 10−5, which is lower than the endangered Chinese crocodile lizard (Shinisaurus crocodilurus, 2.0 * 10−4 – 5.7 * 10−4) (Xie et al., 2021), five‐pacer viper (Deinagkistrodon acutus, 1 * 10−3) (Yin et al., 2016), Swinhoe's soft‐shelled turtle (Rafetus swinhoei, ~ 1.23 * 10−3), and many critically endangered species, and just slightly higher than brown eared pheasant (9.53 * 10−5) (Wang et al., 2021) and Iberian lynx (1.02 * 10−4) (Abascal et al., 2016) (Figure 5a, Table S14). H did not decrease significantly in exons compared to the whole genome. Three populations exhibited no significant difference except the genic and intron regions between ACA and CX (Figure 5b, Tables S15–S16). Genome‐wide π values were remarkably low at both species‐level (1.47 * 10−4) and population‐level: ACA (1.14 * 10−4) < CX (1.15 * 10−4) < XC (1.41 * 10−4) (Figure S12) when compared with crocodile lizard (3.85 * 10−4 ‐ 5.47 * 10−4) (Xie et al., 2021) and tuatara (Sphenodon punctatus) (8 * 10−4 – 1.1 * 10−3) (Gemmell et al., 2020). Several hotspot regions harboured genes associated with MHC class I protein binding, olfactory receptor activity, peptide hormone binding, and ubiquitin protein ligase activity (Tables S17–S18).FIGURE 5Characterization of heterozygosity in Chinese alligators. (a) Comparison of genome‐wide heterozygosity among endangered or extinct species and three other crocodilians. Coloured dots represent each individual and whiskers represent the range in a given species. (b) H of different genomic regions in the three populations.3.7Inference of inbreeding history by ROHs Small and endangered populations are usually threatened by inbreeding, which is reflected in ROHs. Overall, we discovered 20,938 ROH ranging from 0.10 Mb to 36.56 Mb in 23 individuals. The proportion of ROH in the whole genome (FROH) ranged from 46.72% to 69.63%: ACA (55.61 ± 3.49%) 80%) (Wang et al., 2021). Interestingly, the individual with the lowest FROH was the CX1 (46.72%), the wild founder of CX population. ROH longer than 2 Mb potentially indicates mating between closely‐related individuals and not drift alone (Ceballos et al., 2018). All alligator genomes in this study comprised a high proportion (38.88 ± 7.21%) of ROH >2 Mb. Even when considering ROH longer than 10 Mb, FROH was still high (11.76 ± 4.67%). Also, CX1 borne fewer extreme long ROHs than other individuals (Figure 6a).FIGURE 6Investigation of inbreeding history by ROH in Chinese alligator. (a) Distribution of ROH in different length categories for each genome. (b) ROH longer than 5 Mb in the genome of three populations and shared ROH regions between pairwise populations and among three populations. (c) Average genome‐wide heterozygosity and ROH density in the three populations. The colour gradient is scaled according to the ROH density, as shown in the legend. (d) Heat map showing the proportion of genomes in ROH regions (top right) and both ROH and IBD (bottom left) regions between pairwise comparisons among individuals. (e) Distribution of ROH resulting from inbreeding in different generations.In general, the distribution of ROH differed among populations (Figure S13). In total, 1.84 Gb ROH (>0.1 Mb) were found shared by all three populations and 3.92 Mb, 4.30 Mb and 24.42 Mb ROH (>0.1 Mb) were population‐specific in CX, ACA and XC. ROH regions shared by each combination of two populations were also high: FROH>5 Mb CX‐XC = 54.52%, FROH>5 Mb ACA‐XC = 48.29%, FROH>5 Mb ACA‐CX = 44.09% (Figure 6b). The distribution of ROH at the individual level was also investigated (Figures 6c and S14). Although the distribution of ROHs across the genome was various among different individuals, the proportion of shared regions between every two individuals within a certain population was high. Nevertheless, there existed a significant difference when pairwise comparison was conducted on shared ROH in IBD regions within and between populations (within: 12.21% ± 4.74%, between: 4.90% ± 1.73%; wilcox.test, p 11.24 Mb) varied among all individuals, while the founder (CX1) had the least value. FROH of 5–10 generations (5.62–11.24 Mb) ago was higher than that within five generations (FROH>11.24 Mb). FROH from distant time periods before 10 generations fluctuated from 5% to 10%, showing a more stable style compared with 0–5 and 5–10 generations. The distribution of ROHs' expected time indicated that ROHs in Chinese alligator genomes have accumulated gradually since at least 2 ka BP.3.8Mutational loadWhen estimating mutational load in each sample, we firstly found 18.91 ± 3.39 and 12.17 ± 3.91 heterozygous dnsSNPs and LOFs, respectively, without significant differences in the three populations (Figure S15). As expected, there were fewer homozygous dnsSNPs (11.26 ± 4.39) and LOFs (10.09 ± 2.64) than heterozygous ones. However, homozygous dnsSNPs and LOFs were significantly different among populations, with XC presented the highest number (dnsSNP: 14.89 ± 1.52; LOF: 12.33 ± 1.63), compared to the ACA population (dnsSNP: 13.17 ± 2.27; LOF: 9.33 ± 2.36) and the CX population (dnsSNP: 5.75 ± 0.97; LOF: 8.13 ± 1.69) (Figures 7a and S16). After a further inspection of Rnhom, we found that homozygous missense mutations tended to distribute within ROH in sharp contrast to regions outside ROH, with a Rnhom value of 2.24 ± 1.98. Moreover, homozygous missense mutations in three CX individuals were all located within ROH (Figure S17).FIGURE 7Mutational load in three Chinese alligator populations. (a) Comparison of the count of homozygous nonsynonymous mutations, including missense, LOF and dnsSNP mutations. (b) Venn diagram for nonsynonymous mutations. (c) Venn diagram for genes carrying nonsynonymous mutations. (d) GO enrichment of biological process for all genes in (c). (e) Counts of new dnsSNP and LOF mutations introduced by each individual if gene flow occurred.Among all 1235 derived mutations including missense, LOF, and dnsSNP mutations, 38% of them were shared among three populations, whereas 3, 3 and 25% were specific to ACA, CX and XC, respectively (Figure 7b). As to the 613 genes harbouring these mutations, 44% were shared and 29% were population‐specific: CX(2%) < ACA(4%) < XC(23%) (Figure 7c). GO analysis of these 613 genes indicated that these genes are associated with several important biological processes, including cell growth and development (LRP1, MR1, SEMA4G, DBN1, PTPRS), cell morphogenesis (MYO10, ZNF135), bone development (KIT, TNF), lens fibre cell differentiation (SPRED2), reproduction (FSIP2, TEKT2, PLEKHA5, METTL3, MOV10L1), immunity (CD274, MR1) and nervous system (AKAP12, KNDC1). Population‐unique genes also involved similar functions while there were several cell‐cycle‐related genes in XC population, such as TOP2A, which was a classic proliferation marker (Table S20).In the prediction of newly introduced deleterious mutations in cross‐breeding programmes, XC individuals would introduce the most counts to the other two populations, including 0–20 dnsSNPs and 0–13 LOFs (Figure 7e; Table S21), consistent with the fact that XC accounted for the highest proportion of population‐specific deleterious mutations. In pairwise comparison at an individual level, the number of shared deleterious alleles was unsurprisingly higher within a certain population while unique ones were more when the compared samples came from different populations (Figure S18).3.9Signatures putatively under local adaptionBy applying iHS method, we identified 9077, 9520 and 12,766 SNPs under putatively recent positive selection in ACA, CX and XC, corresponding to 260, 224 and 442 genes, respectively. A large proportion of these genes were population‐specific: CX(16%) < ACA (19%) < XC (42%) (Figure S19). For positively selected genes unique to each population, however, GO enrichment did not show strong evidence for overrepresented categories that could be associated with local adaption (Table S22). Finally, PBS analysis showed 28, five and four genes with a signal of positive selection unique to the CX, ACA and XC, respectively (Figure S20). Further investigation of gene function revealed the selection effect on genes relative to immunity, possibly implying different immune genes essential for each population (Table S23).4DISCUSSIONHere, we present the first high‐quality chromosome‐level assembly and population genomic exploration of Crocodilia. Chinese alligator is the most threatened crocodile in the world, thus it is essential to investigate their genomic backgrounds for planing reasonable and scientific strategies for conservation (Supple & Shapiro, 2018).4.1An improved genome assembly and annotationAlthough previous short‐read assembly revealed the possible genetic basis for its biological characteristics (Wan et al., 2013), only a much more improved genome could help to conduct accurate analyses on the genomic characteristics (i.e., ROH and IBD). Thanks to PacBio long‐read sequencing and Hi‐C technology, we assembled the most continuous, complete, and high‐quality reference genome for Chinese alligator so far. Compared with the previously released assembly (GenBank: GCF_000455745.1), the new assembly holds excellent advantages in scaffold N50, gene length, completeness of gene set and the number of annotated genes. This chromosome‐scale assembly and genome annotation would undoubtedly facilitate the assessment of genetic diversity, inbreeding status and mutational load and local adaptation, which are extremely important to isolate and long‐term declining Chinese alligator populations.4.2Population structure analysis revealed genetic diversity extinct in the wildCX and XC are now the last two and largest populations in their original habitats, representing the sole remaining genetic diversity of Chinese alligator in China. A previous study reported that the breeding populations of XC and CX were genetically closely‐related when compared with the wild population from Xuancheng based on MHC class IIb gene analyses (Nie et al., 2013). Individuals in American sanctuaries are actually transferred from China around one century ago (Behler, 1993; Honegger & Hunt, 1990), while their geographical origin and genetic background are ambiguous. Here, we clarify for the first time the genetic relationships of the three populations, proposing that XC is more likely the ancestor population and ACA is distinct from CX and XC. This result is somewhat consistent with Nie et al. , considering that the XC population originated from wild samples from Xuancheng.Pairwise F ST among the three populations was smaller than that between two tuatara populations (Gemmell et al., 2020), comparable to that among three Chinese crocodile lizard populations in China (Xie et al., 2021), but larger than that between the Sichuan and Qinling giant panda subspecies (Guang et al., 2021), the African leopard populations (Pečnerová et al., 2021), and even the human populations of Africa and Asia (Altshuler et al., 2005), further supporting the extent of the geographical isolation of these three populations. We supposed that the split time of ACA with the two Chinese populations is much earlier than the time their founders left China because such a huge genetic difference was unlikely to evolve within one century. And the result of MSMC2 on the divergence was consistent with our thought. Therefore, the geographical origin of ACA ancestors is distinct from CX and XC and ACA may represent genetic diversity of a wild‐extinct population. Animals from ACA could be considered as potential donors for translocation programmes.4.3Causes of long‐term population declinesOur demographic reconstruction for the three populations showed highly consistent trends with a general decline for the recent 20 ka (Figure 3a,b). During a long period before the Song Dynasty, vast areas in Yangtze River region were not exploited by humans (Wen, 2000). The N e decline starting c. 20 ka BP may be caused by the cold climate of LGM (Lambeck et al., 2014), possibly reflecting a drastic reduction in population size. Subsequently, N e kept stable after the 4.2 ka BP aridification event (Zhang et al., 2018) while population divergence occurred at this time point. The serious drought event could constrict the wetland and river systems area, thus partly building geographical barriers and hampering individual migration and gene flow, which would have led to a further decrease in N e. During the Southern Song Dynasty (between 1.5–1.3 ka BP), the economic gravity centre shifted from the Yellow River Basin to the Yangtze River Basin (Wang et al., 2021). This shift was accompanied by deforestation, hunting, intense farming with the introduction of new strains of rice and improved methods of water control and irrigation (Wen, 2000). A destructive anthropogenic disturbance may be important factor in the contraction of Chinese alligator populations from 1.5 ka BP onwards, thus providing possible reasons for the population isolation (Figure 3c). Contemporary N e is still decreasing, at least partly implying a genomic consequence of the declining census population size in recent years (Ding et al., 2001; Ding & Wang, 2004), raising concerns for the long‐term survival of wild populations.4.4Genomic consequences of continuous decliningSevere population declines are likely to lead to a loss of genetic diversity, an increase in inbreeding, exposure of deleterious alleles in homozygous state and strong drift which will lead to fixation of deleterious alleles. The most direct consequence of the long‐term decline in Chinese alligators is the deficiency in genomic heterozygosity, which is lower than most endangered animals. However, heterozygosity in CDS regions was slightly lower compared to intron regions, which has also been observed in A. mississippiensis and G. gangeticus, in stark contrast with other representative species like chicken (G. gallus) and green anole (A. carolinensis) (Green et al., 2014; Wan et al., 2013). This may indicate that maintaining the current remaining low polymorphism in CDS was an evolutionary strategy to keep fitness in struggling with reducing genetic diversity in some fragile species (i.e., narwhal) (Westbury et al., 2019).Long ROHs that have not been broken by recombination are probably the result of recent inbreeding (Mcquillan et al., 2008). Between 19.80%–48.87% of the total ROHs seem to have been generated within the recent 10 generations, while the 100 past generations ranged from 86.93%–92.04%. This result indicates that although three populations have been continuously declining for 20 ka, frequent inbreeding mostly occurred within the past 200 years. We inferred that not only the recent founder effect but also long‐term small population size contributed to their high inbreeding level of them. Interestingly, the FROH generated in the past five generations was lower than that of 5–10 generations (they were generated before breeding programmes [generation time in breeding programmes: 7 years]), indicating that inbreeding level in the wild populations during the 20th century may be comparable with that in breeding centres. Shared ROHs in IBD regions represent four identical haplotypes of the two samples. Optimistically, this index was significantly different when comparing pairwise individuals within and between populations, providing potential possibilities to improve their genetic status by gene flow among populations.Mutational load provides an important way of assessing the exposure to genetic threats in small populations (von Seth et al., 2021). Significant differences were observed in the number of homozygous other than heterozygous deleterious mutations. Furthermore, inbreeding led to the accumulation of homozygous missense alleles, contrary to the result in the brown‐eared pheasant populations (Wang et al., 2021). Several deleterious alleles were located in genes related to osteoclast differentiation, cell development and growth, organ morphogenesis, lens fiber cell differentiation and retinoic acid receptor signalling pathway, which may be candidate SNPs responsible for congenital malformations (Figures S21–S22) (Wu et al., 1999). Yet, further genetic investigation and experimental verification are needed.4.5Implications for conservation and genetic rescue attemptsAlthough species with small N e and low genetic diversity can still survive for thousands of generations (Robinson et al., 2016; Wang et al., 2021), genetic effects can severely reduce the fitness of species and hamper demographic recovery because of the limited adaptive potential in small populations (Willi et al., 2006). Assisted gene flow is regarded as a vital method for the recovery of endangered populations in conservation genetics. From 2001–2006, some individuals from XC and ACA were translocated to Changxing Centre (Ni, 2012). Nevertheless, weighing the risks (i.e., outbreeding depression) and benefits (i.e., genetic rescue) of translocations is essential in such a cross‐breeding programme.We first evaluated the likelihood of introducing new deleterious alleles when selecting different individuals as donors to move to the recipient population. Due to the existence of population‐unique dnsSNPs and LOFs (CX < ACA < XC), a risk of increasing mutational load seems unavoidable, especially when introducing XC individuals into CX population, while it could be alleviated by choosing different donors. Overall, the counts of carried‐over deleterious alleles were lower than that in rhinoceros populations (von Seth et al., 2021). Second, we examined signatures of positive selection to identify potential signatures of adaptation. Even though there was a large genetic distinction among populations, an obvious indication of local adaption was not observed in each population. This result could be explained by recent geographical isolation and similar habitats.5CONCLUSIONSInvestigation of the genetic background of endangered species is essential for their protection and conservation. Here, we assembled the first chromosome‐scale genome of the Chinese alligator and applied whole genome data to examine the genomic consequences of severe declines in this critically endangered species. Extensive investigation across their genomes verified their highly endangered status from a population genetic perspective. Furthermore, this study highlights the need of integrating genetic indices into IUCN classification lists (Hoban et al., 2020). Finally, our investigation of the genetic background of the Chinese alligator populations is a valuable resource and provided recommendations for future conservation and management.AUTHOR CONTRIBUTIONSQiu‐hong Wan and Sheng‐Guo Fang conceived and initiated the project. Mengyuan Hu organized and collected the samples. Jun Cao, Lirong Liu and Jianqing Lin performed DNA library preparation and sequencing. Yi Zhang and Qing Wang assembled the improved genome and conducted comparative genomics analysis. Shangchen Yang, Tianming Lan, Haimeng Li, Minhui Shi and Yixin Zhu performed population genetic analysis. Shangchen Yang wrote the manuscript. Tianming Lan, Sunil Kumar Sahu and Nicolas Dussex coordinated the data analysis and extensively revised the manuscript. Qiu‐hong Wan, Huan Liu, and Sheng‐Guo Fang provided supervision. All authors read and approved the final manuscript.CONFLICT OF INTERESTThe authors declare no conflict of financial interests.Supporting information Appendix S1 Click here for additional data file.

PMC

Neuropsychiatric Disease and Treatment

PMC10588757

10-16-2023

10.2147/NDT.S425509

Subthreshold Depression: A Systematic Review and Network Meta-Analysis of Non-Pharmacological Interventions

Hao Xiaofei, Jia Yuying, Chen Jie, Zou Chuan, Jiang Cuinan

BackgroundSubthreshold depression (StD) is considered to be the “precursor” stage of major depressive disorder (MDD), which could cause higher risk of suicide, disease burden and functional impairment. There have been various non-pharmacological interventions for StD. However, the comparison of their effectiveness still lacks sufficient evidence. We performed a systematic review and network meta-analysis to evaluate and rank the efficacy of multiple non-pharmacological interventions targeting StD.MethodsWe conducted a thorough search across various databases including PubMed, Medline, Embase, Web of Science and PsycINFO from inception to December 2022. All included studies were randomized controlled trials (RCTs) of non-pharmacological interventions for patients with StD compared with control group (CG). Several universal scales for measuring depression severity were used as efficacy outcomes. The surface under the cumulative ranking curve (SUCRA) was used to separately rank each intervention using the “Stata 17.0” software.ResultsA total of thirty-six trials were included, involving twenty-eight interventions and 7417 participants. The research found that most non-pharmacological interventions were superior to controls for StD. In each outcome evaluation by different scales for measuring depression, psychotherapy always ranked first in terms of treatment effectiveness, especially Problem-solving Therapy (PST), Behavioral Activation Therapy (BAT), Cognitive Behavioral Therapy (CBT)/Internet-based CBT (I-CBT)/Telephone-based CBT (T-CBT). Since different groups could not be directly compared, the total optimal intervention could not be determined.ConclusionHere, we show that psychotherapy may be the better choice for the treatment of StD. This study provides some evidence on StD management selection for clinical workers. However, to establish its intervention effect more conclusively, the content, format and operators of psychotherapy still require extensive exploration to conduct more effective, convenient and cost-effective implementation in primary healthcare. Notably, further research is also urgently needed to find the biological and neural mechanisms of StD by examining whether psychotherapy alters neuroplasticity in patients with StD.

IntroductionDepression is highly prevalent and ranked third among the global burden of disease by WHO, expected to rise to being the number one ranked disease by 2030.1 This trend poses a substantial challenge for health systems in both developed and developing countries. Whether in the DSM-IV or ICD-10 diagnostic classification system, a diagnosis of major depressive disorder (MDD) requires the time course, number of symptoms and severity to meet certain threshold requirements.Subthreshold depression (StD) (also called subsyndromal, subclinical or minor depression) was coined by Judd et al2 in 1994 and has been widely accepted by scholars. StD was regarded at a “preclinical” stage of MDD,3 referring to a depressive state with at least two or more depressive symptoms, including mood depression or loss of interest and pleasure, with a duration of at least two weeks, and accompanied by impaired social functioning. This state does not meet criteria for major depressive episodes. In addition, having a higher score than a certain cut-off in self-rated depression scales was also recognized as an alternative definition by researchers.4,5 StD has been a highly prevalent condition, with approximately 2.9%−9.9% incidence rate among adults in primary care, 1.4%−17.2% among adults in community settings,6 and 38.7% among older adults receiving home care.7 And during COVID-19, the proportion of the population meeting the criteria for StD was even as high as 47.8%, suggesting that the epidemic perhaps act as a stressor affecting all population.8 In addition, the state of StD is associated with gender, family status, economic and employment status, disability and impairment associated, health service use and comorbidity,5 the prevalence of StD among patients with diabetes reached 11.6% evaluated by the Patient Health Questionnaire 9-item (PHQ-9).9 Prevention and treatment of cardiovascular diseases also play an important role in preventing depression and other mental disorders.10The pathophysiological features of depressive disorder are complex. The monoamine hypothesis has prevailed for the pathogenesis of depression, holding that depression is caused by the depletion of 5-HT, norepinephrine, or dopamine in the central nervous system (CNS). The other pathogenesis includes stress, neurotrophins and neurogenesis, excitatory and inhibitory neurotransmission, (epi)genetics, inflammation, the opioid system, myelination, and the gut-brain axis, among others. The neural substrate of depression refers to the abnormalities in neuronal activity and neurotransmitters associated with depression in the brain. Battaglia et al11,12 addressed the role of specific neuropharmacological adjuvants that act on neurochemical synaptic transmission and found that depression can be present with neurotransmitter-related abnormalities, including dysregulation of neurotransmitters such as epinephrine, norepinephrine, and dopamine. In addition, the synaptic plasticity in patients with depression may be affected by abnormal release and reuptake of neurotransmitters, changes in synaptic structure, inflammation and stress response, which in turn affects emotional regulation and cognitive function, and deficits in emotion regulation may also lead to mood disorders.13Mitochondria are multifunctional organelles which produce cellular energy and play a major role in other cellular functions including homeostasis, cellular signaling, and gene expression, among others. In recent years, researchers have found that mitochondrial abnormalities may lead to disturbances in energy metabolism, which can lead to depressive symptoms. Abnormal mitochondrial function can lead to both an increase in oxidative stress, inflammation and apoptosis, as well as affecting the synthesis and regulation of neurotransmitters, which in turn can affect mood and cognitive function.14,15 Furthermore, Hakamata et al16 found that the blunted interleukin-6 diurnal rhythm predicts depressive symptoms, modulated by amygdala emotional hyporeactivity and gene-stressor interactions. These findings may indicate a potential mechanism underlying vulnerability to depressive disorders, suggesting their early detection, prevention, and treatment through the understanding of immune system dysregulation. To delve into the new neurobiological mechanisms behind neuropathogenesis, stimulation effects, brain responses, researchers have utilized a variety of neuroimaging techniques such as structural and functional magnetic resonance imaging (s/fMRI), electroencephalography (EEG), diffusion tensor imaging (DTI) etc., all of which provide structural, functional, and chemical brain detailed visualizations and measurements of the brain.17–20 The integration of these techniques enables a deeper understanding of the pathophysiology of these disorders and facilitates the development of more effective therapies.Compared with healthy individuals, StD was associated with a higher risk of suicide, disease burden, and functional impairment.21 Cuijpers et al22 conducted a 6-year longitudinal study on older adults and found that 7.8% of those with StD developed MDD, the rate reached 12% after 3-year follow-up in a Dutch study.23 Early appropriate interventions for StD that can reduce the risk of developing depression have attracted widely attention. Previous meta-analysis indicated that antidepressants did not demonstrate therapeutic advantages for StD over placebos,24 and benzodiazepines were also not determined to their potential therapeutic role.25 Furthermore, according to the Guidelines from the National Institute for Health and Care Excellence (NICE), people with StD should not be recommend pharmacological therapy. Consequently, non-pharmacological treatments currently become important means for StD. Scholars globally were gradually dedicated to exploring in the field. There were various types of methods applied to the treatment of StD, including psychological therapy (ie, cognitive behavioral therapy, problem-solving therapy, behavioral activation, cognitive therapy, mindfulness therapy, etc.), physical activity therapy (ie, aerobic exercise, Chinese Tai Chi, etc.), psychosocial therapy (ie, counseling therapy) and others. In terms of treatment forms, there were not only group face-to-face treatments,26,27 but also self-help or professional guided treatments based on the internet, telephone, email, manual, and so on.28,29A few previous meta-analyses have evaluated the effectiveness of partial measures for StD. He et al30 allocated non-pharmacological interventions among adults into one of two groups: intervention classes (physical activity, psychosocial intervention, psychotherapy and so on) and individual modalities, and showed that psychotherapy demonstrated statistically significant superiority over conventional treatment. A meta-analysis conducted by Spanish researchers assessed the effectiveness of pure or minimal support self-help interventions based on cognitive, behavioural or cognitive-behavioural treatments in older adults, had further been validated to have short-term therapeutic effects, but the long-term preventive effects were not yet clear.31 Moreover, Cuijpers et al3 first presented evidence showing that psychological interventions had a small to moderate but significant effect on reducing depressive symptoms in adolescents with StD. Jiang et al32 suggested that electroacupuncture or bright light therapy appeared to be the better choices in the treatment of StD.However, perhaps there were still some differences between trials those included in patient characteristics, study interventions, outcome assessments or study designs, which make comprehensive comparability remain unclear. Therefore, we decided to conduct a new, comprehensive network meta-analysis (NMA) of non-pharmacological interventions to identify and rank the efficacy of multiple therapies for people aged over 12 years old with StD.MethodsProtocol and RegistrationThe protocol was registered at the International Prospective Register of Systematic Reviews (PROSPERO) (ID: CRD42022355683). It was reported according to Preferred Reporting of Systematic Reviews Incorporating Network Meta-analyses of Health Care Interventions (PRISMA).33Eligibility CriteriaStudies fulfilling the following the “PICOS” inclusion criteria were included in our meta-analysis: Population (P): study participants were patients aged over 12 years old with clinically considered StD, described in the Diagnostic and Statistical Manual of Mental Disorders (DSM), International Classification of Diseases (ICD), or Research Diagnostic Criteria (RDC);Intervention (I): study participants underwent non-pharmacological interventions, including psychotherapy, physical activity therapy, psychosocial therapy, and others explored;Comparison (C): study participants assigned to the control group did not receive active treatment, but only no treatment, treatment as usual (TAU), enhanced TAU, or on the waiting list;Outcome (O): the outcome of efficacy will refer to mean overall change on continuous depression severity scales and included study was required to report at least one of the following indicators: Scores of Depression Scale (CES-D), Beck Depression Inventory scale (BDI/BDI-II), the Patient Health Questionnaire-9 (PHQ-9), the Kessler Screening Scale for Psychological Distress (K-6), Hamilton Depression Scale (HAMD/HRSD), Quick Inventory of Depressive symptomatology (QIDS), Geriatric Depression Scale (GDS), Self-rating Depression Scale (SDS);Study Design (S): published randomized controlled trials (RCTs) in English.Other requirements: articles whose full text could be retrieved and with complete or obtainable data.Databases and Search StrategyWe systematically searched online the following databases from inception to December 2022 for eligible English-language journal RCTs: PubMed, Medline, Embase, Web of Science, and PsycINFO. Combined with the references or studies of previous studies and systematic reviews, some additional records were manually supplemented. The following key terms were used: (“subthreshold depression” OR “subsyndromal depressive symptoms” OR “subsyndromic depression” OR “subclinical depression” OR “Subsyndromal symptomatic depression” OR “minor depression” OR “StD” OR “SD” OR “SDS”) AND (“randomized clinical trial” OR “randomized controlled clinical trial” OR “RCT”) etc. The search strategy was applied to titles and abstracts. We exported the search results into the reference management software Endnote X9.Data ExtractionTwo researchers independently searched and screened the titles and abstracts of records. Full-text papers were retrieved to determine all potentially eligible studies that met the inclusion criteria. For studies published more than once, we included only the trial with the most complete and informative data. Any ambiguous disagreements were settled by consensus or by a discussion with a third researcher.The following data were extracted and collected in Microsoft Excel spreadsheets using a standard data extraction form: article title, publication year, first author’s name, setting, sample size, sex distribution, mean age or age range, details of the interventions in each group, mode of intervention (i.e., in-person or digital, group or individual), duration of intervention, outcome data etc.Assessment of the Risk of Bias in Included StudiesThe risk of bias of all included RCTs was assessed by two reviewers independently using the Cochrane Risk Bias tool (By Review Manager 5.4 Software). The indicators of the tool consists of 7 items including random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting and other biases. The judgment results were expressed as low risk of bias, high risk of bias and bias risk uncertain. Disagreements about quality assessment were resolved by discussion until a consensus was reached.Statistical AnalysisThe network meta-analysis was conducted using Stata 17.0 software program to combine direct and indirect evidence of StD. There were several measures of StD, we divided the articles into different groups based on the type of scale and then analyzed each group separately. The effect size (ES) of continuous variables was expressed as a standardized mean difference (SMD) and 95% confidence interval (95% CI). To note that, although 7 articles used the Hamilton scale, their versions were inconsistent including HAMD, HAMD-14, HAMD-17 and HRSD. To avoid bias in research choices and considering similarity of scale types, we included them in a group after discussion and used weighted mean difference (WMD) to represent the ES. We calculated whether there was a significant difference in the inconsistency model. If P>0.05, we chose a consistency model; if P<0.05, an inconsistency model was selected.34 When there was at least one closed loop in the evidence network (refers to a comparison of multiple interventions included in the study and forming a closed loop), local inconsistency test (node-splitting method) and loop inconsistency were required. We obtained the rank of each intervention through the surface under the cumulative ranking (SUCRA), with 0≤SUCRA≤100% (or represented as 0≤SUCRA≤1), the higher the SUCRA value means the better the rank.35 Performing sensitivity analyses to evaluate the stability of the results, and assessing publication bias in interventions using a funnel plot.ResultsIdentification of StudiesA total of 26,876 records were initially searched, with 19,640 remained after eliminating duplicates. Their titles or abstracts were screened for this systematic review and 4515 articles remained for full-text review. We finally included 36 eligible articles in this systematic review based on the inclusion criteria. The PRISMA flowchart of detailed study selection process is shown in Figure 1. Figure 1Flow diagram for search and selection of the included studies.Study CharacteristicsThe studies were published from 2001 to 2022, and a total of 36 trials were included involving 7417 participants, with 52.22% (n=3873) receiving non-pharmacological interventions, in which 47.78% (n=3544) receiving control treatment. The characteristics of all included studies are summarized in Table 1. Table 1Characteristics of Included Trials (n=36) Ordered ChronologicallyAuthor (Year)CountrySettingNAgeFemale %Inclusion CriteriaModelFormatCourseOutcomeYing et al, 202236ChinaSocial329≥1866.4CES-D≥16, No MDEI-CBTCBTDigital/IndividualIn person/Group5 weeksCES-D, PHQ-9, BDI-IIWang et al, 202237ChinaSchool3012–14NMPHQ-9≥10, No MDDAEIn person/Group12 weeksPHQ-9Sun et al, 202238ChinaHospital65≥1843.1CES-D≥16, 7≤HAMD≤17BATIn person/Individual6 weeksCES-D, HAMD-17Au et al, 202239ChinaCommunity16814, SDS>53No MDDMBSRIn person/Group8 weeksBDI-IIZhang et al, 201845ChinaSchool6416–1964SymptomsMTCCIn person/Group8 weeksPHQ-9Yamamoto et al, 201846JapanSchool3120–3980.6SDS≥39, Self-reportNo mental disorderIPCIn person/Group3 weeksSDSWong et al, 201847ChinaClinic231≥2093.15≤ PHQ-9≤14, No MDDBAMIn person/Group8 weeksBDI-II, SDSTakagaki et al, 201848JapanSchool11818–1938.1BDI-II ≥10, No MDEBATIn person/Group5 weeksBDI-IISinghal et al, 201849AustraliaSchool12013–18NM14≤CDI≤24CBTIn person/Group8 weeksCES-DPols et al, 201850the NetherlandsClinic236≥1845.3PHQ-9≥6, No MDDSTEPEEDStepped/Individual1 yearPHQ-9Ebert et al, 201851GermanySocial204≥1880.4CES-D≥16, No MDDPST+BTDigital/Individual7 weeksQIDS, HRSD, CES-DPan et al, 201752AmericaSocial120≥187814≤BDI-II≤28DI, NIIn person/Individual20 minutesBDI-IILewis et al, 201753BritainClinic705≥6557.7Self-report, No MDDCASPERDigital/Individual7–8 weeksPHQ-9Pibernik et al, 201554CroatiaClinic20918–6554.1Self-report (PHQ-2)No MDDCBT+PSTPEIn person/Group6 weeksCES-DHermanns et al, 201555GermanyHospital21418–7056.5CES-D≥16, No MDDCBTIn person/GroupIn-HospitalCES-D, PHQ-9Buntrock et al, 201556GermanySocial406≥1865.2CES-D≥16, No MDEI-CBTDigital/Individual3–6 weeksCES-DImamura et al, 201457JapanCompany762NM53.3No MDDI-CBTDigital/Individual6–10 weeksBDI-II, K6Kasckow et al, 201458AmericaClinic23≥500CES-D>11, No MDEPSTIn person/Individual6–8 weeksBDI, HRSDMorgan et al, 201228AustraliaSocial132618–7877.6Symptoms (PHQ-9)E-BTDigital/Individual6 weeksPHQ-9Furukawa et al, 201259JapanCompany11820–5722K6≥9, BDI-II≥10T-CBTDigital/Individual8 weeksBDI-II, K6Ullmann et al, 201160ColumbiaCommunity47≥6570.216≤CES-D7T-IPCDigital/Individual9 weeksHAMD-17Willemse et al, 200467The NetherlandsClinic21618–6566.2Symptoms, No MDDCBTDigital/Individual28 weeksCES-DClarke et al, 200127AmericaOrganization9413–1859.6CES-D≥24, No MDDG-CTIn person/GroupNMCES-D, HAMD-14Abbreviations: CES-D, Center for Epidemiologic Studies Depression Scale; PHQ-9, the 9-item Patient Health Questionnaire; BDI, Beck Depression Inventory Scale; HAMD, Hamilton Depression Scale; K-6, Kessler Screening Scale for Psychological Distress; QIDS, Quick Inventory of Depressive Symptomatology; GDS, Geriatric Depression Scale; SDS, Self-rating Depression Scale; HRSD, Hamilton Rating Scale for Depression; CDI, Children’s Depression Inventory; PHQ-2, the 2-item Patient Health Questionnaire; GE9, Geriatric Depression Scale scores; K-SADS-PL, the Schedule for Affective Disorders and Schizophrenia for School-Age Children; MDD, Major Depressive Disorder; MDE, Major Depressive Episode; CBT, Cognitive Behavioral Thera-py; I-CBT, Internet-based CBT; T-CBT, Telephone-based CBT; IT-CBT, Internet- and Telephone-based CBT; BB-CBT, Bibliotherapy-based CBT; G-CBT, Group CBT; AE, Aerobic Exercise; BAT, Behavioral Activation Therapy; VISA-PWS, Video viewing Smartphone Application Intervention involving Positive Word Stimulation; PST, Problem-solving Therapy; CBM, Cogniti-ve Bias Modification; MBSR, Modified Mindfulness-Based Stress Reduction; MTCC, Mindfulness-based Tai Chi Chuan; IPC, Interpersonal Counseling; BAM, Behavioral Activation with Mindfulness; STEPEED, a stepped-care prevention programme; BT, Behavior Therapy; DI, Directive Intervention (Psychoeducation); NI, Non-directive Intervention (Psychoeducation); CASPER, CollAborative care and active surveillance for Screen-Positive EldeRs; PE, Physical Exercise; BT, Behavior Therapy; E-BT, Email-based BT; Feldenkrais, a Mind-Body Intervention; IPT, Interpersonal Psychotherapy; T-IPC, Telephone-administered IPC; G-CT, Group Cognitive Therapy; NM, not mentioned.Population: The age of study participants ranged approximately from 12 to 90 years old, 65.86% of them were female (4 articles with unclear or not obtainable gender information). The included RCTs were conducted in China (n=7), 36–39,44,45,47 the United States (n=5), 27,52,58,62,65 Japan (n=5), 40,46,48,57,59 the Netherlands (n=5), 50,61,63,64,67 Germany (n=4), 29,51,55,56 Australia (n=3), 28,43,49 Columbia (n=2), 60,66 Thailand (n=1), 41 Croatia (n=1), 54 Spain (n=1), 42 Canada (n=1)26 and Britain (n=1).53 Most studies were two-arm trials (n=32), and remaining were three-arm trials (n=4). The diagnostic criteria for StD are not unified among those studies. According to the statistics, there were 25 studies combining severity rating scales with interviews (to exclude MDD or MDE) to get the diagnose of StD,2 articles combining symptoms with interviews, 7 articles only using scales, and 2 articles single relying on symptoms.Interventions: In total, there were 28 different non-pharmacological interventions, summarized into 7 categories, including psychotherapy (n=30), physical activity therapy (n=2), psychosocial therapy (n=3), Feldenkrais (n=1), collaboration therapy (n=2), stepped therapy (n=1) video-viewing therapy (n=1). The network plot of all interventions is shown in Figure 2. Figure 2Evidence network map for the comparison of different non-pharmacological interventions. The numbers represent different intervention measures. The size of the blue nodes relates to the number of participants in that intervention type, and the thickness of lines between the interventions relates to the number of studies for that comparison.Control group: A total of 36 RCTs utilized non-active control conditions, including waitlist (WL; n=3), treatment as usual (TAU; n=20), enhanced TAU (ETAU=10; ie, education, discussion, etc.) and TAU before WL (n=2).Types of outcome assessment: We found that 15 studies used CES-D (n=2), 12 studies used BDI/BDI-II, 9 studies used PHQ-9, 7 studies used Hamilton Depression Scale HAMD/HRSD, 3 studies used K-6 2 studies used QIDS, 2 studies used GDS and 2 studies used SDS.Risk of Bias AssessmentMost studies presented a low risk in the randomization process and allocation concealment. There were 11 (30.6%) studies rated as having a high risk of bias for ‘blinding of participants and personnel; this is, though, typically unavoidable in studies of psychological interventions. There were 2 studies reported a high risk of bias for ‘blinding of outcome assessment (the residents in the treating group occasionally revealed their participation in group to the raters, thus introducing potential bias among assessors), and this was rated as unclear in 13 studies (36.1%). The risk of attrition bias was low: only two studies did not address incomplete data. Three studies were rated as having a high risk of selective reporting bias. None of the studies reported a high risk of bias for other biases. All studies presented a low risk in the attrition bias and reporting bias. In terms of other bias, most included studies are assessed as low risk. The results of the risk bias assessment are shown in Figure 3. Figure 3(A) Risk of bias summary: each risk of bias item for each included study. (B) Risk of bias graph: each risk of bias item presented as percentage.Network Meta-AnalysisCES-DA total of 15 studies reported the results of CES-D, 2 three-arm trials and 13 two-arm trials, with 14 interventions included. The evidence network plot presented two closed loops (see Figure 4). We conducted a global inconsistency test (P=0.84), a local inconsistency test using node-splitting method (P>0.05) and a loop inconsistency (95% CrI included 0), all showing no significant difference between direct and indirect comparisons. Therefore, a consistency model was selected for analysis. We found that most of the treatments show better efficacy on CES-D compared with CG alone. Interventions with significant statistical differences include: PST (MD−12.30, 95% CrI −20.89, −3.71) (P=0.005), I-CBT (MD −7.94, 95% CrI −13.59, −2.29) (P=0.006), CBT (MD−6.51, 95% CrI −10.72, −2.30) (P=0.002). The SUCRA of CES-D is in order from large to small: PST (90%)>I-CBT (74%)>IPT (68%)>CBT (66%)>G-CT (61%)>Feldenkrais (59%)>G-CBT (44%)>VISA-PWS (42%)>BAT (36%)>PE (26%)>BB-CBT >CG (20%) (see Figure 4). Figure 4Results of the network meta-analysis for CES-D scores. (A) The SUCRA value of individual modalities. The area under the curve predicts the efficacy of various treatment measures. The larger the area under the curve is, the better the efficacy is. (B) Evidence network map of eligible comparisons. The numbers represent different intervention measures. The size of the blue nodes relates to the number of participants in that intervention type, and the thickness of lines between the interventions relates to the number of studies for that comparison. (C) Ranking of each intervention based on the SUCRA values and the league table for the relative effects of all treatments.BDI-II and BDIThere were 10 studies involving 11 interventions using BDI-II for the outcome assessment, 2 three-arm trials and 8 two-arm trials. The node cleavage method showed significant difference between direct and indirect comparisons (P=0.04). Thus, we chose an inconsistency model. According to statistics, PST+BAT (MD −9.04, 95% CrI −15.84, −2.24), ICBT (MD −7.04, 95% CrI −12.21, −1.87), BAT (MD −6.24 95% CrI −11.38, −1.10), CBT (MD −5.74, 95% CrI −10.91, −0.57), T-CBT (MD −5.36, 95% CrI −10.41, −0.31) had positive effects relative to the control group (PI-CBT (83%)>BAT (74%)>CBT (67%)>T-CBT (63%)>BAM (51%)>G-CBT (44%)>DI (29%)>MBSR (28%)>CG (12%)>NI (%)> (8%). The network plot and ranking results are presented in Figure 5A. Figure 5Continued.Figure 5Results of the network meta-analysis for BDI-II/BDI scores. (A) Results of the network meta-analysis for BDI-II scores. (a) The SUCRA value of individual modalities. The area under the curve predicts the efficacy of various treatment measures. The larger the area under the curve is, the better the efficacy is. (b) Evidence network map of eligible comparisons. The numbers represent different intervention measures. The size of the blue nodes relates to the number of participants in that intervention type, and the thickness of lines between the interventions relates to the number of studies for that comparison. (c) Ranking of each intervention based on the SUCRA values and the league table for the relative effects of all treatments. (B) Results of the network meta-analysis for BDI scores. (a) The SUCRA value of individual modalities. The area under the curve predicts the efficacy of various treatment measures. The larger the area under the curve is, the better the efficacy is. (b) Ranking of each intervention based on the SUCRA values and the league table for the relative effects of all treatments. (c) Forest plot of the network meta-analysis compared with control group. (A) > (D); (B) > (E); (C) > (F).For BDI, 2 studies and 3 interventions were included to evaluate the outcome. The consistency model was used due to inconsistency test result showing no significant difference (P>0.05). From the direct evidence, when compared to CG, CBT (MD −6.10, 95% CrI −9.45, −2.75) (P0.05) did not. The forest plot and ranking results are presented in Figure 5B.PHQ-9To evaluate the PHQ-9 outcome, 9 studies and 10 interventions were included. From the network plot of Figure 6, we found that I-CBT (MD −3.90, 95% CrI −4.51, −3.29), AE (MD −3.07, 95% CrI −3.98, −2.16), MTCC (MD −2.50, 95% CrI −3.60, −1.40), CBT (MD −2.20, 95% CrI −2.79, −1.61), IT-CBT (MD−1.62, 95% CrI −2.46, −0.78), CASPER (MD−1.50, 95% CrI −2.13, −0.87), CBM (MD−1.14, 95% CrI −2.11, −0.17), EBT (MD −0.80, 95% CrI −1.26, −0.34) had better effects (P0.05) did not. The SUCRA of PHQ-9 is as followed: I-CBT (99%)>AE (87%)>MTCC (74%)>CBT (68%)>IT-CBT (50%) >CASPER (47%) >CBM (35%)>EBT (23%)>STEPEED (14%)>CG (2%) (see Figure 6). Figure 6Results of the network meta-analysis for PHQ-9 scores. (A) The SUCRA value of individual modalities. The area under the curve predicts the efficacy of various treatment measures. The larger the area under the curve is, the better the efficacy is. (B) Evidence network map of eligible comparisons. The numbers represent different intervention measures. The size of the blue nodes relates to the number of participants in that intervention type, and the thickness of lines between the interventions relates to the number of studies for that comparison. (C) Ranking of each intervention based on the SUCRA values and the league table for the relative effects of all treatments.HAMD/HRSDA total of 7 RCTs assessed depression severity for StD in this group, including 3 using HAMD-17, 2 usingHAMD-14, 1 using HAMD and 2 using HRSD. There was no loop structure (see Figure 7) and consistency model was selected (P>0.05). Among 7 interventions, BAT (SMD −0.95, 95% CrI −1.46, −0.43) and IT-CBT (SMD −0.35, 95% CrI −0.58, −0.12) showed better effectiveness compared to CG (P<0.05). No statistical difference was observed in groups of PST+BAT, T-IPC, G-CT and PST+BT. As ranking results and forest plot showed in Figure 7, BAT was the intervention with the highest ranking, with SUCRA 91%. Figure 7Results of the network meta-analysis for HAMD/HRSD scores. (A) The SUCRA value of individual modalities. The area under the curve predicts the efficacy of various treatment measures. The larger the area under the curve is, the better the efficacy is. (B) Evidence network map of eligible comparisons. The numbers represent different intervention measures. The size of the blue nodes relates to the number of participants in that intervention type, and the thickness of lines between the interventions relates to the number of studies for that comparison. (C) Ranking of each intervention based on the SUCRA values and the league table for the relative effects of all treatments. (D) Forest plot of the network meta-analysis compared with control group.K6, GDS, SDS, QIDSThere were 3 trials for outcome evaluation with K-6 and using inconsistency model (P0.05). In the K6 group, T-CBT (MD−3.1, 95% CrI −4.85, −1.35) showed great effectiveness compared to CG and has the highest ranking (SUCRA, 95%). In the GDS group, BAT (MD−3.06, 95% CrI −4.25, −1.87) and G-CBT (MD −3.01, 95% CrI −4.18, −1.84) showed better effectiveness and BAT was the intervention with the highest ranking (SUCRA, 77%), G-CBT the next-highest ranking (SUCRA, 73%). We found no significant difference both in SDS and QIDS group. The ranking results are summarized in Figure 8A and forest map in Figure 8B. Figure 8Continued.Figure 8Results of the network meta-analysis for GDS/K6/SDS/QIDS scores. (A) Results (SCURA values) of the network meta-analysis for GDS/K6/SDS/QIDS scores. SCURA values of individual modalities based on different scales: (a) K6, (b) GDS, (c) SDS, (d) QIDS. The area under the curve predicts the efficacy of various treatment measures. The larger the area under the curve is, the better the efficacy is. (B) Results (forest plot) of the network meta-analysis for GDS/K6/SDS/QIDS scores. Forest plots of the network meta-analysis compared with control group for different scales: (a) K6, (b) GDS, (c) SDS, (d) QIDS. The black horizontal lines represent the confidence interval (CI) of each study. The blue hollow diamond represents the result of the entire study.Publication Bias and Sensitivity AnalysisWhen the effect size of each indicator was taken as the abscissa and the standard error as the ordinate-corrected funnel plot (see Figure 9). Each dot represented a direct comparison of different interventions, and the number of points of the same color represented the number of pairwise comparisons in the study. The results showed that all studies were basically distributed on both sides of the midline in the middle, the left and right distribution was roughly symmetrical, suggesting publication bias risk low. There was a few studies deviated far from the regression line, suggesting that there may be small sample studies. Sensitivity analysis was performed by sequentially omitting one study and our studies showed stable. Figure 9Funnel plots for effect size and standard error of the included studies. Funnel plots for assessing publication bias in the network meta-analysis for different scales: (A) CES-D, (B) BDI-II, (C) BDI, (D) PHQ-9, (E) HAMD/HRSD, (F) K6, (G) GDS, (H) SDS, (I) QIDS.DiscussionIn this network meta-analysis, we compared the effectiveness of 28 non-pharmacological interventions in changing the severity of depression applied 9 kinds of scale. Our study showed evidence that most non-pharmacological treatments for patients with StD had advantages over the control treatment. Furthermore, we found that PST was the best therapy to improve outcome on the CES-D, similarly PST+BAT on the BDI-II, CBT on the BDI, I-CBT on the PHQ-9, BAT on the HAMD/HRSD and GDS, and T-CBT on the K-6.It’s not difficult to notice that the most effective interventions in each group are all classified as psychotherapy. Based on the above evidence findings, we speculated that psychotherapy especially may have the best efficacy for StD, which is consistent with a previous network meta-analysis showing that psychotherapy especially CBT, may be the most effective intervention for StD.30 They compared all included interventions together, the effect size was expressed as SMD because of the consistency of the scales, so there were differences in statistical methods between us. The effectiveness of psychotherapy in depression has been extensively validated in previous studies among adults, the elderly.68,69 In addition, Cuijpers et al3 found that interventions for StD may have positive acute effects in adolescents. Their team also reported that the types of therapy that have been examined best in primary care settings are CBT, BAT, IPT and PST.70 This has also been mostly validated in our research showing that PST, BAT, CBT, I-CBT, and T-CBT performing better effectiveness for StD.Kasckow et al58 conducted a pilot study suggesting that a six-to-eight session version of PST may produce improvements in mental health functioning in primary care veterans with StD, which the sessions of PST focused on seven steps: defining the problem; setting a realistic goal; “brainstorming” on potential solutions; considering the advantages and disadvantages of each solution; choosing the best solution; developing an action plan; and reviewing progress. Similar courses have also been implemented in other studies, PST seems well suited for medically ill the elderly given its collaborative and problem-centered approach to daily living with stressful chronic medical conditions.62 However, the intervention effects were preserved until 1-year but not until 8-year follow-up, may indicating its difficult to achieve long-term prevention and necessary to conduct systematic booster sessions.42BAT is derived from the behavioral model, which was practiced to play an important role in reducing depressive symptoms and increasing behavioral activation for stroke individuals with StD.38 For patients with cognitive and communication impairments, BAT may be an option to consider, which mainly facilitates activities much simpler or even more cost-effective.41CBT, as an widely used individual intervention, had been demonstrated the superiority in treating mental problems. It was described that CBT could modify dysfunctional thoughts, core beliefs and information processing biases, which are preconditions and risk factors for depression.71 According to our study, I-CBT was the second highest intervention on both the CES-D and BDI-II, others also reported its greater reductions on all the outcomes compared to the WL group at post-intervention.72 I-CBT is an online intervention with many advantages, including overcoming some limitations of face-to-face cognitive-behavioral therapy, 73 being conducted anytime and anywhere, having relatively lower costs, providing privacy protection, 72 helping patients avoid the stigma incurred by seeing a therapist.74 Compared to I-CBT, T-CBT is also a remote CBT but needs to be administered by trained psychotherapists rather than self-help. Digital-based interventions are probably more easily disseminated on a large scale, implying that they save a lot of medical resources and costs.Over the recent years, neuroimaging studies have identified structural and functional brain changes in patients with depression, including volume reductions in cortical and subcortical structures,75,76 reduced gray matter volume throughout the brain, enlarged lateral ventricles, and white matter microstructural differences.75,77,78 Synaptic activity plays an important role in the network pathways that emerge from cognitive, emotional, and behavioral functions. Fries et al summarize relatively comprehensively the six major hypotheses for the pathogenesis of depression, elucidating how signaling pathways and molecular systems interact in depression and how each pathway or system relates to synaptic transmission.79There has been a gradual increase in the emphasis on neuroplasticity. It refers to the ability of the nervous system to change its own loops, such as changes in the morphologic structure and functional activity of the central nervous system and peripheral nervous system, i.e., which can readily change the process of information processing. Impaired structural and functional plasticity of neurons in brain regions related to the control of emotions, could in turn affects the clinical manifestations of depression. Based on this, some researchers have proposed the precise modulation of neuroplasticity pathways as a new strategy for antidepressant treatment. Well-designed computerized cognitive training a resulting in activation of the medial prefrontal cortex (mPFC) and improve neural dysfunction in a serious behavioral deficit in schizophrenia.80 The process of synaptic plasticity as LTP-like hippocampal-dependent memory is maintained by fine-tuning mechanisms that control the balance of excitatory and inhibitory neurotransmission.81 Chung et al82 investigated whether cognitive control training (CCT) persistently alters hippocampal neural circuit function. They show that 1) CCT facilitated learning of novel tasks in a new environment for several weeks relative to unconditioned control mice and control mice that avoided the same location when interference was reduced. 2) CCT rapidly altered the function of the entorhinal olfactory cortex-dentate gyrus synaptic circuits resulting in excitatory-inhibitory subcircuitry alterations that persisted for months. 3) CCT increased inhibition by attenuating the dentate response to inputs from the medial entorhinal cortex and, by de-inhibition, by augmenting the response to strong inputs, suggesting an enhanced overall signal-to-noise ratio. These neurobiological perspectives support the neuroplasticity hypothesis that CCT consistently optimizes information processing in neural circuits in addition to storing item-event associations.However, the effect of physical activity therapy, psychosocial therapy, stepped therapy and others explored did not show the best therapeutic effects. We are mainly considering that the proportion of articles included is small and the statistical power may be insufficient due to the limited sample size. Whether non-psychotherapy has a unique benefit for treatment of StD requires further research.LimitationsAdmittedly, there were some limitations in this network meta-analysis. Many RCTs use inconsistent scales, which limited the comprehensive evaluation of the efficacy of all non-pharmacological interventions included. The studies applying non-psychotherapy interventions are relatively insufficient, more multi-center and high-quality RCTs are needed in the future. Most articles we included were of short-term duration and long-term effects that needed further study. The study languages were limited to English, which may have led to language bias and miss other treatments with national characteristics.ConclusionThe network meta-analysis showed that psychotherapy may be the most effective intervention for StD compared to other interventions, especially PST, BAT, CBT, Digital-based CBT. This study provides some evidence on StD management selection for clinical workers. However, to establish its intervention effect more conclusively, the content, format and operators of psychotherapy still require extensive exploration to conduct more effective, convenient, and cost-effective implementation in primary healthcare. Considering the limitations of this study, notably, further research is also urgently needed to find the biological and neural mechanisms of StD by examining whether psychotherapy alters neuroplasticity in patients with StD.

PMC

BMB Reports

38052423

PMC10761751

12-31-2023

10.5483/BMBRep.2023-0190

Nonsense-mediated mRNA decay, a simplified view of a complex mechanism

Carrard Julie, Lejeune Fabrice

Nonsense-mediated mRNA decay (NMD) is both a quality control mechanism and a gene regulation pathway. It has been studied for more than 30 years, with an accumulation of many mechanistic details that have often led to debate and hence to different models of NMD activation, particularly in higher eukaryotes. Two models seem to be opposed, since the first requires intervention of the exon junction complex (EJC) to recruit NMD factors downstream of the premature termination codon (PTC), whereas the second involves an EJC-independent mechanism in which NMD factors concentrate in the 3’UTR to initiate NMD in the presence of a PTC. In this review we describe both models, giving recent molecular details and providing experimental arguments supporting one or the other model. In the end it is certainly possible to imagine that these two mechanisms co-exist, rather than viewing them as mutually exclusive.

INTRODUCTIONExpression of a gene requires perfect correlation between the message contained in the genomic DNA sequence and the resulting RNA or protein. Of course, a given genomic sequence can lead to different RNA or protein isoforms, notably through alternative splicing events. This type of event is extremely well regulated and contributes to the genetic program. On the other hand, some changes can lead to synthesis of aberrant RNA or protein products, which are generally detected by quality control mechanisms. Among these, nonsense-mediated mRNA decay (NMD) is likely the most studied (1-4). NMD is an mRNA surveillance mechanism found in all eukaryotes studied to date: animals, plants, and fungi (5-7). It acts as a premature termination codon (PTC) detector. Once an mRNA carrying a PTC is detected, it is processed by a dedicated machinery which degrades it very quickly in order to prevent its translation to a truncated protein.NMD thus enables cells to eliminate mRNAs liable to cause synthesis of a truncated protein. Such proteins are very often nonfunctional, or they may have acquired a function that is deleterious for the cell. The efficiency of NMD is such that the level of a PTC-carrying mRNA can be reduced by 75-100%. Interestingly, even when the PTC-carrying mRNA is present at a non-zero level, it is not translated to protein: during its translation, the proteasome degrades the peptide being synthesized, notably thanks to one of the central NMD factors, the UPF1 protein . This protein degradation, however, is compatible with presentation at the cell surface, by the major histocompatibility complex class I, of peptides of 8 to 12 amino acids long derived from peptide synthesis during the pioneer round of translation (9, 10). This is precisely the mechanism exploited in the development of anti-cancer therapeutic approaches using NMD inhibition to induce synthesis of neo-epitopes (11, 12).NMD appears as a formidable mechanism for degrading PTC-carrying mRNAs, but that isn’t all: certain genes have diverted this surveillance mechanism into a very powerful gene regulator (13, 14). This implies that NMD must also be tightly regulated by different mechanisms , notably in a tissue-specific manner . For example, COL10A1 mRNA carrying a PTC is strongly degraded by NMD in cartilage cells unlike in non-cartilage cells . The cell status also influences NMD efficiency. For example, a gene like GADD45b which has been shown to be a pro-apoptotic gene has its expression repressed by NMD when cells are alive unlike when cells move towards cell death by apoptosis (17-19). It is thus easy to understand that NMD must be activated or inhibited at specific times or locations, and this involves extremely complex machinery. Since its discovery in 1979 NMD has been dissected, and although there are still gray areas, it is certainly the best-studied quality control mechanism. This review aims to take stock of the knowledge accumulated on mRNA degradation through NMD in human cells. By presenting the most convergent and established data, we hope to make this mechanism accessible, although in certain aspects it still remains quite confusing.RECOGNITION OF PTCsFor an mRNA to be degraded by NMD, the first step is its recognition as a PTC-carrying mRNA. PTCs arising from a nonsense mutation, from a frameshift mutation, or from a modification of the splicing profile, are recognized during translation by the ribosome. Just to remind, a nonsense mutation is a point mutation in DNA that changes a codon specifying an amino acid into a translational stop codon. A frameshift mutation could be caused by a DNA sequence insertion or deletion in the open reading frame of a gene. The ribosome, by pausing, will trigger initiation of NMD. Fig. 1 illustrates what happens at a stop codon, according to whether it’s a physiological one or a PTC. When the ribosome arrives at a stop codon, no tRNA enters the A site. Instead, the site accepts the translation termination complex, within which the eukaryotic Release Factor 1 (eRF1) mimics the tRNA. At the physiological stop codon at the end of the open reading frame, the polyA binding protein C1 (PABPC1), bound to the polyA tail, recruits the translation termination complex to the A site of the ribosome (Fig. 1). On a PTC, PABPC1 is distant from the ribosome located on the stop codon. Recruitment of the translation termination complex occurs through an NMD factor, the UPF3X protein (also called UPF3B), and indirectly through another NMD factor, the UPF1 protein, shown to accumulate in the 3’UTR of mRNAs (20, 21). The role of UPF3X would be to slow down recognition of the PTC by eRF1 and to promote release of the nascent peptide and the ribosome . Delayed PTC recognition is certainly very important, and could lead to a kinetic difference between translation termination at a physiological stop codon via the PABPC1 and translation termination at a PTC via UPF proteins. After departure of the ribosome, UPF2 may join UPF3X to stimulate the 5’-to-3’ helicase activity of UPF1 (23, 24). This promotes removal of protective proteins from the downstream region of the PTC, the ribosome having already removed the proteins upstream of the PTC when reading the mRNA. The exposed mRNA then becomes a substrate for RNases as described in the next paragraph.mRNA DEGRADATION BY NMDAn mRNA can be degraded via different pathways, including degradation from either the 5’ or the 3’ end or through endonucleolytic cleavage generating free 5’ and 3’ ends for exoribonucleases (25-29). NMD involves all these degradation pathways, as if to ensure that as few as possible PTC-carrying mRNAs escape degradation (Fig. 2). Activation of the 5’-to-3’ pathway is initiated by interaction of the phosphorylated NMD factor UPF1 with the SMG5/SMG7 or SMG5/PNRC2 heterodimer . The involvement of PNRC2 in NMD is controversial, however this protein might simply have a general role in the decapping step, since knocking it down does not increase the level of NMD substrates . Both SMG5/SMG7 and SMG5/PNRC2 interact with the decapping complex, particularly with DCP1 and DCP2, so that the cap is removed and a free 5’ end, accessible to exoribonucleases such as XRN1, is generated.The SMG5/SMG7 heterodimer has also been shown to interact with the exosome and with the CCR4-NOT deadenylase complex . In the latter case, direct interaction has been shown between the C-terminal proline-rich region of SMG7 and the POP2 catalytic subunit of the CCR4-NOT complex. On the other hand, the involvement of the exosome has been shown by evidencing protein interactions between UPF factors and exosome subunits such as RRP4 and RRP41, and more recently by demonstrating the involvement of DIS3L2 (RRP44), a subunit of the exosome, in NMD (33, 34).The endonucleolytic degradation pathway is induced by the SMG6 protein which, in interaction with the phosphorylated UPF1 protein, induces a cleavage in the vicinity of the PTC (28, 29). The consequence is the appearance of an unprotected 5’ end and an unprotected 3’ end, which will be targeted, respectively, by exoribonucleases such as XRN1 or by the exosome. It is impossible at the moment to exclude either the hypothesis that all three degradation pathways might be activated on the same PTC-carrying mRNA or that each pathway is activated exclusively. A recent study could answer this question since it suggests that all these degradation pathways are closely linked since the presence of the SMG5/SMG7 heterodimer is necessary for the endonucleolitic activity of SMG6 . Finally and to complete the description of this degradation pathway, it seems that the departure of the ribosome from the mRNA occurs later than initially thought since the ribosome is still detected in the vicinity of the PTC when endonucleolitic cleavage by the SMG6 protein take place .THE DIFFERENT ACTIVATION PATHWAYS OF NMDThis is certainly the most debated part of the NMD mechanism (37-40). There exist at least two competing models of NMD activation for cells of higher eukaryotes, based on published experimental data and described in the following paragraphs. These models attempt notably to explain how a stop codon is recognized as a PTC and how proteins such as UPF1 are recruited to the translation termination complex to activate NMD.EJC-dependent NMDThe first model relates the position of the PTC to splicing events having occurred downstream of this position. By moving the position of a stop codon on a construct coding for triosephosphate isomerase, it was possible to transform the physiological stop codon located in the last exon into a PTC, when an intron was introduced more than 50-55 nucleotides downstream of the stop codon position . The link between NMD and splicing has been confirmed by multiple studies and represents an autoregulatory pathway for certain genes, such as the splicing factor SRSF2 gene (SC35), which activates splicing in the 3’UTR region of is own mRNA so as to transform the physiological stop codon into a codon recognized as a PTC . The splicing reaction results in deposition of a protein complex called the EJC, for Exon Junction Complex. This complex is deposited 20-24 nucleotides upstream of exon-exon junctions as a consequence of a splicing event (43, 44). These are the splicing factors Complexed With Cef1 (CWC) 22 and 27 present in the spliceosome which recruit the EJC core proteins (eIF4A3, MAGOH, Y14 and MLN51) and position them upstream of the splicing event (45, 46). EJC composition evolves from the time of its deposition on the mRNA, at the end of splicing, to the moment of translation. This enables it to play several roles, and notably a role in NMD. The view that this complex is involved in NMD is based on diverse experimental data. First of all, downregulation of EJC components, notably with siRNA, has been shown to inhibit NMD. Or on the contrary, EJC components can induce accelerated degradation when they are tethered to an mRNA (47, 48). Secondly, interactions between EJC components and NMD factors have been demonstrated notably by immunoprecipitation (49-53). This recruitment of NMD factors, particularly UPF3X, by the EJC appears to occur thanks to the protein interactor of little elongation complex ELL subunit 1 (ICE1) .Following recruitment of UPF3X by the EJC, the UPF2 protein joins the complex before the possible arrival of UPF1, if a PTC is detected. The molecular modalities of UPF1 recruitment are not yet fully understood. According to one study, UPF1 is recruited to the EJC as a complex, the SURF complex, with the proteins SMG1, SMG8, SMG9, eRF1, and eRF3 . According to another, UPF1 interacts directly with cap-located CBP80, and CBP80 then facilitates interaction between UPF1 and EJC-located UPF2 . To reconcile these two studies, we could imagine that CBP80 places UPF1 not only on the EJC but also on the SURF complex (Fig. 3A). This possibility is supported by the fact that the ARS2 protein, which interacts with the CBP80/20 heterodimer, also interacts with UPF1, SMG1, and eRF1, thus facilitating interaction of the SURF complex with the proteins carried by the cap . The fact that these studies show an interaction between the SURF complex and the CBP20/80 proteins on the cap indicates that this event takes place during the pioneer round of translation . Once positioned on the EJC, UPF1 is then phosphorylated by the SMG1 kinase (58, 59). Until recently, SMG1 was the only kinase known to phosphorylate UPF1, but two studies have now shown that the protein kinase AKT1 can also phosphorylate UPF1 (60, 61). It has not yet been clarified, however, whether AKT1 can replace SMG1 by acting identically or whether its intervention in NMD activation results from another cascade of interactions.EJC-independent NMDIn this model, the EJC is not required to induce the NMD response. Hence, nor is any splicing event required downstream of a PTC. What comes into play is competition between the UPF proteins located downstream of a stop codon and PABPC1 attached to the polyA tail of the mRNA for recruitment of the eRF1 and eRF3 proteins, leading to translation termination. Thus, the longer the 3’UTR, the greater the number of UPF proteins bound downstream of the stop codon and the greater the probability that recruitment of the translation termination complex will be done by the UPF proteins and not by PABPC1. Moreover, PABPC1 is located furthest from the ribosome paused on the PTC and therefore has less chance to recruit the translation termination complex . This is a model similar to the mode of activation of NMD in other organisms than Human such as Yeast, Drosophila, and the worm C. elegans (62, 63).This model, however, suffers from several experimental contradictions, as it has clearly been demonstrated that a splicing event in the 3’UTR of an mRNA leads to transforming a physiological stop codon into a PTC. Yet a splicing event tends to bring PABPC1 closer to the physiological stop codon, which should oppose induction of NMD. Another paradox stems from the fact that, according to this model, the closer the PTC to the translation initiation codon, the greater the efficiency of NMD, since PABPC1 is very far away and the probability that termination of translation will be induced by UPF proteins is higher (Fig. 3B). Yet no gradient in NMD efficiency has been observed, generally, in higher eukaryotic cells . On the other hand, the EJC-independent NMD activation model fits very well with certain experimental data that cannot be explained by the EJC-dependent model. For example, PTCs located only about fifteen nucleotides upstream of the last splicing event elicit NMD of T cell receptor (TCR) β and immunoglobulin mRNAs . Yet it must be remembered that these mRNAs very often carry PTCs because of rearrangement of the VDJ domains at the gene locus and that the corresponding mRNAs must absolutely be degraded so as not to induce an erroneous immune response. This is also almost certainly the reason why NMD of these mRNAs appears much more efficient than that of mRNAs from genes not involved in the immune response . Everything thus seems to indicate that the mRNAs coding for the T-cell receptor or for immunoglobulins belong to a particular category of mRNAs requiring such high-efficiency NMD that the process can also be activated via an EJC-independent mechanism. Although sequences bordering introns in TCR RNA or sequences in the V segment in immunoglobulin RNA have been identified (65, 66), the molecular mechanism inducing this greater NMD efficiency is still relatively poorly understood. Overall, the EJC-independent mechanism of NMD activation has been less studied than the EJC-dependent model. A distinguishing feature of EJC-independent NMD activation is that it can occur during any translation round, not just the first. It has indeed been shown that mRNAs whose cap is bound by the eIF4E protein can be subject to NMD (67, 68).The steps following UPF1 recruitment are a priori similar in the two models. An important question is why the cell has evolved two pathways of NMD activation. At present, everything suggests that these two pathways are complementary and on the basis of experimental data, we cannot exclude that EJC-independent NMD activation might be limited to certain specific mRNAs or be initiated after EJC-dependent NMD.CONCLUSIONNMD is certainly the most studied of all the quality control mechanisms taking place during gene expression. Inevitably it is also the most debated, given the mass of knowledge accumulated on NMD and the great diversity of models used to study it. Generally speaking, everyone agrees that NMD is not only an mRNA surveillance mechanism that rapidly detects and degrades mRNAs carrying a PTC, but also a gene regulation pathway. The estimated proportion of genes, particularly human, using NMD to regulate their expression ranges from 5 to 10% . One should remember, however, that many of these genes might be only indirectly regulated by NMD. In addition, this estimate was based on the use of siRNA, which can possibly lead to off-target effects. A small-scale study has led to the conclusion that this percentage could be an overestimate .One of the greatest complexities of NMD lies in the number of different proteins involved in the mechanism, whether for PTC recognition or for mRNA degradation. Factors thought to be central may appear non-essential, at least for some NMD reactions. For example, it has been clearly shown that whether certain NMD factors, such as UPF3X and UPF2, are necessary or not depends on the composition of the EJC . Certain EJC proteins are likewise thought to be required in some but not all NMD reactions. For example, the protein MLN51/CASC3, shown to belong to the core of the EJC (71, 72), might not constitutively be a component of this complex (73, 74). The EJC itself has been questioned, particularly in the EJC-independent model of NMD activation: it might simply act as an activator of NMD but not be absolutely necessary for PTC detection. All this information suggests that NMD is a very flexible and certainly evolving process, mediated by protein complexes whose composition varies over time, according to kinetics that has not yet been completely established . This variability of the protein composition of NMD complexes might also reflect modes of regulation that need to be further investigated. For example, SMG1 has long been presented as the only kinase to phosphorylate the UPF1 factor, but very recently a second has been discovered: the protein kinase AKT1. This opens new possibilities for regulation, particularly in cancer cells, where AKT1 is very often overexpressed (60, 61). Clearly the NMD mRNA surveillance mechanism, although theoretically simple in its roles and activation, appears much more complex in its mode of operation. How it interacts with the various cellular metabolic pathways, remains to be studied in much more detail. Such data will certainly make it possible to clarify the parameters necessary for NMD activation and the functional specificities linked to the composition of the protein complexes involved.NMD appears to be a central player in numerous biological processes and in the development of pathologies due to its involvement in the elimination of mutant mRNAs carrying PTCs and in the regulation of numerous genes. Concerning the biological processes in which NMD plays an essential role, we can first mention embryonic development since the absence of expression of the UPF1 gene leads to the death of the embryo at 3.5 days p.c. . The central nervous system seems all particularly dependent on NMD since neurological disorders are observed in patients in which the NMD factors UPF3X, UPF3, UPF2, SMG6, RNPS1 or eIF4A3 are mutated . The differentiation of myoblasts into myotubes has been shown to require inhibition of NMD in order to allow the expression of myogenin . The involvement of NMD in many other mechanisms has been described in recent reviews (1, 3). In a pathological context, NMD can also play a determining role either by preventing the pathology or, on the contrary, by inducing it. In fact, around 10% of cases of genetic diseases are linked to the presence of a nonsense mutation . The consequence of this mutation is the absence of gene expression due to NMD. However, depending on the position of the PTC in the reading frame, some truncated proteins if synthesized in the absence of NMD could partially or completely retain the function of the wild-type protein. For example, in Duchenne muscular dystrophy, all nonsense mutations located from exon 71 could lead to a functional dystrophin . Inhibition of NMD also represents an interesting anti-cancer therapeutic approach, particularly for inducing the expression of neo-antigens on the surface of tumor cells (11, 12, 80). Finally, inhibiting NMD could make PTC readthrough more effective by increasing the quantity of substrate mRNA for readthrough (81, 82). Therefore, although so far no NMD inhibitor has reached the clinical trial phase, NMD inhibition could represent a future therapeutic development.

PMC

Gut

38129101

PMC10958257

4-01-2024

10.1136/gutjnl-2023-331335

ForePass endoscopic bypass device for obesity and insulin resistance—metabolic treatment in a swine model

Angelini Giulia, Galvao Neto Manoel, Boskoski Ivo, Caristo Maria Emiliana, Russo Sara, Proto Luca, Previti Elena, Olsson Lisa, Aggarwal Hobby, Pezzica Samantha, Ferrari Elisa, Bove Vincenzo, Genco Alfredo, Bornstein Stefan, Tremaroli Valentina, Gastaldelli Amalia, Mingrone Geltrude

WHAT IS ALREADY KNOWN ON THIS SUBJECTMetabolic surgery (MS) is a highly effective treatment for obesity and type 2 diabetes, leading to sustained weight loss over time. Notably, MS can reverse several obesity-related comorbidities such as chronic inflammation, hypertension and non-alcoholic fatty liver diseases, including nonalcoholic steatohepatitis (NASH).WHAT ARE THE NEW FINDINGSWe showed that ForePass influences glucose kinetics, enhancing insulin-mediated whole-body glucose uptake, hepatic insulin sensitivity and insulin clearance. Moreover, ForePass modifies plasma metabolites and the variety and structure of faecal microbiota, increasing bacteria that beneficially impact glucose metabolism.HOW MIGHT IT IMPACT ON CLINICAL PRACTICE IN THE FORESEEABLE FUTUREAs a highly effective procedure, ForePass can be considered an incisionless alternative to traditional surgical procedures. ForePass can also be used for high-risk patients who are ineligible for MS, reject a surgical approach, prefer a bridge to MS, or as a complement or substitute to new anti-obesity and anti-diabetes medications. The use of ForePass may provide a much-needed alternative to MS.MessageMetabolic surgery (MS) causes long-lasting type 2 diabetes (T2D) remission through mechanisms that are beyond the mere weight loss being linked to the bypass of the upper gut. The ForePass device combines a channelled gastric balloon with an intestinal sleeve and aims at reducing simultaneously food intake and nutrients’ absorption. In an experiment in four pigs, ForePass significantly lowered blood glucose and powerfully increased insulin-mediated glucose uptake, insulin clearance and reduced endogenous glucose production (EGP) over an observation period of 4 weeks without relevant complications. The weight gain was 79% lower than that observed in 4 sham-operated pigs. ForePass modified the composition of faecal microbiota raising the proportions of bacteria associated with metabolic health. Clinical studies are warranted.In more detailA dramatic increase in T2D rates has been observed over the past 40 years.1 T2D is closely associated with obesity, with over 80% of individuals with T2D having also obesity.1 Standard treatments for T2D and obesity include lifestyle interventions, medical therapy and MS. Nevertheless, lifestyle interventions and anti-obesity medications are only partially effective in determining long-term weight loss.2 In contrast, MS has the potential to achieve long-lasting remission of T2D and reversal of several obesity complications.3 The ForePass device is an endoscopic alternative to MS that links the stomach to the jejunum via a gastric funnel connected to an intestinal sleeve. The balloon, which reduces the gastric volume by approximately 2/3, is traversed by a central channel that connects to the sleeve, which extends through the duodenum and proximal jejunum (figure 1A–C). Hence, ingested foods bypass the duodenum and proximal jejunum arriving directly into the mid-jejunum.Figure 1Characteristics of the Forepass device. (A) The overall structure of the ForePass device, which is composed of a silicone gastric balloon and an expanded polytetrafluoroethylene (EPTFE) intestinal sleeve. A nitinol stent-like funnel, which traverses the balloon, connects to the sleeve. The transplyloric stent, coated by EPTFE, helps to improve device stability. (B) An endoscopic image of the proximal end of the ForePass device, including the inflated gastric balloon. The balloon’s colour is due to methylene blue added to the saline solution used to inflate the device. (C) An X-ray fluoroscopy image of the ForePass device positioned in the stomach and proximal gut. The balloon component of the device is placed in the stomach, while the transpyloric stent and intestinal sleeve are located further down, past the pylorus.We hypothesised that the ForePass, which limits food intake and bypasses the upper gut, significantly improves glucose disposal and reduces weight gain in pigs relative to controls.To this end, we assessed glucose disposal, weight gain, metabolomics and faecal microbiota in eight pigs that were assigned to either Sham-operation (controls) or Forepass. Experimental procedures are shown in detail in the online supplemental appendix.10.1136/gutjnl-2023-331335.supp1Supplementary data After 4 weeks, we observed a large reduction (79%) in the overall weight gain in part due to reduced food intake (22%) and in part to incomplete food digestion with increased faecal nutrient loss in the group with ForePass as compared with sham operation (table 1). We did not observed macroscopical or microscopical lesions of the stomach and duodenal mucosa and submucosa.Table 1Upper part: weight gain and food intakeWeight gain and food intakeSham-OpForePassP value Basal weight (kg)46.88±1.9546.50±1.51NS Final weight (kg)56.38±1.7748.5±1.670.029 Food intake (kg/day)2.00±0.011.77±0.040.028 Glucose minimal model Sham-OpForePassP value SG∙102 (per min)1.28±0.151.57±0.06NS p∙102 (per min)0.37±0.130.17±0.03NS SI∙104 (pm/min)0.41±0.0310.65±0.0320.029 Stable isotope glucose kinetic Sham-OpForePassP value EGP AUC ∙insulin AUC (µmol*pmol*min)10.91±0.594.24±0.450.029 Insulin clearance (l/min)2.61±0.0092.96±0.060.029 Rd AUC/insulin AUC (µmol/pmol*min)0.024±0.0030.039±0.0040.029Upper part: weight gain and food intake. art: basal, final weight and food intake. Middle part: minimal model analysis of glucose, insulin and C-peptide time courses following glucose administration via gastric gavage after Sham-Operation or ForePass. Lower part: stable isotope glucose kinetics. Data are expressed as mean±SEM.SG, glucose effectiveness; p, minimal model parameter; SI, insulin sensitivity; AUC, area under the curve; EGP, endogenous glucose production; Rd, rate of glucose disappearance.We observed a significant decrease in plasma glucose, insulin and C-peptide levels in response to an intragastric glucose load (figure 2A–C) in ForePass. Accordingly, insulin sensitivity was significantly higher in the ForePass than in the sham group (table 1).Figure 2Metabolic shifts after ForePass. (A–C) Time courses and areas under the curve (AUCs) of blood glucose (A), plasma insulin (B), and plasma C-peptide (C) during an intragastric glucose administration (75g) using a combination of ingested and infused stable isotopically labelled glucose tracers in both ForePassTM and sham-operated pigs (Sham-Op). (D–F) Time courses and AUCs of the rate of appearance of exogenous glucose (Ra) (D), glucose rate of disappearance (Rd) (E), and endogenous glucose production. (G) Principal component analysis explains 85.8% of the variance of metabolites that significantly differ between ForePass and Sham-operation. (H) Heat map of polar metabolites 4 weeks after the interventions. Data are presented as mean values±SEM (n=4 pigs per group). Statistical significance values were calculated by Mann-Whitney U test and repeated measure analysis of variance were appropriate.Intragastric glucose administration combined with U-13C-glucose and 6,6-deuterated glucose infusion resulted in a significantly lower glucose rate of appearance and disappearance in the ForePass than in the sham-operated group (figure 2D and E). Moreover, EGP was markedly suppressed with the ForePass (figure 2F) indicating a better hepatic insulin sensitivity. Accordingly, we observed a higher hepatic insulin-sensitivity and insulin clearance as well as higher whole-body insulin-mediated glucose uptake in the ForePass versus the sham group (table 1).To gain further insight into the mechanisms responsible for the improvement of insulin sensitivity following the ForePass implant, we performed polar metabolite analysis using GC/MS/MS. Figure 2G shows that the first two components of the principal component analysis explain 85.8% of the variance in plasma metabolites that significantly differ between sham-operation and Forepass. As observed in other studies testing markedly reduced energy intake,4 5 we found increased circulating levels of amino acids and their metabolites in pigs with ForePass as compared with sham-operated animals (figure 2H, online supplemental table 1, online supplemental figure 1). Among amino acids, we found a surge of branched chain amino acids, valine, isoleucine and leucine, which are essential amino acids provided only with food. In agreement with the reduction of EGP, we observed a decrease in gluconeogenesis precursors with ForePass. Specifically, alanine, glutamine and glycine, but also lactate, which contributes from 7%6 to 18%7 to plasma glucose levels after an overnight fast.10.1136/gutjnl-2023-331335.supp2Supplementary data 10.1136/gutjnl-2023-331335.supp3Supplementary data To understand how caloric restriction and upper gut bypass could affect gut microbiota composition, we profiled the V4 region of the 16 S rRNA gene in faecal samples. Faecal microbiota beta diversity, estimated both as weighted UniFrac (online supplemental figure 2A, p=0.026, R2=0.42) and unweighed UniFrac (online supplemental figure 2B, p=0.023, R2=0.32), was strongly affected by the ForePass device, indicating an effect on dominant faecal taxa (online supplemental figure 2C). The abundance of the genera Treponema and Prevotella decreased, while Akkermansia, Christensenellaceae R-7 group, Bifidobacterium and the Archaea Methanobrevibacter significantly increased (online supplemental figure 3A-F). The taxa increased with ForePass correlated significantly with the reduction of the area under the curve of EGP and insulin and with fasting C-peptide (online supplemental figure 4).CommentsOur study shows that the ForePass device reduces body weight gain by 79% in rapidly growing pigs due to reduced food intake and increased faecal energy loss. Glucose absorption was also reduced by 67%. Whole-body and hepatic insulin sensitivity were significantly increased with consequent reduction of insulin secretion and improvement of insulin clearance.Other endoscopic procedures, such as duodenal-jejunal bypass liner (DJBL) and duodenal mucosal resurfacing (DMR) have attempted to mimic the effects of MS on T2D and insulin resistance. DJBL improves glycaemic control in people with T2D, with an average HbA1c reduction of 0.9% and a weight loss of 11.3 kg at 1-year follow-up as compared with controls.8 However, in people with insulin resistance and T2D, DJBL does not suppress EGP.9 DMR improves glycaemic control in T2D10 likely via a weight-independent mechanism, since weight loss was not significant at 6-month follow-up.10 In women with insulin resistance, obesity and polycystic ovary syndrome, DMR did not improve significantly hepatic insulin sensitivity as shown by the lack of EGP suppression at 6 months after the procedure.11 With due precaution when comparing human and swine data, ForePass significantly reduces EGP showing its beneficial effect in improving hepatic insulin resistance and potential in the treatment of both T2D and non-alcoholic fatty liver disease, where EGP is a key element.12 Moreover, ForePass reduces major gluconeogenic substrates and, consequently, gluconeogenesis.ForePass had an effect not only on glucose homeostasis but also on the composition of gut microbiota. The literature provides evidence that the gut microbiota has a substantial impact on metabolism and can modulate various aspects of the metabolic syndrome beyond obesity, including insulin resistance and glycaemic control.13 14 In this study, we found that the ForePass device promotes the formation of a microbiota pattern that is known to be associated with a better metabolic outcome.In conclusion, the ForePass device reduces glucose absorption and EGP, enhances whole-body insulin-mediated glucose uptake and hepatic insulin sensitivity with increased insulin clearance, reduces gluconeogenic substrates and improves the abundance and composition of faecal microbiota promoting a configuration that positively affects glucose metabolism. ForePass is an endoscopic procedure that, contrary to MS, is completely reversible. It could be used for those hesitant to undergo MS, high-risk patients who are ineligible for MS, as a bridge to bariatric surgery or MS, as well as a complement or substitute to new anti-obesity and anti-diabetic medications for these lifelong diseases.

PMC

RSC Advances

38348294

PMC10859695

10.1039/d3ra07771c

Performance and mechanism of a bioelectrochemical system for reduction of heavy metal cadmium ions

Wang XiaXia, Zhao Yu, Jin Li'E., Liu Bin

This study explores the removal of Cd(ii) from wastewater using a microbial electrolysis cell (MEC) to investigate the electrochemical performance and removal kinetics of an anodic polarity reversal biocathode and the mechanism of action of electrochemically active bacteria. Comparative electrochemical methods showed that using an anodic polarity reversal biocathode resulted in greater than 90% removal of different concentrations of Cd(ii) within three days, which may be related to the catalytic effect of anodic electrochemically active bacteria. However, due to the ability of bacteria to regulate, up to nearly 2 mg L−1 of Cd(ii) ions will remain in solution. As shown by the linear fitting relationship between scanning speed and peak current, the removal process was dominated by adsorption control for 20–80 mg L−1 Cd(ii) and diffusion control for 100 mg L−1 Cd(ii). The analysis of raw sludge and sludge containing Cd(ii) showed that Arcobacter and Pseudomonas were the primary cadmium-tolerant bacteria, and that the ability to remove Cd(ii) was the result of a synergistic collaboration between autotrophic and heterotrophic Gram-negative bacteria. This study explores the removal of Cd(ii) from wastewater using a microbial electrolysis cell (MEC) to investigate the electrochemical performance and removal kinetics and the mechanism of action of electrochemically active bacteria.

1.IntroductionPollution caused by Cd(ii) metal is a severe threat to ecological stability and human health. Cd is commonly used in industries such as electroplating and mining, and it often accumulates in industrial wastewater in various forms, including ions and compounds. From wastewater, Cd accumulates in the soil and eventually throughout the food chain, which results in organisms becoming the ultimate bearers of heavy metals. Cd(ii) is not an essential element in the human body, and it can cause chronic poisoning of the kidneys and chondromalacia.1 However, because the half-life of Cd(ii) is 10–30 years, Cd will continue to be hazardous to life and health even if its enrichment is stopped.2Bioelectrochemical systems (BESs) utilize anodic microorganisms to degrade organic matter and release the resulting electrons to the surface of a solid anode, where the electrons are transported through an external circuit and are received at the cathode for reduction reaction.3 The BES consists of a microbial fuel cell (MFC) that generates electrical energy and a microbial electrolysis cell (MEC) that produces hydrogen energy. Based on the MFC, the MEC has been developed for applications such as hydrogen and methane production, heavy metal ion reduction, and coupling to other devices. In addition, the performance of the cathode material in the MEC affects the overall power reduction efficiency.Biocathodes are currently a new technology whereby pollution treatment and energy saving are combined. Electroactive microorganisms (membranes) on low-cost electrode carriers are a cathodic reaction catalyst, with living microbial cells (electrical nutrients) as the basis to reduce overpotential with accompanying high-performance operation. There are various advantages to using microorganisms as catalysts, such as self-regeneration, adaptability, mild operating conditions, and low cost.4–6 Although early research on the biocathode MEC focused on hydrogen production, one of the most promising applications of the MEC is to combine hydrogen production with the removal of heavy metal ions from wastewater by simultaneously acquiring electrons and generating a green and clean energy source, which is hydrogen.7Yiran1et al. discovered that an increase in voltage led to an increase in Cd(ii) removal for biocathodes domesticated at different voltages. Aradhana31et al. explored the role of using biocathodes for the removal of mixed metal ions in an MFC facility, and showed that the removal of nickel(ii) and cadmium(ii) was high at low concentrations versus high concentrations because the toxic environment at high concentrations exceeded the tolerance of the microbial cells and caused damage to the biocathodes. Therefore, adding an appropriate concentration of metal ions to the solution is crucial for the survival of microorganisms and the proper functioning of the biocathode. The application of MEC devices in metal removal or recovery has become a promising technology, such as the use of NaHCO3 as a carbon source to recover various heavy metal ions on a biocathode in a double-chamber electrochemical system.8 Additionally, there were greater advantages with single-chamber bioelectrochemistry in the treatment of a combination of organics and heavy metal wastewater.9Electroactive microorganisms are indispensable components of BESs, and the species composition varies from system to system. However, most microorganisms originate from the same phylum, such as Proteobacteria, Acidobacteria, and Bacteroidetes. According to previous studies, the Proteobacteria phylum contains several types of electroactive microorganisms, most of which are metal-reducing organisms that can be effectively used in wastewater treatment.10,11 Alternatively, there are two pathways for electron transfer: intracellular and extracellular, where intracellular transfer refers to the simultaneous transfer of electrons by electrochemically active bacteria utilizing their specific metabolic pathways (i.e., the release of extracellular polymeric substances). Extracellular transfer refers to redox reactions within the MEC system relying on the conductance of electrons over long distances (several centimeters) by the microorganism's flagella and cytochromes, which enables the diffusion of electrons from the cell to the solution.12,13The polarity-reversal domestication of a band into a biocathode has also been employed. Wu5 compared in situ and ex situ domestication methods for biocathodes. The ex situ domestication method accelerated the domestication time of the biocathode and enhanced the treatment capacity of the biocathode. The biocathode was domesticated in individually acclimatized Cd(ii) in a double-chamber MEC to achieve nearly complete removal of the metal, and the performance of the reaction system was evaluated based on metal removal and coulombic efficiency. An investigation of Cd(ii) removal kinetics and high-throughput 16S rDNA gene sequencing were conducted to characterize the microbial communities of the original and Cd(ii)-containing sludge for comparing the metal removal rates of the biocathodes at different times and at various initial Cd(ii) concentrations.2.Materials and methods2.1MEC configuration and start-upThe double-chamber MEC reactor was constructed from Plexiglas using a cation exchange membrane (CMI-7000S Hangzhou Hua Membrane Technology Co., Ltd) to separate the anode and cathode chambers. The effective volume of the cathode and anode chambers was 80 mL (diameter: 5 cm, height: 8 cm, Tianjin Gosh Rui Co., Ltd), and the cathode and anode electrode material consisted of carbon felt (2.5 cm × 5.0 cm, 0.8 cm thick, Tianjin Haitian Muzi Graphite Products Factory). A saturated Ag/AgCl reference electrode (0.241 V vs. SHE) was placed in the cathode chamber of the reactor to collect cathode potential data.2.2Inoculation and handlingThe MEC (Fig. 1) reactor was started using the same inoculum in all experiments, which was collected from the Yang Jia Bao Wastewater Treatment Plant, Taiyuan, China. The reactor electrolyte consisted of mixed bacteria (20 mL) and buffer solution (60 mL), where the mixed bacteria consisted of aerobic and anaerobic bacteria in a 1 : 1 ratio, and the buffer solution contained the following components: NaH2PO4·2H2O (3.32 g L−1), Na2HPO4·12H2O (10.32 g L−1), NH4Cl (0.31 g L−1), KCL (0.13 g L−1), CH3COONa (1 g L−1), and trace minerals (10 mL L−1). In addition, the initial pH in the electrolyte varied from 6.8 to 7.2. The circuit current was monitored by applying an external voltage of 1.0 V and subsequently connecting a multimeter (UT52 Multimeter) and recording the current every hour until it exceeded 1 mA and stabilized at a maximum value for three consecutive cycles. This indicated that the removal process in the anodic domestication of anhydrous sodium acetate biofilm carbon felts was well established. All experiments were performed using the same inoculum for the reactor inoculation start-up process.Fig. 1Diagram of the double-chamber microbial electrolysis cell device.A double-chamber MEC was constructed to domesticate the biocathode by reversing the polarity of the abovementioned biological anode, and the electrolyte simulating cadmium-containing wastewater as a biocathode inoculum was based on the same inoculum as described above for the anode with the addition of Cd(ii). CH3COONa was used as the only carbon source for the cathode microorganisms. The initial Cd(ii) concentration in the solution was increased in gradients of 5, 10, and 20 mg L−1, and the length of each cycle was four days. Biofilm cathodes are tolerant of the toxicity of a single heavy metal, and were obtained when the circuit current stabilized. All experimental MECs were domesticated in a constant temperature shaker at 35 °C.2.3Methods of testing and analysisCd(ii) was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Thermo Fisher CAPPRO) in all experiments. The removal rate of Cd(ii) (mg L−1), coulometric efficiency (CEca, Cd), energy efficiency per unit (ηE, Cd), and energy consumption per unit (kW h kg−1) were calculated for the cathode of the MEC device, as shown in the ESI.†The electrochemical behavior of the biocathode and abiotic cathode was determined by cyclic voltammetry (CV) using a three-electrode configuration with an electrochemical workstation (CS2350M, CORRTEST, Wuhan, China) at scanned potentials of −1.2 V to 0.2 V (vs. SHE) and a scan rate of 1.0 mV s−1. CV is an electrochemical method used to characterize redox reactions on the surface of a reactive electrode, and it was used to analyze the response of the cathodic electrode to the presence of Cd(ii), with the cathode as the working electrode, Ag/AgCl as the reference electrode, and the anode as the counter electrode. The Tafel slope is an essential parameter for determining the catalytic activity of the cathodic hydrogen precipitation reaction, and we used a scan rate of 1–10 mV s−1 to study the cathodic reaction kinetics. The kinetics of the electrode process were analyzed by electrochemical impedance spectroscopy (EIS). The change in impedance with sinusoidal frequency was measured to study the mechanism of the electrode material, and used an equivalent circuit fitted with the electrochemical workstation.In sampling the original sludge and the Cd(ii)-containing sludge for microbial identification, 16S rDNA can be used as a characteristic nucleic acid sequence for revealing biological species.13–15 First, the genomic DNA of the samples was extracted by the sodium dodecyl sulfate (SDS) method and then tested for purity and concentration. Second, PCR amplification of selected V3–V4 variable regions, which are highly variable regions of bacterial genes, was performed using specific primers containing the barcode and Phusion® High-Fidelity PCR polymerase. Then, PCR products were detected by 2% agarose gel electrophoresis, and cut gel recovery was then performed using an AxyPrepDNA Gel Extraction Recovery Kit (Axygen). The PCR-amplified recovered products were detected and quantified using a QuantiFluor™-ST Blue Fluorescence Quantification System (Promega).Next, library construction was performed using the NEB Next®Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, USA), and the quality of the built libraries was confirmed by an Agilent Bioanalyzer 2100 system and Qubit. Lastly, based on the characteristics of the amplified 16S region, a small fragment library was constructed for paired-end sequencing using the Illumina MiSeq sequencing platform. Through the clustering of operational taxonomic units (OTUs), species annotation and abundance analysis can reveal the species composition of the samples. It can also explore the differences between the samples to identify the differential bacterial communities.3.Results and discussion3.1Performance of the abiotic and biotic cathodesAbiotic and polarity-reversed biological cathodes were compared under open-circuit (OC) and closed-circuit (CC) conditions for three initial concentrations of Cd(ii) ranging from 20 mg L−1 to 60 mg L−1 after 24 h of treatment in the MEC. The Cd(ii) removal rate was significantly higher in the CC-biotic than in the OC-biotic under CC-abiotic conditions at all three concentrations (Fig. 2A). Therefore, this indicates that electrophilic microorganisms and circuit currents play a significant role in Cd(ii) removal.Fig. 2(A) Comparative plots of Cd(ii) removal rates under CCC, OCC, or abiotic control conditions. Electrochemical diagrams of (B) biocathode and non-biocathode CV, (C) Tafel curve, and (D) EIS (initial Cd(ii): 40 mg L−1, 60 mg L−1; applied voltage: 0.5 V).Under OC-biotic conditions, Cd(ii) removal can be related to the physical adsorption of carbon felt and the presence of bacteria, such as biosorption and bioreduction of Cd(ii). The addition of sodium acetate as a carbon source for microorganisms, ammonium ions in the cathodic electrolyte medium,16 and metal-containing trace elements may also serve as electron donors for trophic bacteria (chemo-living bacteria).17In this experiment, the high efficiency of Cd(ii) removal by OC organisms may be attributed to the fact that the biocathode used has been subjected to extensive experiments to convert toxicity-tolerant microorganisms. Moreover, the removal of Cd(ii) from the simulated wastewater by acetate was unchanged, which excluded the effect of Cd(ii) and acetate complexation on the removal process, and acetate was found to facilitate the growth of heterotrophic microorganisms on the biocathode of the MEC.17This study compared the electrochemical properties of abiotic and polarity-reversed abiotic cathodes under CC conditions. The CV (Fig. 2B, S1 and S2†) analyses of the abiotic and biotic cathodes showed a Cd(ii) reduction peak potential of approximately −0.4 V. In contrast, the reduction peak potentials and reduction peak currents in a 20 mg L−1 Cd(ii) solution of abiotic and biotic cathodes did not indicate a significant difference. Therefore, this study utilized Cd(ii) concentrations of 40 and 60 mg L−1 as an example for analysis.The reduction peak potential of the biocathode was negatively shifted compared with that of the abiotic cathode (Fig. 2B), which indicates that more negative peak potentials of the biocathode were more favorable for the reduction reaction and reduced the total free energy required for electron transfer during electrochemical processes. The biocathode also exhibited a more positive reduction onset potential than the abiotic cathode [biotic/abiotic: 0.124 V/0.100 V at 40 mg L−1; biotic/abiotic: 0.128 V/0.091 V at 60 mg L−1] (Fig. S1†) and a small reduction peak current. These differences can be attributed to electrochemically active bacteria on the electrode surface, increased mass transfer resistance, and decreased peak current.18 However, the electrochemically active bacteria shifted the peak and reduction onset potential to the left, confirming the catalytic activity of polar inversion biofilms for metal reduction.The kinetic parameters were determined using Tafel polarization curves, as shown in Fig. 2C. The Tafel curves for different concentrations and electrodes exhibited similarities, and the intercept and slope of the Tafel curves were obtained by linear fitting, with the slope = (1 − α)F/2.3RT.18 The transfer coefficient α is 0.98 for the abiotic cathode and 0.99 for the biotic cathode. The lower Tafel slope [biotic/abiotic: 14.25 mV dec−1/24.89 mV dec−1 at 40 mg L−1; biotic/abiotic: 13.94 mV dec−1/21.29 mV dec−1 at 60 mg L−1] (Fig. 2C) indicated a favorable electrochemical performance, which suggested that the biocathode exhibited superior electrochemical performance compared to the abiotic cathode. Based on the intercepts of the fitted curves, the exchange current density (j0) was higher at the abiotic cathode than at the biotic cathode. This observation was in accordance with the results of the CV analysis, and emphasized that the catalytic role of electrochemically active bacteria and the kinetic reaction rate of electrodeposition are related to the speed of electron transfer.The equivalent circuit of the cathode resistance consists of an ohmic resistor Rs, a double-layer charge transfer resistor Rct, a capacitor Cd, and diffusion impedance Zw.19 From the Nyquist plots of the different cathodes and the corresponding equivalent circuit diagrams, the EIS curves for both electrodes are characterized by semicircular arcs and diagonal lines, with the charge transfer impedance (Rct) derived from the diameter of the semicircle, which represents the resistance to electron transfer between the electrode surface and the adsorbed material on the electrode. Diffusion impedance Zw is the concentration impedance of reactants diffusing from the solution to the electrode reaction interface, and the combination with Rct is helpful for understanding the kinetics of the electrochemical reactions with the redox reactions on the electrode.Based on the data in Fig. 2D and Table 1, it is evident that there was lower ohmic resistance, charge transfer resistance, and diffusive impedance for the biocathodes with polarity reversal as compared to the non-biocathodes. The biological cathodes exhibited a lower ohmic resistance, resulting in increased conductivity and more rapid electron transfer. As noted by Ha et al.,20 the charge transfer resistance of the electrode is linked to the activation energy of the cathode, and serves as an indicator of the kinetics of the cathodic reaction. Moreover, electrodes with low charge transfer resistance were more conducive to reduction reactions, and the magnitude of the diffusive impedance is proportional to the slope of the oblique line.Data fitting comparison between EIS and Tafel curve of the abiotic cathode and biological cathodeCd(ii) mg L−1 R s/Ω R ct/Ω W/ΩSlopeInterceptAbiotic405.94714.64739.6070.248930.63613605.42643.989.9760.212890.35052Biotic402.73462.687226.2550.142500.31638603.33841.764320.5440.139410.22284The polarity of the reversed bioelectric polarographic line indicated an angle greater than 45° from the real axis, signifying rapid ion diffusion toward the electrode. In contrast, the non-biological polarographic line fell below 45° from the real axis, which may have occurred due to the electrode surface roughness and the variability of the solution state, indicating that the biocathode surface absorbed more electroactive bacteria and reduced the impedance for solution diffusion to the electrode surface. However, the charge transfer resistance for both electrodes was higher in the 40 mg L−1 solution as compared to the 60 mg L−1 solution, and this difference was mainly due to the increased concentration of metal ions in the cathode electrolyte, which led to facilitated electron movement, lower resistance, and decreased charge transfer resistance. These phenomena indicate a greater driving force for kinetics, more rapid charge transfer, and stronger reducing capabilities at the electrode.21,223.2Effect of Cd(ii) concentrationWe investigated the influence of Cd(ii) concentration on system performance and circuit current magnitude across a concentration range of 20–100 mg L−1. The data showed that the removal of five initial Cd(ii) concentrations from biofilms at the same applied voltage of 0.5 V ranged from 90.80% to 98.08% after four days (Fig. 3A), suggesting that heterotrophic microorganisms within the biofilm gradually adapted to the Cd(ii)-tolerant bacteria in the replicated experiments at progressively higher concentrations. These Cd(ii) removal rates were higher than those of 69.2 ± 1.5% in the non-ex situ domestication of biocathodes at the same applied voltage.1 The Cd(ii) removal rates were also higher than 70% for the previous biocathodes under the same carbon source.31 However, different regularities were observed at different times in the removal rates for Cd(ii) at low (≤40 mg L−1) and high (≥60 mg L−1) concentrations in wastewater.Fig. 3Comparison of the (A) removal rates, (B) current change plots, and (C) CV with different concentrations of Cd(ii), and (D) plot of peak current vs. scan rate or square root of scan rate with biocathodes (initial acetate: 1.0 g L−1; applied voltage: 0.5 V).For instance, the removal rate in the cathodic solution with an initial concentration of 20 mg L−1 Cd(ii) gradually increased from 73.03% to 92.57% within a day and reached the highest removal rate on the third day. By contrast, a decrease in the removal rate (90.80%) was observed on the fourth day of the treatment process (Fig. 3A). This trend was also observed for Cd(ii) wastewater with an initial concentration of 40 mg L−1. On the contrary, the rate of removal of high-concentration Cd(ii) (60 mg L−1) from wastewater increased from 89.19% to 96.93% within four days (Fig. 3A).This trend is also observed from Fig. S3 (ESI),† where the standard deviation of the removal rate on the third day was smaller. In addition, combined with the standard deviation graph of different Cd(ii) concentrations, the relative error at higher concentrations was small, illustrating that an enhanced Cd(ii) removal rate by the electrochemical bacteria and the biological cathode achieved optimal removal capacity under this repeated selective pressure with elevated Cd(ii) levels and prolonged acclimation time.The decreased removal rate at low concentrations may be due to accumulated or adsorbed Cd(ii) within the microorganisms being re-released in solution, which occurred because microorganisms can regulate and maintain the internal balance of the system. The residual Cd(ii) ions in high- and low-concentration wastewater solutions are approximately 2.00 ± 0.2 mg L−1, which indicates that the removal of Cd(ii) by the biocathode is unlikely to reach 100%. Nevertheless, following extensive domestication, the cathode exhibited a breakthrough capacity for applicability beyond environments characterized by low concentrations and low toxicity.The circuit currents at different concentrations were observed for four days of operation at 0.5 V (Fig. 3B). There were two patterns exhibited by the circuit currents of the biocathode: one was a curve that rapidly decreased from a higher point at the beginning and then gradually increased and subsequently decreased, and the other was a curve that gradually increased from the lowest point to the highest point and then decreased. The initial rise to the highest point represents the oxidation and reduction of organic material by microorganisms on the electrodes. The subsequent gradual increase signifies the progressive decomposition of the organic material due to substrate depletion.In the study by Colantonio and Kim,23 the magnitude of the current generated conferred no direct effect on the removal rate of cadmium from wastewater during the operation of the MEC unit. There were trends in output currents caused by different concentrations of Cd(ii), as shown in Fig. 3B, and the removal rates on the third day were all 90% (Fig. 3A). This finding excludes a direct impact of the circuit current and the removal rate for different concentrations of Cd(ii).Table S2† shows the comparison of cathodic electrons (CEca, Cd) and energy efficiency (ηE, Cd) consumed for the cathodic reduction of Cd(ii) and cathodic energy consumption per unit (kW h kg−1) for different initial Cd(ii) concentrations, as described in detail in the ESI and Table S2.† As a result, the cathodic CEan, Cd and ηE, Cd of the MEC device tended to decrease with the increase in removal time for the five Cd(ii) concentrations, while the power consumption gradually rose [CEan, Cd: 1.60% (1D)–0.79% (4D); ηE, Cd: 1.29% (1D)–0.63% (4D)].The example in Table S2† [energy consumption: 14.91 (1D)–30.37 (4D) kW h kg−1; applied voltage: 0.5 V 20 mg L−1 Cd(ii)] also reflects the progressive decrease of Cd(ii) in solution with the increase in removal time. Conversely, the initial Cd(ii) concentration increased from 20 mg L−1 to 100 mg L−1; the cathodic CEan, Cd rose from 0.79% to 4.83%; and ηE, Cd improved from 0.63% to 3.89% in a gradual manner. In contrast, the electrical energy consumption gradually decreased from 30.37 kW h kg−1 to 4.93 kW h kg−1 (Table S2†) on the fourth day of treatment conditions in the MEC unit. Accordingly, the electron utilization and energy utilization efficiencies for the reduction in Cd(ii) ions increased with the increase in concentration, and the electrical energy consumption decreased with the decrease in concentration, which indicates that higher concentrations are favorable for the rapid reduction in Cd(ii) ions. Fig. 3C illustrates the CV analysis conducted across a spectrum of initial Cd(ii) concentrations, ranging from 20 mg L−1 to 100 mg L−1. A reduction peak appeared at the biocathode with acetate as the carbon source, and the reduction peak on the cathode progressively shifted to the left as the concentration increased. This finding indicates that the presence of the Cd(ii) electron acceptor significantly favored the electron transfer of the cathode, although the reason for this favorable effect was unclear because bacterial cells catalyze the reduction in Cd(ii) and the production of acetate and hydrogen through several possible mechanisms.24,25For example, respiratory processes in bacterial cells may be a physiological behavior of Cd(ii)-tolerant EABs in the limited environment of the MEC system or may be a significant channel for them to transfer cathodic electrons to the cathodic solution for facilitating the reduction in metal ions.26 From Fig. 3D and Table 2, peak currents were systematically examined at varying scan rates, spanning from 1–10 mV s−1, across different initial Cd(ii) concentrations to elucidate the rate-limiting step governing the behavior of the biocathode. As the scan rate increased, it led to incremental peak current, accompanied by shifts in positive and negative potential, signifying that the biocathode biocathode was reversible.Linear analysis of CV and peak current vs. scan rate or the square root of the scan rate with biocathodes20 mg L−140 mg L−160 mg L−180 mg L−1100 mg L−1Rv120.993860.995200.995260.994440.98871Rv1/220.984440.984180.989330.972280.99684The peak current displays a linear relationship with the scan rate (ν), which indicates adsorption control, and the phenomenon is known as ‘thin-film behavior’; when the peak current is linearly related to the square root of the scan rate (ν1/2), it indicates diffusion control.11,27 The fitting analysis showed that the linear relationship between the peak current and scan rate (ν) was superior to that of the square root of the scan rate (ν1/2) for initial Cd(ii) concentrations from 20 mg L−1 to 80 mg L−1, and the removal of Cd(ii) by the biocathode for this concentration range was controlled by adsorption. When the initial concentration of Cd(ii) was 100 mg L−1, the linear relationship between the peak current and the scan rate (ν1/2) was inferior when compared to the square root relationship for diffusion control (Table 2).The voltage loss during electrodeposition at a standstill is presumed to be due to the charge transfer resistance of the electrode.19 However, in the current study, the diffusion impedance of the biocathode and abiocathode electrodes was higher than the charge transfer resistance in both Cd(ii) solutions (Fig. 2D and Table 1); the diffusion impedance was also higher than the charge transfer in solutions with different concentrations of Cd(ii) (Fig. S4B and Table S1†).The analysis suggested that diffusive resistance may be the primary resistance to the electrodeposition process of metals due to the difference between the supply and demand for metal ion reduction and diffusion control, and that this phenomenon is more pronounced when there is a reduction in the amount of metal ions. Equivalent circuit fitting showed that the diffusion impedance of the system gradually decreased when the Cd(ii) ion concentration increased from 20 mg L−1 to 80 mg L−1. In comparison, it tended to increase at a high Cd(ii) ion concentration of 100 mg L−1. This result was consistent with the analyses of the plots of the relationship between the peak currents and the sweeping speeds at the primary and one-half power, so that the electrochemical kinetics of the system for Cd(ii) removal up to 100 mg L−1 was controlled by adsorption. In contrast, the removal process at a concentration of 100 mg L−1 Cd(ii) was likely to shift towards diffusion control, as it becomes challenging for microorganisms to eliminate Cd(ii) from the water. Thus, an appropriate increase in the concentration of the cathodic electrolyte may accelerate the motion of metal ions toward the cathodic electrode, and a decrease in the concentration of metal ions in the solution may lead to an increase in the diffusive resistance.193.3Cd(ii) removal and kinetic properties of the MECThe Cd(ii) reduction reaction in the MEC described above occurs by removal kinetics.Pseudo first-order kinetics:1In C0/C = Kobstwhere Kobs denotes the pseudo-first-order rate constant (h−1), C0 denotes the initial Cd(ii) concentration (mg L−1), C denotes the dissolved Cd(ii) concentration at time t (mg L−1), and t denotes the running time (h). A linear fit to the data yielded an equation with the slope of as the rate constant Kobs in the pseudo-first-order kinetics (Kobs = 0.027 h−1 at 20 mg L−1, Kobs = 0.022 h−1 at 60 mg L−1) (Fig. 4A and B). The greater rate constant Kobs indicates a higher rate of removal, which suggests that the biocathode was more suitable for the survival of cadmium-containing wastewater at low concentrations. By contrast, fittings at low concentrations more closely matched the pseudo-first-order kinetics (R2 = 0.97595 at 20 mg L−1, R2 = 0.93980 at 60 mg L−1) (Fig. 4A and B). Therefore, the kinetic reaction of Cd(ii) ion reduction in the MEC biocathode follows the pseudo-first-order kinetic equation dominated by adsorption.28,29 Wei4et al. reported that the removal was consistent with pseudo-first-order kinetics for a two-chamber MEC for p-bromoaniline-containing wastewater.Fig. 4Trend plot of Cd(ii) removal under (A) optimum conditions and (B) its kinetic fit.3.4Microbial community analysisIn this experiment, microbial community analyses of the original sludge and the Cd(ii)-containing sludge were conducted to further explore the effect of Cd(ii) on the biological communities. The results of the OTU number (CA_1_Cd:1923, OR_1:1658) (Table S3†) and Shannon's index (CA_1_Cd:7.59, OR_1:5.75) (Table S3†) for the two sludges indicated that Cd(ii) was selective for the survival of microorganisms. The similarities and differences of microorganisms in the two silts are shown using a Venn diagram (Fig. 5A),29 which showed 691 and 426 different species. However, most of these strains are identical because the same slime had been inoculated, and a few changed under the pressure of Cd(ii) and adapted to the toxic living environment. In addition, the species distributions of 16S rRNA gene sequences were analyzed at the phylum, class, and genus level in conjunction with single-sample multilevel species composition maps (Fig. S5 and S6†) and community structure component plots (Fig. 5B–D).Fig. 5(A) Venn diagrams of bacterial communities in the original and Cd(ii)-containing sludge, and species classification diagrams of bacterial communities at the (B) phylum, (C) class, and (D) genus levels (CA_1_Cd: Cd(ii)-containing sludge; OR_1: original sludge).Remarkable differences in the structure of microbial communities were observed between different samples. According to the results of 16S rRNA gene analysis, Proteobacteria, Bacteroidetes, Acidobacteria, and Actinobacteria were the predominant phyla in the two sludge communities at the phylum level (Fig. 5B).30 However, their abundance distributions were quite different, with an increase in the relative abundance of Proteobacteria from 28.00% in the original sludge to 45.22% in the Cd(ii) solution. Conversely, the relative abundance of Bacteroidetes decreased from 44.28% in the original sludge to 8.00% in the Cd(ii) solution. The relative abundance of new Epsilonbacteraeata was analyzed from the graph, and it increased from 0% in the original sludge to 16.66% in the Cd(ii)-containing sludge.This increase indicated that Proteobacteria and Epsilonbacteraeata were the phyla that were tolerant of heavy metals, and were also the main electron-producing phyla.31 At the class level (Fig. 5C), compared with the original sludge, the reactor with Cd(ii)-containing sludge increased the abundance of Campylobacteria and Gammaproteobacteria while decreasing the number of Lactobacillus, as compared to the original sludge. As reported, Campylobacteria and Proteobacteria promoted the reduction of Cd(ii) and electricity generation using nanoconductors (flagella) or conductivity-related cytochrome in electron generation and transport.32,33 These results showed that Gammaproteobacteria, Alphaproteobacteria, and Campylobacteria were more characteristic of cadmium tolerance. However, most of the Bacteroidia gradually disappeared under prolonged operational time, which suggests that they were not involved in the reduction reaction of the heavy metal Cd(ii) ions or the absence of anti-cadmium properties.Further study at the genus level (Fig. 5D) provided additional detailed information on the microbial community, and it was determined that the biodiversity of the sludge containing Cd(ii) was more complex than that of the original sludge. After domestication by prolonged addition of Cd(ii), the dominant bacteria Citreitalea in the original sludge gradually disappeared, and two absent species of bacteria, Arcobacter and Lentimicrobium, were simultaneously added under Cd(ii) stress. In addition, the relative abundances of the species Pseudomonas, Sphingomonas, Nitrospira, and Thiobacillus increased from 0.62%, 0.80%, 0.34%, and 3.77% in the original sludge to 7.14%, 4.45%, 4.50%, and 4.23%, respectively, in the Cd(ii)-containing sludge.As reported, Arcobacter, Pseudomonas, and Sphingomonas are Gram-negative bacteria that can survive in microaerobic and anaerobic environments. Pseudomonas can consume various carbon sources and participate in metal reduction and electron production.34Sphingosphinomonas is an endophytic bacterium with efficient metabolic regulation and can repair the toxicity damage in plants caused by Cd.35,36 Lentibacterium is a fermenting Gram-negative bacterium that may metabolize organic matter by apoptosis in an anaerobic environment. Nitrospira oxidizes nitrite to nitrate, and Thiobacillus oxidizes thiosulfate and elemental sulfur to sulfate,37 and both are autotrophic Gram-negative bacteria. Gradually disappearing Citreitalea are strictly aerobic Gram-negative bacteria, indicating that aerobic bacteria were not involved in Cd(ii) removal.The community analysis showed that the original bacterial community was transformed into an organic biological community with higher power generation efficiency and anaerobic fermentation by drug-resistant bacteria, which were numerically dominant during the acclimatization to the heavy metal ion Cd(ii). Gram-negative bacteria possess an outer lipid membrane that protects them from toxic and harmful environments, while the flagellum facilitates bacterial motility and electron transport. In Cd(ii)-containing sludge, Gram-negative bacteria are classified as autotrophic and heterotrophic bacteria, where autotrophic bacteria derive their metabolic energy from the oxidation of electrons, and heterotrophic bacteria are responsible for transferring electrons along the electron transport chain to the cell membrane.38These results confirmed that the removal of Cd(ii) ions in the MEC system is the result of a synergistic mechanism of multiple parthenogenetic anaerobic bacteria in anaerobic and microaerobic environments,39 and that the diversity of microbial communities contributes to maintaining the balance within the biological system. Arcobacter and Lentimicrobium were the dominant Cd-tolerant bacteria in the MEC system.4.ConclusionIn this investigation, we examined the influence of biocathodes and abiotic cathodes on the performance of microbial electrolysis cells (MECs) operating under open-circuit (OC) and closed-circuit (CC) conditions. Our findings revealed that the polarity-reversing biocathode is more favorable for Cd(ii) removal in MEC devices. Then, the removal rate and reaction mechanism for cadmium(ii) were investigated for different concentrations of cadmium(ii) at an applied voltage of 0.5 V under CC conditions. Remarkably, the removal rate exceeded 90% within three days, with the removal reaction in the MEC reactor adhering to the pseudo-first-order kinetic equation. The microbial population analysis showed a gradual decrease in the numbers of Bacteroidetes and Epsilon in the raw sludge after the addition of Cd(ii), with changes in Alphaproteobacteria and Gammaproteobacteria in the Proteobacteria. These findings highlight the close relationship between Cd(ii) removal and the composition of cathodic bacterial communities.Author contributionsAuthor 1 (first author): conceptualization, data curation, formal analysis, methodology, software, investigation, writing – original draft; author 2 (corresponding author): conceptualization, funding acquisition, resources, supervision, writing – review and editing, visualization, investigation; author 3 (corresponding author): resources, supervision; author 4: writing – review and editing, data curation.Conflicts of interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Supplementary MaterialRA-014-D3RA07771C-s001

PMC

Annals of Translational Medicine

PMC10009575

2-28-2023

10.21037/atm-22-6481

Exploration of prognostic biomarkers in head and neck squamous cell carcinoma microenvironment from TCGA database

Li Ying, Bi Jianping, Pi Guoliang, He Hanping, Li Yanping, Han Guang

BackgroundImmune checkpoint blockade (ICB) therapies have redefined human cancer treatment, including for head and neck squamous cell carcinoma (HNSCC). However, clinical responses to various immune checkpoint inhibitors are often accompanied by immune-related adverse events (irAEs). Therefore, it is crucial to obtain a comprehensive understanding of the association between different immune tumor microenvironments (TMEs) and the immunotherapeutic response.MethodsThe research data were obtained from The Cancer Genome Atlas (TCGA) database. We applied RNA-seq genomic data from tumor biopsies to assess the immune TME in HNSCC. As the TME is a heterogeneous system that is highly associated with HNSCC progression and clinical outcome, we relied on the Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) algorithm to calculate immune and stromal scores that were evaluated based on the immune or stromal components in the TME. Then, the Tumor Immune Dysfunction and Exclusion algorithm (TIDE) was used to predict the benefits of ICB to each patient. Finally, we identified specific prognostic tumor-infiltrating immune cells (TIICs) by quantifying the cellular composition of the immune response in HNSCC and its association to survival outcome, using the CIBERSORT algorithm.ResultsUtilizing the HNSCC cohort of the TCGA database and TIDE and ESTIMATE algorithm-derived immune scores, we obtained a list of microenvironment-associated lncRNAs that predicted different clinical outcomes in HNSCC patients. We validated these correlations in a different HNSCC cohort available from the TCGA database and provided insight into the prediction of response to ICB therapies in HNSCC.ConclusionsThis study confirmed that CD8+ T cells were significantly associated with better survival in HNSCC and verified that the top five significantly mutated genes (SMGs) in the TCGA HNSCC cohort were TP53, TTN, FAT1, CDKN2A, and MUC16. A high level of CD8+ T cells and high immune and stroma scores corresponded to a better survival probability in HNSCC.

Highlight boxKey findings• A high level of CD8+ T cells and high immune and stroma scores corresponded to a better survival probability in HNSCC.• The top five significantly mutated genes in the TCGA HNSCC cohort were TP53, TTN, FAT1, CDKN2A, and MUC16.What is known and what is new?• Tumor-infiltrating immune cells were found to be significantly associated with prognosis and the identification of immunotherapy targets.• This study is the first to utilize the HNSCC cohort of the TCGA database and TIDE and ESTIMATE algorithm-derived immune scores to obtain a list of TME-associated lncRNAs to predict clinical outcomes in HNSCC patients.What is the implication, and what should change now?• A comprehensive investigation of immune profiling in the progression of HNSCC tumors may promote the development of new immunotherapeutic agents and novel treatment regimens.IntroductionHead and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide, accounting for approximately 4% of all cancers (1-3). Despite significant treatment progress in surgery, radiotherapy, and concomitant chemoradiotherapy, the mortality rate remains as high as 40–50% . Over the last decade, immune checkpoint inhibitors (ICIs) have emerged as a promising therapy for various cancers due to their prominent antitumor efficacy. Programmed death-1 (PD-1) inhibitors, such as pembrolizumab and nivolumab, programmed death-ligand 1 (PD-L1) inhibitors (durvalumab, avelumab, and atezolizumab) and Ipilimumab, a CTLA-4 inhibitor, soon followed, presenting promising results . However, clinical responses after anti-PD-1/PD-L1 treatment are heterogeneous; the majority of HNSCCs are resistant to immunotherapy ab initio, and serious immune-related adverse events (irAEs) are seen in patients experiencing ICI therapy (6,7). Malignant carcinomas, including HNSCC, are characterized not only by the intrinsic activities of cancer cells but also by the immune cells recruited to, and activated in, the tumor microenvironment (TME) . The sophisticated mechanistic basis remains largely unclear. According to the previous study, this may be due d to factors in the TME, such as a lack of proper rejection antigens, lack of immune surveillance, or the presence of immunosuppressive mediators . Therefore, a comprehensive investigation of immune profiling in the progression of HNSCC tumors may promote the development of new immunotherapeutic agents and novel treatment regimens. In this study, we mined The Cancer Genome Atlas (TCGA) database for genes with prognostic value and responses to various ICIs in the HNSCC microenvironment.CD8+ T cells have an affinity for major histocompatibility complex (MHC) class I molecules and are key anticancer immune cells (10,11). Additionally, CD8+ T cells are the main effectors in PD-1 blockade-induced antitumor responses, and reinvigoration of exhausted CD8+ T cells is one of the major determining factors of responsiveness to PD-1 blockade (12,13). For instance, in breast cancer, previous studies have shown a significantly increased number of CD8+ T cells at tumor sites, which was negatively correlated with advanced tumor stages and positively correlated with clinical outcomes (14,15). CD8+ T cells were also significantly associated with the clinical outcomes of HSCNN. In the study of Zhang et al., they found that CD8+ T cells exhaustion in the TME of HNSCC determines poor prognosis . Moreover, CD8+ T cells differentiate to cytotoxic T cells, traffic into the TME, and exhibit cytotoxicity against tumor cells . Upon arrival in tumor beds, CD8+ T cells inevitably face many obstacles, including the intrinsic checkpoint regulators PD1-PD-L1 and CD28-CTLA-4; an abnormal TME contain protumor inflammation; antigen loss and immune evasion of tumor targets; and tissue-specific alterations .The TME is a complex microenvironment where the tumor cells are generated and located, consisting of immune cells, stromal cells, endothelial cells, surrounding blood vessels, inflammatory mediators and extracellular matrix (ECM) molecules . Cytotoxic T lymphocytes (CTL), natural killer (NK) cells, myeloid-derived suppressor cell (MDSC), regulatory T cell (Treg) and tumor-associated macrophage are immune cells which play vital roles in tumor biological process . Cancer-associated fibroblasts (CAFs) construct the main bulk of stromal cells in the TME to promote the growth of cancer cells (21,22). Also, the TME in HNSCC is comprised of heterogeneous non-malignant cells that integrated in a complex ECM . Increasing evidences indicated that tumors can be classified by the components of TME and stromal cell proportions or activations status (24,25). TME of different types of tumors have their own characteristics and also commonalities. However, the TME of HNSCC is characterized by some unique features compared to other cancer types . HNSCC patients have decreased absolute T cells count in tumor and circulation . Moreover, the interaction of various cellular components in the TME along with the production of cytokines and chemokines profoundly impact the function of T cells (26,28,29). Decreased number of NK cells is also common in HNSCC, leading to seriously immunodeficient tumors (30,31). YTHDF1 expression was significantly associated with the TME in HNSCC, which was correlated with CD4+ T cells and CD8+ T cells infiltration . In addition, HNSCC is characterized by desmoplastic stromal fibroblasts that promote tumor invasion and progression through autocrine and paracrine factors (33,34). In the TME, immune and stromal cells are the two main types of non-tumor components and have been suggested that they are valuable for the diagnostic and prognostic biomarkers of tumors. In recent year, algorithms have been developed to predict tumor purity using gene expression data based on the TCGA database. For example, Yoshihara et al. designed the Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) algorithm . Immune and stromal scores to predict the infiltration of non-tumor cells by analyzing the specific gene expression signatures of immune and stromal cells were calculated based on the ESTIMATE algorithm. Many studies have applied the ESTIMATE algorithm to prostate cancer (36,37), breast cancer (38,39), colorectal cancer (40-42) and hepatocellular carcinoma , which demonstrated that such big-data-based algorithms are effective, although its utility on immune and/or stromal scores in glioblastoma multiforme (GBM) has not been explored in detail.Mounting research has revealed that long noncoding RNAs (lncRNAs) play a pivotal role in cancer progression (44-46). However, their potential involvement in HNSCC remains to be elucidated. In this paper, we investigated the CD8+ T cell profiles in the TME and their clinical value in HNSCC patients using the TCGA RNA sequencing data. Signature lncRNAs combined with their coefficients in an elastic net model were used to calculate the risk score for every patient. The “ConsensusClusterPlus” R package clustered patients by the expression level of 22 selected lncRNAs. We further predicted the benefits of immune checkpoint blockade (ICB) to each patient using the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm ( Moreover, mutation patterns of significantly mutated genes (SMGs) in relation to HNSCC subtypes and immune score and stromal score were calculated to reveal the relationship between tumor immune status and prognosis. Finally, tumor-infiltrating immune cells (TIICs) were found to be significantly associated with prognosis and the identification of immunotherapy targets.This study is the first to utilize the HNSCC cohort of the TCGA database and TIDE and ESTIMATE algorithm-derived immune scores to obtain a list of TME-associated lncRNAs to predict clinical outcomes in HNSCC patients. Notably, we validated these correlations in a different HNSCC cohort available from the TCGA database. And were able to provide insight into the prediction of response to ICB therapies in HNSCC. We present the following article in accordance with the REMARK reporting checklist (available at profilingThe lncRNA expression microarray data and clinical characteristics, including medication history, histologic grade, pathological TNM stage, and survival information for 546 TCGA-HNSC samples from 528 TCGA-HNSC patients was retrieved from the TCGA database using the TCGA “biolinks” R package. Patients with complete survival information and genetic expression data at that time point were included in this study. The detailed clinicopathological information for the TCGA data sets are shown in Table S1. The lncRNA annotation information was downloaded from Ensemble (ftp://ftp.ensembl.org/pub/release-99/gtf/homo_sapiens/Homo_sapiens.GRCh38.99.gtf.gz). The lncRNA annotations were extracted from the Ensemble gtf annotation file based on genecode-defined lncRNA biotypes ( The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).lncRNA differential expression analysisThe lncRNA expressions (fpkm values matrix) were extracted from the total RNA fpkm matrix by the Ensemble ID of the lncRNAs. Samples were classified into normal or tumor groups according to tissue definition. Differentially expressed lncRNAs between the normal and tumor samples were qualified using the “DESeq2” R package. lncRNAs with an adjusted P value <0.0001 were selected to plot the heatmap and volcano plot.Construct and validation of survival-related lncRNA signaturesTo find lncRNAs that were most relevant to the prognosis of patients, we first selected the top 1,000 most significant genes using univariate Cox models (genes were ranked by the Wald test P value). We then selected the most related lncRNAs using an elastic net model in the “glmnet” R package with an alpha =0.1 and family = “Cox”. These selected signature lncRNAs combined with their coefficients in the elastic net model were used to calculate the risk score for every patient. The risk score was defined as the sum of products from all signature lncRNAs and their coefficients. After calculating the risk score for every patient, these patients were then divided into two groups by the median value of all risk scores. The survival difference was calculated using the “survival” R package and plotted with the “survminer” R package. Risk score =∑i=1n(Coefi∗xi) Consensus clusteringThe “ConsensusClusterPlus” is an open resource, Bioconductor-compatible R software package for unsupervised class discovery . The algorithm starts by subsampling a proportion of items and a proportion of features from a data matrix. In this study, we performed consensus clustering using “ConsensusClusterPlus”, where the lncRNAs for each HNSCC sample were divided into three subgroups and measured by the median absolute deviation (MAD). The most variable genes were used for subsequent clustering.Differential expression level of 22 selected lncRNAsAfter selecting the signature lncRNAs using the elastic net model, we clustered patients by the differential expression level of 22 selected lncRNAs using the “ConsensusClusterPlus” R package (50 iterations, 80% resampling rate, Pearson correlation, The “PCA” R package (R v3.5.1) was used to investigate gene expression arrays in the HNSCC sample groups.Estimation of tumor mutational burden (TMB)TMB has been recognized as an emerging therapeutic measure of sensitivity to predict immunotherapy response in clinical oncology. The TMB score of each patient with HNSCC was calculated as previously described .Immunotherapeutic response predictionThe programmed cell death 1 PD-1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) pathways are associated with tumor immune evasion; therefore, ICIs targeting PD-1 and CTLA-4 enhance the antitumor immunity. To predict clinical responses to ICIs, we used the TIDE algorithm and subclass mapping as previously described (49,50).Correlations between prognoses and stromal/immune scoresTo investigate the relationship between tumor immune status and prognosis, we calculated the immune and stromal scores and identified the optimal score cutoff for grouping patients most significantly to the maximally selected rank statistics using the “maxstat” R package . Before calculating the scores, we first filtered genes that showed an fpkm value smaller than 5 in all samples. Patients were divided into 4 stages based on the stromal and immune scores estimated from each HNSCC sample and the prognoses for each group were examined. The log-rank tests were used to compare the survival outcomes.Immune TME analysisTo explore the differences in immune cell subtypes for each HNSCC sample, the CIBERSORT package was used to value the fraction of 22 immune cell subtypes, based on an expression file as previously described . We set the number of permutations to 1,000. A total of 22 types of TIICs were quantified for each sample, together with CIBERSORT metrics, including CIBERSORT P value, Pearson's correlation coefficient and root mean square error. The “Genefilter” R package was used to screen each sample, and the threshold was set at a P value 1.0, P1, P1, P<0.01).Figure 5TMB score and mutation status of HNSCC patients. (A) TMB for every patient. (B) Oncoplot for the top 50 mostly mutated genes. TMB, tumor mutational burden; HNSCC, head and neck squamous cell carcinoma.Immune and stromal scores were significantly associated with HNSCC subtypesTo investigate the relationship between tumor immune status and prognosis, we calculated the immune and stromal scores using the “estimate” R package After excluding patients without complete clinical information and the normal control group, 508 patients with HNSCC were enrolled in this research. Relying on the pathological diagnosis given in the TCGA database, HNSCC was divided into four stages. The stromal scores among the different HNSCC stages decreased from stage I to stage III, and increased from stage III to stage IV (Figure 6A). However, the distribution of the immune scores among the different HNSCC stages was quite stable from stage I to stage IV (Figure 6B).Figure 6The relationship between HNSCC tumor immune status and prognosis. (A) Distribution of stromal score among different stage of tumors. (B) Distribution of immune score among different stage of patients. (C) The effects of stromal score on the survival of patients. (D) Prediction of HNSCC patients’ prognosis corresponding with different immune score. HNSCC, head and neck squamous cell carcinoma.Association of stromal and immune scores with HNSCC patient tumor stages and prognosisAfter excluding patients without complete clinical information and the normal control group, 508 HNSCC patients were enrolled in this research. Relying on the pathological diagnosis given in the TCGA database, HNSCC was classified into four stages. The association between the stromal and immune scores with HNSCC patient pathologic characteristics was qualified by comparing the score distributions in different tumor stages (Figure 6A,6B). A roughly increased stromal and immune scores with the advancing of tumor stage could be seen. There was a significant association between stromal (P=0.015) and immune scores (P=0.00088) and HNSCC patients survival probability (Figure 6C,6D).Composition difference and prognosis value of TIICs in HNSCC samplesThe CIBERSORT analytical tool allowed us to specifically analyze the intrinsic fractions of 22 subpopulation TIICs in the bulk tumor samples. Insight into TIICs may be helpful in explaining the initiation and development of HNSCC. The total value of the 22 subset immune cells in each sample as one, and the fraction of all 22 subpopulations of immune cells in each sample is depicted in Figure 7A. Calculating the average fraction of each subset of TIICs among the HNSCC samples, the top five average fractions were M0 macrophages, CD8 T cells, M2 macrophages, CD4 resting memory T cells, and M1 macrophages. Detailed information is shown in Moreover, as presented in Figure 7B, we used unsupervised hierarchical clustering based on the above identified cell subsets to divide the HNSCC samples into two discrete groups. It was clear that the fractions of immune cells in the different HNSCC samples were significantly varied. Hence, we inferred that variation in TIIC fractions might be an essential characteristic of HNSCC. We further identified the prognostic subsets of TIICs in HNSCC. The Kaplan-Meier plots and log-rank tests for the above-identified TIIC subpopulations showed that high expression levels of immune cells, including CD8 T cells (P=0.0014), follicular helper T cells (P=0.0024), and regulatory T cells (P=0.00034) were associated with a better OS; whereas high expression levels of CD4+ memory T cells (P=0.00035) corresponded to a poor OS (Figure 7C-7F).Figure 7The landscape of immune infiltration in HNSCC. (A) The difference of immune infiltration in HNSCC samples. (B) Heat map of the 22 immune cell fractions in TCGA-HNSCC cohort. The vertical axis depicts the clustering data of samples which were divided into two discrete groups. (C) Survival plot of median of CD8 T cells. (D) Survival plot of median of CD4+ memory T cells. (E) Survival plot of median of follicular helper T cells. (F) Survival plot of median of regulatory T cells. The P values are from log-rank tests. HNSCC, head and neck squamous cell carcinoma; TCGA, The Cancer Genome Atlas.DiscussionAs increasing application of immunotherapy in cancer, the information of individual’s immune status could help identify those who will resist to ICB therapies. TME has been reported to seriously affect gene expression of tumor cells, thus the clinical outcomes. The TME is a complex microenvironment where the tumor cells are generated and located. It is comprised of mesenchymal cells, stromal cells, immune cells, endothelial cells, surrounding blood vessels, as well as inflammatory moderators and ECM molecules (24,54). In the TME, immune and stromal cells are two notable kinds of non-tumor components, which have magnificent value for tumors diagnosis and prognosis assessment. HNSCC is one of the most common malignant tumors in the world, with high metastasis rate, low survival rate and poor prognosis . Importantly, most HNSCCs have significant immune infiltration, and the close interaction between tumor cells and their microenvironment significantly impairs the development and progression of malignant tumors, therefore influences the prognosis of patients .In this study, we identified TME related genes that contribute to HNSCC OS and conducted prediction of immunotherapeutic response using data from TCGA database. Specifically, by comparing lncRNA expression level of 546 TCGA-HNSC samples from 528 TCGA-HNSC patients, we then extracted 258 genes involved in prognoses and stromal/immune Scores and immunotherapeutic response prediction. We used the ESTIMATE algorithm to calculate immune and stromal scores in the TME. Therefore, we demonstrated that patients with low stroma risk score in line with a relatively higher survival probability, and low immune risk score corresponds a relatively higher survival probability.In our study, the average TMB was 3.48 mutants per Mb from 508 HNSCC patients, which was similar to background TMB value in HNSCC . We verified top five mutation SMGs in TCGA HNSCC cohort were TP53, TTN, FAT1, CDKN2A and MUC16. Early studies verified that TP53 is a ubiquitous tumor suppressor which is critically mutated in different malignant tumors, with about two-thirds of HNSCC accompanying mutations in exons (58,59), and these alterations were intimately associated with resistance to radiation and cisplatin-based chemotherapeutics (60,61). Many studies have revealed that TP53 is one of the most frequently mutated genes in HNSCC (62-64). The TTN was included in old age specific clusters and pathway enrichment in HNSCC SMGs . Based on previous studies, FAT1 plays different roles in different tissues or cancer types and its expression level is downregulated in HNSCC and other cancers, which has been considered as a tumor suppressor (66,67). The highest mutation rate of FAT1 was approximately 23% in HNSCC, ranking as the second most mutated gene after TP53 (68,69). Several studies have shown that suppression of CDKN2A expression by methylation is involved in the development of HNSCC, which could be a potential diagnosis and prognosis biomarker for HNSCC (70-72). Although not much is known about the specific effects of MUC16 in HNSCC, many studies have been verified that deregulated expression of MUC16 was associated with several cancers, such as breast cancer , ovarian cancer , non-small-cell lung cancer and pancreatic cancer .Furthermore, both CIBERSORT and TIDE algorithm outcome agree that a high level of CD8+ T cells was significantly corresponding to a preferable survival in HNSCC, which was in a line with that high CD8+ intratumoural counts exhibited a remarkable association with relapse free survival in breast cancer and Triple-negative breast cancer (14,15). And this tendency is consistent with previous study that increased numbers of intraepithelial CD8+ TIL was associated with favorable outcome in HNSCC . Besides, the CD8+ T cells would experience a declination from stage I to stage IV. Though there are contradictory study point out, instead of CD8+ T cell, CD4+ T cells in cancer stroma are closely related to a favorable prognosis in human non-small cell lung cancers , We speculate that exogenous reactivation of CD8+ T cells might be theoretically feasible to alleviate HNSCC patients suffering using rational immunotherapy strategies (17,79).Over the recent years, the TME was characterized as a pivotal role in determining tumor progression and treatment outcomes. As an indispensable part of the TME, the tumor stroma greatly affects tumorigenesis, cancer progression, metastasis, and therapy resistance in various cancers . In our study, even though the survival difference between high immune score group and low immune score group was quite slight, the high immune score corresponding to a high survival probability (P=0.015). Moreover, there are a huge survival gap between high stroma score group and low stroma score group, high stroma score group has obvious better survival than low stroma score group as depicted in Kaplan-Meier OS analysis (P=0.00088).ConclusionsIn conclusion, we performed a comprehensive analysis of the TME in HNSCC using RNA-seq genomic data from TCGA database. Immune and stromal scores were calculated by ESTIMATE algorithm. The responses of ICB to each HNSCC patient were predicted by TIDE algorithm. Finally, we used CIBERSORT algorithm identified specific prognostic TIICs by quantifying the cellular composition of the immune response in HNSCC and its association to survival outcome, using the CIBERSORT algorithm. The results showed that high level of CD8+ T cells was significantly corresponding to a preferable survival and high level of CD4+ T cells was significantly associated with poor survival in HNSCC. Furthermore, high immune score and stroma score corresponded to a better survival probability in HNSCC. In addition, we verified that top five SMGs in TCGA HNSCC cohort were TP53, TTN, FAT1, CDKN2A and MUC16. Therefore, the expression level of CD8+ T cells and CD4+ T cells could be used as prognostic biomarkers for HSCNN patients in clinical applications. The expression levels of characteristic genes that used by CIBERSORT and ESTIMATE algorithms, or the expression levels of characteristic lncRNAs of LASSO Cox regression model in this study can be detected through high-throughput sequencing methods, and then the relative T cells proportion, immune score or stromal score can be calculated based on the algorithm models. Hence, the prognosis of patients can be stratified using the comprehensive immune approaches at the transcriptome level. Our study results consolidate the role TME in the progression of HNSCC and keep investigating the mechanisms of TME that preciously mediate tumorigenesis will augment the new targets for cancer therapy.SupplementaryThe article’s supplementary files as10.21037/atm-22-648110.21037/atm-22-648110.21037/atm-22-648110.21037/atm-22-6481

PMC

iScience

PMC10518488

9-09-2023

10.1016/j.isci.2023.107883

Innate mechanism of mucosal barrier erosion in the pathogenesis of acquired colitis

Yang Won Ho, Aziz Peter V., Heithoff Douglas M., Kim Yeolhoe, Ko Jeong Yeon, Cho Jin Won, Mahan Michael J., Sperandio Markus, Marth Jamey D.

SummaryThe colonic mucosal barrier protects against infection, inflammation, and tissue ulceration. Composed primarily of Mucin-2, proteolytic erosion of this barrier is an invariant feature of colitis; however, the molecular mechanisms are not well understood. We have applied a recurrent food poisoning model of acquired inflammatory bowel disease using Salmonella enterica Typhimurium to investigate mucosal barrier erosion. Our findings reveal an innate Toll-like receptor 4-dependent mechanism activated by previous infection that induces Neu3 neuraminidase among colonic epithelial cells concurrent with increased Cathepsin-G protease secretion by Paneth cells. These anatomically separated host responses merge with the desialylation of nascent colonic Mucin-2 by Neu3 rendering the mucosal barrier susceptible to increased proteolytic breakdown by Cathepsin-G. Depletion of Cathepsin-G or Neu3 function using pharmacological inhibitors or genetic-null alleles protected against Mucin-2 proteolysis and barrier erosion and reduced the frequency and severity of colitis, revealing approaches to preserve and potentially restore the mucosal barrier. Graphical abstract Highlights•Recurrent Salmonella infection models acquired colitis and inflammatory bowel disease•Colonic mucosal barrier erosion with Mucin-2 proteolysis is a hallmark of colitis•Inductions of host Neu3 and Cathepsin-G are linked to increased Mucin-2 proteolysis•Loss of Neu3 or Cathepsin-G function protects against Mucin-2 proteolysis and colitis Biochemistry; Molecular biology; Immunology; Microbiology

IntroductionThe maintenance and renewal of biophysical barriers within the body are essential for maintaining health. The mucosal barrier of the colon for example provides a physical separation between host cells and the microbes present in the intestinal lumen. This separation is essential to prevent host cell infection and tissue ulceration; consequently, the degradative breakdown of this barrier is a pathological hallmark of colitis and human inflammatory bowel disease (IBD).1 Composed primarily of the sialylated Mucin-2 (Muc2) glycoprotein, the colonic mucosal barrier is produced by goblet cells that secrete processed mature Muc2 monomers as a disulfide-linked net-like polymer that expands with hydration at the lumenal surface of epithelial cells.2 In the absence of Muc2, bacteria are observed in physical contact with epithelial cells and signs of inflammation and severe colitis develop early in life.3,4 Nascent Muc2 polymers first contribute to the stratified (inner) mucin layer which forms the physical barrier between host epithelial cells and bacteria in the lumen.5 As new Muc2 polymers are produced, previously secreted mucin layers are displaced outward from the epithelial cell surface and eventually undergo a progressive breakdown by glycolytic and proteolytic processes in the lumen becoming what is termed the loose (outer) mucin layer in which bacteria reside, forage, and thrive.6 To maintain the mucosal barrier, the production and secretion kinetics of Muc2 polymers by goblet cells must closely match the rate of Muc2 polymer breakdown in the lumen.As the major protein contributing to the colonic mucosal barrier, Muc2 is also a target of proteases that can be implicated in barrier erosion and the onset of colitis. Proteases of bacterial and protozoan origin have been reported capable of cleaving Muc2 in cell culture systems.7,8 Additionally, proteases produced by the colonic microbiome or perhaps by host cells in response to infection and inflammation may participate in colonic mucosal barrier breakdown. We investigated among these possibilities using a recurrent food poisoning model of human IBD involving repeated low dose and non-lethal gastric infections of mice with Salmonella enterica Typhimurium (ST), a prevalent human foodborne pathogen.9 Herein, we report the discovery of an innate host response induced by recurrent ST infection that elevates the rate of Muc2 proteolysis in the absence of a compensatory increase in Muc2 protein production. This pathogenic mechanism involves the sequential action of host neuraminidase and protease enzymes induced in disparate cell types. Inhibition of this mechanism blocks the elevation of Muc2 proteolysis, preserves the structure and function of the mucosal barrier, and reduces the frequency and severity of colitis.ResultsMuc2 sialylation and desialylation in mucin layer proteolysisRecurrent monthly low dose gastric ST infection of adult laboratory mice results in the development and progression of an enduring colitis (Figures S1A–S1E).9 Prior to the 4th gastric ST infection, the stratified inner mucin layer was significantly eroded with reduced Muc2 protein levels coincident with bacterial invasion of the host epithelium and elevated permeability of the mucosal barrier (Figures 1A–1D and S1F). The induction of Muc2 RNA has been reported among goblet cells in response to various stimuli; however, goblet cell numbers were decreased as was previously observed, and Muc2 RNA levels were not altered among colon tissue (Figure 1E).9 Extracellular Muc2 was isolated by immunoprecipitation separately from stratified and loose mucin layers and analyzed using reducing and non-reducing gel electrophoresis to measure the abundance and integrity of Muc2. Induced proteolysis of Muc2 was found in the stratified and loose mucin layers following recurrent ST infection with the appearance of Muc2 fragments of approximately 100 and 200 kDa in reducing and non-reducing gel analyses, respectively (Figures 1F and 1G).Figure 1Recurrent ST infection is linked to colonic Muc2 desialylation, Muc2 proteolysis, and the erosion of the protective stratified mucin layerWild-type (WT) mice at 8 weeks of age were infected with ST (2 × 103 cfu) or uninfected (PBS) every 4 weeks for 6 consecutive months.(A) Colon sections isolated from WT mice were incubated with antibodies to Muc2 (red), and then FISH staining for bacteria (green) was performed with EUB-338 probe that detected intact bacteria (white arrows) and a negative control probe (non-EUB-338). DNA is stained with DAPI (blue). In lower panels, colon sections were stained with Alcian blue and nuclear fast red, and separately, H&E. S, stratified inner mucin layer; L, loose outer mucin layer. Scale bars, 50 μm. The thickness of the stratified inner mucin layer and FISH fluorescent (EUB-338+) cells in mucosa was quantified from 4 fields of view each from 6 wild-type mice including littermates with each condition.(B) Relative abundance of bacterial 16S rDNA detected in colon tissues by real-time PCR using 16S universal primers (n = 4 mice with each condition).(C) Intestinal epithelial barrier function (n = 8 mice with each condition).(D) Immunoblot analysis of Muc2 protein in the colonic stratified (S) and loose (L) mucin layer (n = 8 mice with each condition).(E) Muc2 mRNA expression in colon tissue (n = 6 mice with each condition).(F and G) Immunoblot analysis of reduced and non-reduced Muc2 protein samples analyzed by corresponding polyacrylamide gel electrophoresis from the colonic stratified (S) and loose (L) mucin layers (n = 4 mice with each condition). Arrowheads to the right of the gels denote the positions of two major Muc2 protein bands detected by anti-Muc2 antibody.(H) Lectin blot analysis from identical amount of Muc2 in the colonic stratified (S) and loose (L) mucin layer (n = 6 mice with each condition). Glycan linkages attached to Muc2 were analyzed using lectins including Erythrina cristagalli agglutinin (ECA) and Ricinus communis agglutinin (RCA-I/RCA) that both bind exposed galactose linkages, peanut agglutinin (PNA) that selectively binds the unsialylated Core 1 O-glycan, the Maackia amurensis lectin II (MAL-II) that detects a subset of α2-3 sialic acid linkages, and the Sambucus nigra agglutinin (SNA) that detects a subset of α2-6 sialic acid linkages.(A–H) All mice were analyzed at 20 weeks of age prior to the fourth ST infection. Muc2 was visualized following denaturing (D, F, H) or non-denaturing (G) polyacrylamide gel electrophoresis. Plots are presented as means of biological replicates ±SD (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).The Muc2 mucin domain is heavily sialylated during its synthesis and this posttranslational modification plays a role in modulating Muc2 proteolysis. Multiple sialyltransferases modify Muc2 during its biosynthesis and trafficking to the cell surface. For example, sialylation by the ST6GALNAC1 sialyltransferase produces α2-6 linkages of sialic acid that reduced Muc2 susceptibility to proteolytic degradation in the context of colonic microbial dysbiosis.10 Other sialyltransferases produce α2-3 sialic acid linkages also found at high levels on Muc2. Glycoprotein sialylation levels can be compared using analytical lectins such as the Sambucus nigra agglutinin lectin that binds a subset of α2-6 sialic acid linkages, the Maackia amurensis lectin II that binds a subset of α2-3 sialic acid linkages, and the Erythrina cristagalli agglutinin, Ricinus communis agglutinin, and peanut agglutinin lectins that bind subsets of galactose linkages exposed upon the removal of sialic acids by neuraminidases.9,11,12 Comparing lectin binding among mucin samples from healthy uninfected mice, Muc2 sialylation was significantly lower in the outer loose mucin layer compared to the inner stratified mucin layer, consistent with a progressive desialylation during Muc2 aging in the lumen. Recurrent ST infection further reduced the presence of sialic acid linkages on Muc2 as was also noted by elevated galactose exposure among Muc2 in both mucin layers (Figure 1H). Our results indicated that not all sialic acids were eliminated from Muc2 during this in vivo response; instead, these findings suggested an increase in the normal rate of lumenal Muc2 proteolysis in the context of previous ST infections.We identified the mouse St3gal6-encoded ST3Gal6 sialyltransferase expressed among colon epithelial cells in the normal modification of nascent Muc2 with α2-3 sialic acid linkages. Reduced Muc2 sialylation in the absence of ST3Gal6 and among uninfected mice was linked to spontaneous Muc2 proteolysis and mucin barrier breakdown with the onset of colitis (Figure S2).9 These findings reveal that the St3gal6 sialyltransferase adds α2-3 sialic acid linkages that protect Muc2 from premature proteolysis, while elevated Muc2 desialylation observed following ST infection among wild-type animals suggests the induction of one or more neuraminidases/sialidases. Because the ST pathogen used herein does not encode a neuraminidase enzyme, these findings infer the involvement of a neuraminidase originating from the commensal microbiota and/or the host.Neu3 neuraminidase in Muc2 modification and proteolysisAmong mammalian neuraminidases, Neu3 protein levels were significantly induced at the colon epithelial cell surface in response to recurrent ST infection (Figure 2A). This induction quantitatively accounted for the majority of neuraminidase activity measured in colon tissue and was linked to the reduction of glycoprotein sialylation with exposure of underlying galactose at the colonic epithelial cell surface (Figures 2B and S3). In the absence of Neu3, Muc2 expression was not significantly altered, and erosion of the stratified layer was reduced, thereby maintaining stratified layer integrity and separation of bacteria to the loose mucin layer with improved barrier function (Figures 2C–2G). These findings were linked to the retention of Muc2 sialylation with reduced Muc2 proteolysis in both stratified and loose mucin layers (Figures 2H–2K). In comparing the protective effect of Neu3 deficiency to oral treatment with the neuraminidase inhibitor zanamivir (Figure S4), we found that both approaches similarly maintained Muc2 sialylation, reduced Muc2 proteolysis, and preserved stratified mucin layer integrity and function.Figure 2Colonic Neu3 neuraminidase induction in Muc2 desialylation and proteolysis(A) In situ localization of Neu3 protein in colon sections of littermates of indicated genotypes and conditions. Neu3 protein is visualized using anti-Neu3 antibodies (green). Quantitation was analyzed from the average of 3–4 fields of view each from 6 independent mice each including littermates of each genotype and condition. Scale bars, 100 μm.(B) Neuraminidase (Neu) activity measured from total tissue homogenates of the colon at multiple time points prior and subsequent to recurrent ST infection (n = 6 mice of each genotype and condition).(C) Colon sections isolated from indicated genotypes were incubated with antibodies to Muc2 (red), and then FISH staining for bacteria (green) was performed with the EUB-338 probe. White arrows denote bacterial penetration in the colonic mucosa. Colon sections were also stained with Alcian blue and nuclear fast red, or H&E. S, stratified inner mucin layer; L, loose outer mucin layer. Scale bars, 50 μm. The thickness of the stratified inner mucin layer and FISH fluorescent (EUB-338+) cells in mucosa were quantified from the average of 3–4 fields of view each from 6 independent mice including littermates of each genotype and condition.(D) Relative abundance of bacterial 16S rDNA detected in colon tissues by real-time PCR using 16S universal primers (n = 4 mice per each genotype and condition).(E) Intestinal epithelial barrier function (n = 8 mice per each genotype and condition).(F) Immunoblot analysis of Muc2 protein in the colonic stratified (S) and loose (L) mucin layer (n = 6 mice per each genotype and condition).(G) Muc2 mRNA expression in colon tissue (n = 4 mice per each genotype and condition).(H and I) Lectin-binding analyses of glycan linkages from identical amounts of Muc2 in the colonic stratified and loose mucin (n = 6 mice per each genotype and condition).(J and K) Immunoblot analyses of reduced and non-reduced Muc2 protein samples analyzed by corresponding polyacrylamide gel electrophoresis from the colonic stratified (S) and loose (L) mucin layers (n = 4 mice per each genotype and condition). Arrowheads to the right of the gels indicate two major Muc2 protein bands detected.(A and C–K) Neu3-deficient mice and WT littermates at 20 weeks of age prior to the fourth ST infection. Muc2 was visualized following denaturing (F, H–J) or non-denaturing (K) polyacrylamide gel electrophoresis. Plots are presented as means of biological replicates ±SD (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).Desialylation renders Muc2 susceptible to an increased rate of proteolysisNascent Muc2 desialylation by Neu3 hydrolyzes a subset of Muc2 sialic acid linkages in elevating Muc2 susceptibility to proteolysis. We tested the proteolytic susceptibility of desialylated Muc2 among non-proteolyzed Muc2 immunoprecipitates isolated from the colonic stratified layer of wild-type uninfected mice. Isolated intact Muc2 was incubated with neuraminidase ex vivo either prior to or at the same time a protease source was added to the assay. The protease source was provided using Muc2-depleted intestinal lumenal extracts obtained from uninfected or previously infected cohorts. Muc2 proteolysis was detectable and measurable in this assay and was significantly increased among Muc2 preparations that had been previously desialylated (Figure 3A). A time course using this assay revealed that the rate of Muc2 proteolysis was doubled with desialylated Muc2 and was further elevated in the presence of lumenal extracts from previously infected animals (Figure 3B).Figure 3Prior Muc2 desialylation in altering the rate of Muc2 proteolysisIn vitro Muc2 proteolysis assay by pretreatment or posttreatment with Arthrobacter urefaciens sialidase (neuraminidase) (n = 4 mice of each condition).(A and B) Muc2 immunoprecipitates isolated from the colonic stratified layer of WT mice at 12 weeks of age were treated with Arthrobacter urefaciens sialidase before or after incubation for 24 h (A) or indicated times (B) with Muc2-depleted lumenal extracts prepared from WT mice at 20 weeks of age prior to the fourth ST infection or PBS treatment. Muc2 was visualized following denaturing polyacrylamide gel electrophoresis. Arrowheads represent major Muc2 protein bands detected. Plots are presented as means of biological replicates ±SD (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).Serine protease activity with induction of Cathepsin-G in Muc2 proteolysisTo identify the protease(s) involved in increased Muc2 proteolysis, we similarly tested the effect of Muc2-depleted lumenal extracts in the presence of various protease inhibitors. We found that a combination of protease inhibitors was able to block Muc2 proteolysis; therefore, we separately applied selective inhibitors of different protease classes spanning cysteine proteases, serine proteases, and metalloproteases. Only the serine protease inhibitor blocked Muc2 proteolysis (Figure 4). The investigation of serine proteases within the intestinal tract included three from host sources, namely Cathepsin-G (CatG), neutrophil elastase (NE), and proteinase 3 (PR3). NE and PR3 are produced mainly by neutrophils while CatG is also produced by Paneth cells of the small intestine.13,14 From comparative studies of lumenal contexts of the total intestinal tract, we found that CatG was elevated 6-fold during recurrent ST infection, while NE and PR3 were elevated approximately 2-fold (Figure 5A). RNA transcripts encoding these proteases were similarly induced in small intestine tissue; however, CatG RNA was very low in the colon and was not elevated (Figure 5B). During the progression of disease in this colitis model, CatG protein levels in the lumen tracked upward with repeated ST infection and remained elevated following the cessation of recurrent infection (Figure 5C).Figure 4Selective inhibition of Muc2 proteolysisMuc2 proteolysis was assayed in the absence or presence of various general protease inhibitors including a protease inhibitor cocktail (PIC), the cysteine protease inhibitor (E−64), the serine protease inhibitor (PMSF), and the metalloprotease inhibitor (EDTA/EGTA). Muc2 immunoprecipitates isolated from the colonic stratified layer of uninfected WT mice at 12 weeks of age were incubated with protease inhibitors in the presence of Muc2-depleted lumenal extracts prepared from either uninfected WT mice (PBS) or from infected littermates (ST) prior to the fourth ST infection or PBS treatment. Representative result is shown in left panel, while results from multiple animals is plotted on the right (n = 4 mice at 20 weeks of age with each condition). Muc2 was visualized following denaturing polyacrylamide gel electrophoresis. Plots are presented as means of biological replicates ±SD (∗∗p < 0.01; ∗∗∗p < 0.001).Figure 5Serine protease expression is induced with Muc2 proteolysis(A) Immunoblot analysis of different serine proteases including Cathepsin-G (CatG), neutrophil elastase (NE), and proteinase 3 (PR3) in the luminal content (n = 4 mice per each condition).(B) mRNA expression of serine proteases in small intestine and colon tissue (n = 4 mice per each condition).(C) Alterations in CatG protein abundance in the lumenal content of WT mice during ST reinfection (n = 4 mice per each condition).(D) In situ localization of CatG protein in duodenum, jejunum, ileum, and colon sections stained with H&E or in fluorescent analyses with antibody to CatG (green) and DAPI staining of cell nuclei (blue). Quantitation was analyzed from the average of 3–4 fields of view each from 6 independent mice including littermates of each treatment condition. Scale bars, 100 μm.(E) In situ localization of CatG and lysozyme in duodenum sections using antibodies to CatG (green) and lysozyme (red). Quantitation was analyzed from 3 to 4 fields of view each from 6 independent mice including littermates of each treatment condition. Scale bars, 100 μm.(F) Immunoblot analyses of CatG protein levels present in intestinal contents isolated from indicated compartments of the intestine (n = 4 mice per each condition).(G) Immunoblot analysis of CatG in the colonic stratified (S) and loose (L) mucin layers, and in colon tissue preparations following the removal of both mucin layers (n = 4 mice per each condition).(A, B, D–G) WT mice at 20 weeks of age prior to the fourth ST infection.(A, F, G) Proteins were visualized following denaturing polyacrylamide gel electrophoresis. Plots are presented as means of biological replicates ±SD (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).Histological analyses of the intestinal tract revealed CatG protein induction among epithelial cells within the crypts of the duodenum, jejunum, and ileum of the small intestine, with the noted absence of CatG protein in colon tissue (Figure 5D). The cell source of CatG was further identified using the Paneth cell marker lysozyme that colocalized with induced CatG (Figure 5E). Following secretion of CatG by Paneth cells, CatG was elevated in the lumenal contents of the small intestine and colon (Figure 5F). CatG protein induction was further detected in both the loose and stratified mucin layers of the colon, but not in tissue extracts of the colon (Figure 5G). The induction of CatG by Paneth cells was dependent upon Toll-like receptor 4 (Tlr4) function. Tlr4 deficiency abolished Neu3 induction on the colonic epithelium and protected against Muc2 proteolysis and erosion of the colonic mucosal barrier (Figure S5).Cathepsin-G is required for induced Muc2 proteolysis and erosion of the mucosal barrierThe potential roles of CatG, NE, and PR3 in Muc2 proteolysis were investigated initially in our ex vivo assay using selective small-molecule inhibitors of these proteases. Only the CatG inhibitor significantly reduced Muc2 proteolysis (Figure 6A). A more definitive assignment of CatG as responsible for the induction of Muc2 proteolysis was sought by comparing mice lacking a functional CatG-encoding Ctsg gene. Ctsg-null mice appear normal without reported developmental abnormalities or spontaneous disease.15 In our studies, the absence of CatG in Ctsg-null mice protected against colonic mucosal barrier erosion caused by recurrent ST infection with preservation of Muc2 expression and abundance (Figures 6B–6G). Muc2 proteolysis was significantly reduced in the absence of CatG in both stratified and loose mucin layers even though recurrent ST infection continued to desialylate Muc2 in the presence of elevated neuraminidase activity (Figures 6H, and S6A–S6C). The previously recorded 50% elevation of commensal microbial load in the intestinal tract following recurrent ST infection continued in CatG deficiency with increased abundance of gram-negative Enterobacteriaceae (Figure S6D).9 The overgrowth of gram-negative Enterobacteriaceae following recurrent ST infection was also detected in CatG deficiency, indicating that this microbial dysbiosis was not responsible for significantly elevated Muc2 proteolysis or the severity of disease signs of acquired colitis in the presence of CatG further including fecal blood, diarrhea, and inflammatory cytokine expression (Figures 6H–6K).Figure 6Pharmacological and genetic inhibition of Cathepsin-G(A) Muc2 proteolysis using Muc2-depleted lumenal extracts in the absence or presence of selective protease inhibitors of CatG, NE, and Trypsin. Left panel provides a representative result (n = 4 mice per each condition). Arrowheads to the right of the gels denote positions of major Muc2 protein bands detected.(B) Immunoblot analysis of CatG protein levels in the small intestine tissue and in the lumenal intestinal contents of WT and CatG-deficient Ctsg-null littermates (n = 4 mice per each genotype and condition).(C) Colon tissue sections were analyzed using antibody to Muc2 (red); FISH staining of bacteria (green) was performed with the EUB-338 probe. White arrows denote intact bacterial penetration into the colonic mucosa. Colon sections were also stained with Alcian blue and nuclear fast red. S, stratified inner mucin layer; L, loose outer mucin layer. Scale bars, 50 μm. The thickness of the stratified inner mucin layer and FISH fluorescent (EUB-338+) cells in mucosa were averaged from 4 fields of view each from 6 independent mice including littermates of each genotype and condition.(D) Relative abundance of bacterial 16S rDNA detected in colon tissues by real-time PCR using 16S universal primers (n = 4 mice per each genotype and condition).(E) Intestinal barrier function (n = 4 mice per each genotype and condition).(F) Immunoblot analysis of Muc2 protein in the colonic stratified (S) and loose (L) mucin layer (n = 4 mice per each genotype and condition).(G) Muc2 mRNA expression in colon tissue (n = 4 mice per each genotype and condition).(H) Immunoblot analyses of Muc2 proteolysis in the colonic stratified (S) and loose (L) mucin layer (n = 4 mice per each genotype and condition).(I) Inflammatory cytokine mRNA expression in isolated colon tissue (n = 4 mice per each genotype and condition).(J and K) Body weight and colon length (n = 5 male mice per each genotype and condition).(L) Frequency of diarrhea (n = 12) and frequency of fecal blood detected (n = 12) were plotted throughout 48 weeks of age of adult life in the course of recurrent ST infections (arrows).(B–I) Ctsg-null mice and WT littermates at 20 weeks of age prior to the fourth ST infection.(J and K) Ctsg-null mice and WT littermates at 48 weeks of age.(A, B, F, H) Proteins were visualized following denaturing polyacrylamide gel electrophoresis. Plots are presented as means of biological replicates ±SD (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).DiscussionThe pathogenesis of colitis includes the proteolytic erosion of the protective colonic mucosal barrier. This process has been difficult to study and relatively little understanding exists of the mechanism(s) involved in barrier homeostasis. Using a repeated human food poisoning model of IBD originating from recurrent gastric ST infections, and with disease signs similar to human Ulcerative Colitis, we have identified an innate mechanism of colonic mucosal barrier erosion contributing to the onset and severity of acquired colitis. This sequential glycoproteolytic mechanism encompasses Tlr4-dependent induction of host Neu3 neuraminidase at the colonic epithelial cell surface resulting in the partial desialylation of nascent Muc2 glycoprotein produced by goblet cells. This desialylated Muc2 is more susceptible to proteolysis by lumenal CatG. Neu3 absence or prolonged inhibition reduced Muc2 proteolysis preserving barrier maintenance with a pre-infection trend toward increased barrier thickness at steady state that may contribute to protection. Concurrently, host Tlr4 was required for the induction of CatG among Paneth cells of the small intestine, resulting in the secretion of CatG into the intestinal tract where it was found at elevated levels throughout the lumen including the colon. The basal rate of extracellular Muc2 proteolysis measured in healthy animals was significantly elevated in this model of acquired IBD. No compensatory increase in Muc2 protein level was detected likely because goblet cells were reduced in number and function as typical in chronic intestinal inflammation.9,16 This contributes to a disequilibrium affecting Muc2 homeostasis consisting of an increased rate of Muc2 degradation in the absence of a compensatory increase in production, causing the progressive erosion of the colonic mucosal barrier.Muc2 resistance to CatG proteolysis in the mouse colon is provided by the posttranslational modification of Muc2 by sialic acid linkages during its biosynthetic trafficking through the Golgi and prior to secretion by goblet cells. The critical dependence of mucosal barrier preservation on Muc2 sialylation by multiple sialyltransferases is indicated from our studies and from those reported among human IBD patients bearing an inherited deficiency of the ST6GalNAc1 sialyltransferase.10 Similarly, inborn deficiency of the ST3Gal6 sialyltransferase, which produces only α2-3 sialic acid linkages, resulted in elevated Muc2 proteolysis, spontaneous barrier erosion, and colitis in the mouse.9 The degree of Muc2 sialylation deficiency in one or more peptide sequence contexts may modulate the rate of Muc2 proteolysis and barrier breakdown. Compared with the absence of CatG, we found that oral treatment with the neuraminidase inhibitor zanamivir resulted in similar protection from elevated Muc2 proteolysis and barrier erosion. CatG was observed in the lumen of the colon at basal levels in wild-type mice, and may also operate as an antimicrobial factor. While CatG induction was the major source of the increased proteolysis of Muc2, the absence of CatG did not completely inhibit the induction of Muc2 proteolysis. Other host proteases including NE and PR3, or perhaps proteases originating from commensal microbes, may contribute more prominently in other contexts. The increased number of gram-negative Enterobacteriaceae observed has been linked to an acquired deficiency of host alkaline phosphatase.9 In contrast with the present studies, CatG deficiency did not prevent the expansion of Enterobacteriaceae and was not as effective as alkaline phosphatase supplementation at blocking the induction of inflammatory cytokine expression. Therefore, the colonic microbial dysbiosis that involves Enterobacteriaceae does not contribute substantially to Muc2 proteolysis and barrier erosion in our repeated food poisoning model of acquired IBD.Sialic acid linkages are posttranslational protein modifications bearing a negative charge in physiological conditions. The structure of Muc2 includes multiple domains including two “PTS” domains, often called mucin domains, rich in tripeptide repeats of proline, threonine, and serine. The two PTS/mucin domains of Muc2 harbor the greatest degree of glycosylation, predominantly O-glycosylation on multiple threonine and serine residues.17,18 The O-glycans of nascent Muc2 are sialylated and the resulting high density of negative charge in the PTS domains is believed to contribute to the extended conformation of the Muc2 glycoprotein appearing as a “bottlebrush.”19 Deficiency in the formation of Core 1 and Core 3 O-glycans, which are mostly sialylated, resulted in a deficient and defective mucosal barrier with the development of colitis.20,21 The partial elimination of sialic acid linkages and perhaps those normally residing on Core 1 and Core 3 O-glycans may occur at the cell surface where the majority of Neu3 induction is observed in proximity to nascent Muc2, and may occur during secretion. Deficient sialylation can occur from inherited genetic defects of sialyltransferases or the induction of neuraminidases, each decreasing Muc2 sialylation and an increased rate of proteolysis.The colon is a tissue under constant threat from environmental toxins and infection. Muc2 is a key component of the stratified mucin layer protecting the colon from damage and disease. Recurrent ST infection induced Muc2 proteolysis resulting in mucosal barrier erosion in the onset of an acquired colitis that appears similar to human Ulcerative Colitis. The pathogenic mechanism includes innate Tlr4-dependent induction of colon epithelial cell Neu3 and Paneth cell CatG of the small intestine that act sequentially by removing Muc2 sialic acids that protect against increased Muc2 proteolysis and barrier degradation. Tlr4 has been shown essential also for the inflammation produced in this food poisoning model of IBD by causing the acquired depletion of the anti-inflammatory intestinal alkaline phosphatase (IAP).9 CatG deficiency has a lesser effect on inflammatory cytokine induction than does IAP deficiency comparing with previous studies, which may reflect the efficacy of augmented IAP treatment in reducing LPS-P levels. Whether this mechanism operates in response to infection by other microbial species and in the context of other changes in the colonic microbiome remains to be known. In humans, this mechanism of disease may exist among some IBD patients with increased Neu3 neuraminidase and CatG serine protease levels reported.22,23,24 This glycoproteolytic mechanism of colonic mucosal barrier erosion begins with the innate inflammatory response of the host to repeated gastric infection by the common foodborne ST bacterial pathogen and identifies potential therapeutic targets for inhibition to maintain and possibly restore the protective colonic mucosal barrier.Limitations of this studyThe structure of nascent secreted Muc-2 and its net-like polymeric organization in the stratified mucin layer under normal and pathogenic conditions remains under intense investigation. The murine Muc2 protein translated (accession number NP_076055.4) is composed of 4576 amino acids. Our findings using polyacrylamide gel electrophoresis demonstrate that colonic murine Muc2 isolated from the stratified mucin layer and visualized with commercially available antibody reagents migrates above the highest molecular weight marker used of 250 kDa in reducing polyacrylamide gel electrophoresis, and significantly larger in non-reducing conditions. Absent standard molecular weight markers are above 250 kDa; we do not know the precise molecular weight of Muc2 in these gel migration analyses. Other studies using polyacrylamide gel electrophoresis reported Muc2 migration at 200 kDa and above, or above 450 kDa.25,26 Studies using agarose gel electrophoresis have reported Muc2 migration above the highest molecular weight markers used in each study, either 225, 250, or 460 kDa.20,27,28 Another study reports Muc2 molecular weight above 540 kDa although the gel system used was not indicated.29 There are multiple possible reasons for these differences that we cannot distinguish at present. First, agarose gel electrophoresis may more closely represent Muc2 polymeric structure in the stratified layer, perhaps similar to our findings of Muc2 migration using non-reducing polyacrylamide electrophoresis. Second, secreted Muc2 may be normally processed into smaller forms by removal of terminal domain(s) during oligomerization and residence within in the stratified layer, rendering subsequent molecular weight analyses unrepresentative of primary amino acid sequence. Third, protein sialylation with the attendant negative charge can affect electrophoretic determinations of molecular weight. Finally, our findings as well as those of some others referenced may be detecting a processed fragment of the full-length translated Muc2 product that primarily contributes to the normal structure and functioning of the mucin barrier. Nevertheless, using pharmacological inhibition and genetic-null alleles, we have demonstrated that the induction of Muc2 proteolysis and erosion of the colonic mucosal barrier are linked to the functions of Neu3 and Cathepsin-G. Moreover, Neu3 and Cathepsin-G induction are further linked to an increased frequency of onset and severity of colitis using this repeated food poisoning model of acquired colitis and IBD.STAR★MethodsKey resources table REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesRabbit polyclonal Muc2 antibodySanta Cruz BiotechnologyCat #: sc-15334; RRID: AB_2146667Rabbit polyclonal Muc2 antibodyGeneTexCat #: GTX100664; RRID: AB_1950958Goat polyclonal Muc2 antibodySanta Cruz BiotechnologyCat #: sc-13312; RRID: AB_2146672Rabbit polyclonal Neu3 antibodySanta Cruz BiotechnologyCat #: sc-134931; RRID: AB_10609640Rabbit polyclonal cathepsin G antibody, biotinylatedBiorbytCat #: orb460740Rabbit polyclonal cathepsin G antibodyAbcamCat #: ab197354Rabbit monoclonal lysozyme antibodyAbcamCat #: ab108508; RRID: AB_10861277Goat polyclonal αTubulin antibodySanta Cruz BiotechnologyCat #: sc-31779; RRID: AB_2210217Rabbit polyclonal neutrophil elastase antibodyCell Signaling TechnologyCat #: 44030Rabbit polyclonal proteinase 3 antibodyLSBioCat #: LS-C296141Erythrina cristagalli lectin, biotinylatedVector LaboratoriesCat #: B-1145; RRID: AB_2336436Ricinus Communis Agglutinin-I lectin, biotinylatedVector LaboratoriesCat #: B-1085; RRID: AB_2336707Peanut Agglutinin lectin, biotinylatedVector LaboratoriesCat #: B-1075; RRID: AB_2313597Maackia amurensis-II lectin, biotinylatedVector LaboratoriesCat #: B-1265; RRID: AB_2336569Sambucus nigra lectin, biotinylatedVector LaboratoriesCat #: B-1305; RRID: AB_2336718HRP-conjugated Erythrina cristagalli lectinEY LaboratoriesCat #: H-5901-1HRP-conjugated Ricinus Communis Agglutinin-I lectinEY LaboratoriesCat #: H-2001-1HRP-conjugated Peanut Agglutinin lectinEY LaboratoriesCat #: H-2301-1HRP-conjugated Maackia amurensis-II lectinEY LaboratoriesCat #: H-7801-1HRP-conjugated Sambucus nigra lectinEY LaboratoriesCat #: H-6802-1Texas Red-conjugated goat anti-rabbit IgGSanta Cruz BiotechnologyCat #: sc-2780; RRID: AB_ 649006FITC-conjugated goat anti-rabbit IgGSanta Cruz BiotechnologyCat #: sc-2090; RRID: AB_ 641179Texas Red-conjugated rabbit anti-goat IgGSanta Cruz BiotechnologyCat #: sc-3919; RRID: AB_ 654579FITC-conjugated streptavidinVector LaboratoriesCat #: SA-5001; RRID: AB_ 2336462HRP-conjugated streptavidinBD BiosciencesCat #: 550946Bacterial and virus strainsSalmonella enterica serovar TyphimuriumCDC 6516-60ATCC 14028Chemicals, peptides, and recombinant proteins10% buffered formalinSigma-AldrichCat #: HT5014MethanolSigma-AldrichCat #: 179337ChloroformSigma-AldrichCat #: 34854Acetic acidSigma-AldrichCat #: 695092SucroseSigma-AldrichCat #: S0389Tissue-Tek OCT compound, Sakura FinetekVWRCat #: 25608-930Mayer’s hematoxylin solutionSigma-AldrichCat #: MHS16Eosin Y solutionSigma-AldrichCat #: HT110116TRIzol ReagentInvitrogenCat #: 15596026Dextran-FITCSigma-AldrichCat #: 68059BD Microtainer Serum Separator TubeBD BiosciencesCat #: 365967Protein A/G PLUS agaroseSanta Cruz BiotechnologyCat #: sc-2003Amersham ECL Western Blotting Detection ReagentGE HealthcareCat #: RPN2109Coomassie brilliant blue G250Bio-RadCat #: 1610406Precision Plus Protein™ Dual Color StandardsBio-RadCat #: 1610394Bovine Serum Albumin (BSA)Jackson ImmunoResearchCat #: 001-000-162Sulfo-NHS-LC-LC-BiotinThermoFIsher ScientificCat #: 213353,3′,5,5′-Tetramethylbenzidine (TMB) Liquid SubstrateSigma-AldrichCat #: T0440ZanamivirSigma-AldrichCat #: SML0492Complete protease inhibitor cocktailRocheCat #: 11697498001E−64Sigma-AldrichCat #: E3132PMSFSigma-AldrichCat #: P7626EDTASigma-AldrichCat #: E9884EGTASigma-AldrichCat #: E3889Cathepsin G inhibitorSigma-AldrichCat #: 219372Elastase inhibitorSigma-AldrichCat #: M0398Trypsin inhibitorSigma-AldrichCat #: T7254Neuraminidase (from Arthrobacter ureafaciens)EY LaboratoriesCat #: EC-32118-5Critical commercial assaysHemoccult Fecal Blood Slide Test SystemBeckman CoulterCat #: 60151Alcian Blue Stain KitVector LaboratoriesCat #: H-3501Ulysis Alexa Fluor 488 Nucleic Acid Labeling KitMolecular ProbesCat #: U21650SuperScript III reverse transcriptase kitInvitrogenCat #: 18080093Brilliant SYBR green qPCR master mix kitAgilent TechnologiesCat #: 600830Amplex Red Neuraminidase Assay KitThermo Fisher ScientificCat #: A22178QIAamp DNA Mini KitQiagenCat #: 51304Experimental models: Organisms/strainsC57BL/6J miceThe Jackson LaboratoryN/ANeu3–/– miceYamaguchi et al.30N/ASt3gal6Δ/Δ miceYang et al.31N/ACtsg–/– miceMacIvor et al.15N/ATlr4–/– mice (B6(Cg)-Tlr4tm1.2Karp/J)The Jackson LaboratoryStock #: 029015OligonucleotidesEUB-338: GCTGCCTCCCGTAGGAGTGerlach et al.33N/ANon-EUB-338: CGACGGAGGGCATCCTCAGerlach et al.33N/ACCL5-forward: TCGTGTTTGTCACTCGAAGGYang et al.9N/ACCL5-reverse: CTAGCTCATCTCCAAATAGTYang et al.9N/AIL-1β-forward: GCCCATCCTCTGTGACTCATYang et al.9N/AIL-1β-reverse: AGGCCACAGGTATTTTGTCGYang et al.9N/ATNFα-forward: CATCTTCTCAAAATTCGAGTYang et al.9N/ATNFα-reverse: TTTGAGATCCATGCCGTTGGYang et al.9N/AMuc2-forward: GCTGACGAGTGGTTGGTGAATGMan et al.37N/AMuc2-reverse: GATGAGGTGGCAGACAGGAGACMan et al.37N/ACatG-forward: CAAGGAGATGAGGCAGGGAAThis paperN/ACatG-reverse: TGAGCTGCTGTTAGGACGAAThis paperN/ANE-forward: TGGCCTCAGAGATTGTTGGTThis paperN/ANE-reverse: TACCTGCACTGACCGGAAATThis paperN/APR3-forward: AATGACGTGCTTCTCCTCCAThis paperN/APR3-reverse: GTGACGTTCAGTTCCTGCAGThis paperN/AGAPDH -forward: TGGTGAAGGTCGGTGTGAACYang et al.12N/AGAPDH -reverse: AGTGATGGCATGGACTGTGGYang et al.12N/ATotal bacteria-forward: GTGCCAGCMGCCGCGGTAAFuhrer et al.36N/ATotal bacteria-reverse: GACTACCAGGGTATCTAATFuhrer et al.36N/AClostridiaceae-forward: TTAACACAATAAGTWATCCACCTGGFuhrer et al.36N/AClostridiaceae-reverse: ACCTTCCTCCGTTTTGTCAACFuhrer et al.36N/ALactobacillaceae-forward: AGCAGTAGGGAATCTTCCFuhrer et al.36N/ALactobacillaceae-reverse: CGCCACTGGTGTTCYTCCATATAFuhrer et al.36N/ABacteroidaceae-forward: CCAATGTGGGGGACCTTCFuhrer et al.36N/ABacteroidaceae-reverse: AACGCTAGCTACAGGCTTFuhrer et al.36N/AEnterobacteriaceae-forward: CATTGACGTTACCCGCAGAAGAAGCFuhrer et al.36N/AEnterobacteriaceae-reverse: CTCTACGAGACTCAAGCTTGCFuhrer et al.36N/ASoftware and algorithmsGraphPad Prism 7.0GraphPad Software ExcelMicrosoft imaging software 3.5TissueGnostics imaging solution Analysis software 4.0TissueGnostics imaging solution Analysis software 4.0TissueGnostics imaging solution Image Acquisition and Analysis SoftwareUVP Resource availabilityLead contactFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jamey D. Marth ([email protected]).Materials availabilityThis study did not generate new unique reagents.Experimental model and study participant detailsLaboratory animalsAnimal experiments were used equal numbers of male and female mice, unless otherwise indicated. Inbred C57BL/6J mice were used (Jackson Laboratory). Neu3-deficient, St3gal6-deficient, and Ctsg-deficient mice were backcrossed six or more generations into the C57BL/6J background prior to study.15,30,31 Tlr4–/– mice (B6(Cg)-Tlr4tm1.2Karp/J) were purchased from the Jackson Laboratory. Littermates of the indicated genotypes were used as controls. Adult mice 8 weeks of age or older as indicated were used in all studies. Mice of both sexes were also used in approximately equal fractions. We found that the phenotypes and results obtained in our studies were not significantly different among adult males and females. All mice analyzed were provided sterile pellet food and water ad libitum. Institutional Animal Care and Use Committees of the University of California Santa Barbara and the Sanford-Burnham-Prebys Medical Discovery Institute approved the mouse studies undertaken herein. All mice were housed with their littermates typically in groups of four or five animals per cage in a specific pathogen-free barrier facility at the University of California Santa Barbara and the Sanford-Burnham-Prebys Medical Discovery Institute. Every effort was made to minimize and eliminate animal suffering and to reduce the number of animals needed.Bacterial strains and culture conditionsMT2057, a kanamycin-resistant derivative of Salmonella enterica serovar Typhimurium (ST) reference strain ATCC 14028 (CDC 6516-60) was used.9,32 ST was streaked from frozen stocks onto Luria-Bertani (LB) agar plates and incubated overnight at 37°C. Single colonies were inoculated into LB broth and incubated overnight with shaking at 37°C. ST was pelleted by centrifugation, washed, and suspended in sterile 0.2M sodium phosphate buffer (pH 8.1).Method detailsBacterial infectionsFor the induction of chronic colitis, Adult 8-week-old mice were infected with ST (2 × 103 cfu) via gastric intubation at 4-week intervals successively up to five times during adult life after the initial infection.9 Mice were weighed bi-weekly and further assessed for overt signs including the presence of diarrhea and fecal blood (Hemoccult Fecal Blood Slide Test System, Beckman Coulter).9,21HistologyMouse intestinal tissues were fixed in 10% buffered formalin (Sigma-Aldrich) or Methanol-Carnoy’s fixative, transferred to 30% sucrose/PBS, and embedded in Tissue-Tek OCT compound (Sakura Finetek).5,21,33 Three-micron frozen sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich) or incubated with 1 μg/ml of antibodies to one or more molecules including Muc2 (H-300, Santa Cruz Biotechnology), Neu3 (M-50, Santa Cruz Biotechnology), cathepsin G (orb460740, Biorbyt), lysozyme (EPR2994, Abcam), or αTubulin (P-16, Santa Cruz Biotechnology), or 5 μg/ml of biotinylated lectins including Erythrina cristagalli (ECA), Ricinus Communis Agglutinin-I (RCA), Peanut Agglutinin (PNA), Maackia amurensis-II (MAL-II), or Sambucus nigra (SNA) (Vector Laboratories). Tissue sections were also stained with Alcian blue, followed by counterstaining with nuclear fast red (Vector Laboratories). Muc2 or lysozyme was visualized with 0.4 μg/ml of Texas Red-conjugated goat anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology); Neu3 was visualized with 0.4 μg/ml of FITC-conjugated goat anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology); αTubulin was visualized with 0.4 μg/ml of Texas Red-conjugated rabbit anti-goat IgG secondary antibodies (Santa Cruz Biotechnology); and cathepsin G or biotinylated lectins were visualized with 1 μg/ml of FITC-conjugated streptavidin (Vector Laboratories). These primary antibody or lectin incubations were performed at 4°C overnight and secondary antibody or streptavidin incubations were performed at room temperature for 1 h. For fluorescence in situ hybridization (FISH), colon sections fixed with Methanol-Carnoy’s fixative were incubated with of 250 μg of Alexa Fluor 488–conjugated EUB-338 (5′-GCTGCCTCCCGTAGGAGT-3′; bp 337–354 in bacteria EU622773) or a control nonspecific probe complementary to EUB-338 (Non-EUB-338; 5′-CGACGGAGGGCATCCTCA-3′) in 100 μl hybridization buffer (20 mM Tris-HCl (pH 7.4), 0.9 M NaCl, and 0.1% SDS) at 50°C overnight.33 The sections were rinsed in wash buffer (20 mM Tris-HCl (pH 7.4) and 0.9 M NaCl) at 50°C for 15 min and co-immunostained with 1 μg/ml of antibody to Muc2 (H-300, Santa Cruz Biotechnology) at 4°C. Analyses by microscopy was performed using a TissueGnostics workstation equipped with Zeiss AxioImager Z1, Hamamatsu C13440-20C camera, PixeLINK PL-D673CU camera, and Lumen Dynamics X-Cite XLED1 illuminator. Images collected were analyzed using TissueFAXS (Version 3.5), TissueQuest (Version 4.0), HistoQuest software (Version 4.0) (TissueGnostics USA Ltd.), and ImageJ software (Version 2.0.0) (NIH).mRNA preparation and quantification by real-time PCRTotal RNA was isolated from tissues using Trizol (Invitrogen) and subjected to reverse transcription (RT) using SuperScript III (Invitrogen). Quantitative real-time PCR was performed using Brilliant SYBR Green Reagents with the Mx3000P QPCR System (Stratagene). Primers used for real-time PCR in the mouse were: CCL5-RT-F (5′-TCGTGTTTGTCACTCGAAGG-3′), CCL5-RT-R (5′-CTAGCTCATCTCCAAATAGT-3′), IL-1β-RT-F (5′-GCCCATCCTCTGTGACTCAT-3′), IL-1β-RT-R (5′-AGGCCACAGGTATTTTGTCG-3′), TNFα-RT-F (5′-CATCTTCTCAAAATTCGAGT-3′), TNFα-RT-R (5′-TTTGAGATCCATGCCGTTGG-3′), Muc2-RT-F (5′-GCTGACGAGTGGTTGGTGAATG-3′), Muc2-RT-R (5′-GATGAGGTGGCAGACAGGAGAC-3′), CatG-RT-F (5′-CAAGGAGATGAGGCAGGGAA-3′), CatG-RT-R (5′-TGAGCTGCTGTTAGGACGAA-3′), NE-RT-F (5′-TGGCCTCAGAGATTGTTGGT-3′), NE-RT-R (5′-TACCTGCACTGACCGGAAAT-3′), PR3-RT-F (5′-AATGACGTGCTTCTCCTCCA-3′), PR3-RT-R (5′-GTGACGTTCAGTTCCTGCAG-3′), GAPDH-RT-F (5′-TGGTGAAGGTCGGTGTGAAC-3′), and GAPDH-RT-R (5′-AGTGATGGCATGGACTGTGG-3′). Relative mRNA levels were normalized to expression of Gapdh RNA.In vivo intestinal barrier functionDextran-FITC (Sigma-Aldrich) was administered via oral gavage (600 mg/kg), blood was collected from anesthetized animals into Microtainer Serum Separator Tubes (BD Biosciences) at 4 h with no anticoagulant, and allowed to clot for 30 min at room temperature.34 Serum was collected after centrifugation at 10,000 × g for 10 min. The amount of FITC in each sample was measured by using a Spectra Max Gemini EM fluorescent plate reader (Molecular Devices) at 490 and 530 nm for the excitation and emission wavelengths, respectively.Separation of loose and stratified mucus layer Muc2Mucus from the colon was removed from an identically measured epithelial surface by suction (loosely adherent) or scraped (firmly adherent).5 The samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) supplemented with complete protease inhibitor cocktail per instructions (Roche). Muc2 proteins in loose and stratified mucus layer were analyzed by immunoblotting using 1 μg/ml of antibody to Muc2 (C3, GeneTex) or immunoprecipitated with 2 μg/ml of antibody to Muc2 (R-12, Santa Cruz Biotechnology).Immunoprecipitation, immunoblotting, and lectin blottingTissue samples were homogenized in RIPA buffer supplemented with complete protease inhibitor cocktail per instructions (Roche) and incubated overnight at 4°C on a rotating wheel with 2 μg/ml of antibody to Muc2 (R-12, Santa Cruz Biotechnology), followed by 2 h of incubation in the presence of protein A/G PLUS agarose (Santa Cruz Biotechnology). Immunoprecipitates were washed five times with RIPA buffer and eluted with SDS sample buffer. Protein samples eluted or total tissue homogenates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and incubated with 3% BSA in Tris-buffered saline (TBS). They were then analyzed by immunoblotting using 1 μg/ml of antibody to Muc2 (C3, GeneTex), cathepsin G (ab197354, Abcam), neutrophil elastase (44030, Cell Signaling Technology), or proteinase 3 (LS-C296141, LSBio) or by lectin blotting with HRP-conjugated ECA (0.5 μg/ml), RCA (0.1 μg/ml), PNA (1 μg/ml), MAL-II (0.2 μg/ml), or SNA (0.1 μg/ml) (EY Laboratories). Signals detected by chemiluminescence (GE Healthcare) were analyzed by integrated optical density using LabWorks Image Acquisition and Analysis Software (UVP Bioimaging Systems). Parallel protein samples were visualized with Coomassie brilliant blue G250 staining (Bio-Rad). Protein standards prestained, broad range 250–10 kDa were used as molecular weight markers (1610394, Bio-rad).ELISAELISA plates (Nunc) were coated with 2 μg/ml of antibodies to Muc2 (R-12, Santa Cruz Biotechnology) and blocked by incubation at room temperature for 1 h with 5% BSA in PBS (Jackson ImmunoResearch). To generate biotinylated antigens, 500 μl of total protein extracts isolated from mouse tissue (1 mg/ml) were incubated with 500 μl of sulfo-NHS-LC-LC-biotin (1 mg/ml) (Thermo Fisher Scientific) on ice for 2 h and the biotinylation reaction was stopped with the addition of 15 mM glycine (pH 8.0; final concentration). After washing the ELISA plates, 20 μg of biotinylated protein extracts was added to each well and incubated at room temperature for 2 h. Antigens were detected following the addition of 1:5000 dilution of HRP-streptavidin (BD Biosciences) and 3,3′,5,5′ tetramethylbenzidine (TMB, Sigma-Aldrich). Lectin binding was determined in parallel by the addition of HRP-conjugated ECA (0.5 μg/ml), RCA (0.1 μg/ml), PNA (1 μg/ml), MAL-II (0.2 μg/ml), or SNA (0.1 μg/ml) (EY Laboratories), followed by TMB, and changes in glycan linkages were detected by comparing lectin binding among identical amounts of biotinylated Muc2.35Neuraminidase activity and inhibitionNeuraminidase activity was measured in tissue extracts in RIPA buffer supplemented with complete protease inhibitor cocktail per instructions (Roche) using the Amplex Red Neuraminidase Assay Kit according to the manufacturers’ instructions (Molecular Probes). For inhibition of neuraminidase activity in the colon, Zanamivir (0.5 mg/ml; Sigma-Aldrich) was provided in the drinking water immediately following the initial ST infection at 8 weeks of age and continued for the duration of study as indicated.In vitro Muc2 proteolysisFor obtaining Muc2 immunoprecipitates, 1 mg of tissue lysates in RIPA buffer from the colonic stratified layer of indicated genotypes at 12 weeks of age was incubated overnight at 4°C with 2 μg/ml of antibody to Muc2 (R-12, Santa Cruz Biotechnology), followed by 2 h of incubation in the presence of protein A/G PLUS agarose and washed five times with RIPA buffer. For producing the Muc2-depleted intestinal content solution, 100 mg of intestinal content from wild-type mice at 20 weeks of age following ST re-infection or PBS administration was diluted in 1 ml of TBS buffer and vortexed thoroughly to make an intestinal content solution. After centrifugation at 14,000 rpm, the supernatants was incubated with 2 μg/ml of Muc2 antibody (R-12, Santa Cruz Biotechnology) and protein A/G PLUS agarose for 2 h at room temperature, centrifuged at 2,000 rpm to remove agarose beads, and then applied to Poly-Prep gravity-flow columns (Bio-rad). The flow-through fractions were incubated with Muc2 antibody and protein A/G PLUS agarose, centrifuged at 2,000 rpm to remove agarose beads, and then applied to Poly-Prep gravity-flow columns and these steps were repeatedly performed until Muc2 proteins were not detected in the final flow-through fractions. For in vitro Muc2 proteolysis assay, Muc2 immunoprecipitates isolated from the colonic stratified layer of indicated genotypes at 12 weeks of age were mixed with 1 ml of Muc2-depleted lumenal extract solution generated from 100 mg of lumenal content of WT mice at 20 weeks of age prior to the fourth ST infection or PBS treatment. After incubation in a shaking incubator at 37°C for the indicated times otherwise 24 h, Muc2 immunoprecipitates were washed five times with RIPA buffer, eluted with SDS sample buffer, and subjected to immunoblotting. For protease activity inhibition, complete protease inhibitor cocktail per instructions (Roche), 1 mM E-64 (Sigma-Aldrich), 1 mM PMSF (Sigma-Aldrich), 1 mM EDTA/EGTA (Sigma-Aldrich), 100 μM cathepsin G inhibitor (CAS 429676-93-7; Sigma-Aldrich), 100 μM neutrophil elastase inhibitor (CAS 65144-34-5; Sigma-Aldrich), or 100 μM trypsin inhibitor (CAS 4272-74-6; Sigma-Aldrich) was added to the in vitro Muc2 proteolysis assay.23 For in vitro sialidase treatment, Muc2 immunoprecipitates were incubated with 0.3 U/ml of Arthrobacter urefaciens sialidase (EY lab) or PBS at 37°C for 2 h in the absence or presence of a sialidase inhibitor zanamivir (0.5 mg/ml; Sigma-Aldrich) and washed with TBS buffer prior to or after in vitro Muc2 proteolysis assay.Comparative studies of intestinal microbiotaTotal DNA was extracted from 1 mg of intestinal content or colon tissue per individual mouse using QIAamp DNA Mini Kit according to the manufacturer’s instructions (Qiagen). Total DNA was quantified and used as the template for quantitative real-time PCR (qPCR). A subset of commensal microbial populations were analyzed with Brilliant SYBR Green Reagents and the Mx3000P QPCR System (Stratagene). Oligonucleotide primers for total bacterial DNA were (Total-F-5′-GTGCCAGCMGCCGCGGTAA-3′, Total-R-5′-GACTACCAGGGTATCTAAT-3′; while those to measure individual populations included Clostridiaceae-F-5′-TTAACACAATAAGTWATCCACCTGG-3′, Clostridiaceae -R-5′- ACCTTCCTCCGTTTTGTCAAC-3′; Lactobacillaceae-F-5′-AGCAGTAGGGAATCTTCC-3′, Lactobacillaceae-R-5′-CGCCACTGGTGTTCYTCCATATA-3′; Bacteroidaceae-F-5′-CCAATGTGGGGGACCTTC-3′, Bacteroidaceae-R-5′-AACGCTAGCTACAGGCTT-3′; and Enterobacteriaceae-F-5′-CATTGACGTTACCCGCAGAAGAAGC-3′, Enterobacteriaceae-R-5′-CTCTACGAGACTCAAGCTTGC-3′).36 Serial dilutions of total DNA were used to generate standard curves in acquiring each measurement. Relative levels of bacterial DNA obtained per mg of intestinal content from each mouse were calculated in plotting comparisons to wild-type littermates.Quantification and statistical analysisAll data were analyzed as mean ± SD. unless otherwise indicated. Student’s unpaired t test was used to compare the means of two groups. One-way Analysis of Variance (ANOVA) with Tukey’s multiple comparisons test was used to compute statistical significance between multiple groups. GraphPad Prism software (Version 7.0) was used to determine statistical significance among multiple studies. P values of less than 0.05 were considered significant. Statistical significance was denoted by ∗P < 0.05, ∗∗P < 0.01, or ∗∗∗P < 0.001. The exact value of n, representing the number of mice in the experiments depicted, was indicated in the figure legends.

PMC

Journal of Morphology

36533735

PMC10107104

1-01-2023

10.1002/jmor.21540

Incipient morphological specializations associated with fossorial life in the skull of ground squirrels (Sciuridae, Rodentia)

Gomes Rodrigues Helder, Damette Mathilde

AbstractAnatomical and biological specializations have been studied extensively in fossorial rodents, especially in subterranean species, such as mole‐rats or pocket‐gophers. Sciurids (i.e., squirrels) are mostly known for their diverse locomotory behaviors, and encompass many arboreal species. They also include less specialized fossorial species, such as ground squirrels that are mainly scratch diggers. The skull of ground squirrels remains poorly investigated in a fossorial context, while it may reflect incipient morphological specializations associated with fossorial life, especially due to the putative use of incisors for digging in some taxa. Here, we present the results of a comparative analysis of the skull of five fossorial sciurid species, and compare those to four arboreal sciurids, one arboreal/fossorial sciurid and one specialized fossorial aplodontiid. The quantification of both cranial and mandibular shapes, using three dimensional geometric morphometrics, reveals that fossorial species clearly depart from arboreal species. Fossorial species from the Marmotini tribe, and also Xerini to a lesser extent, show widened zygomatic arches and occipital plate on the cranium, and a wide mandible with reduced condyles. These shared characteristics, which are present in the aplodontiid species, likely represent fossorial specializations rather than relaxed selection on traits related to the ancestral arboreal condition of sciurids. Such cranial and mandibular configurations combined with proodont incisors might also be related to the frequent use of incisors for digging (added to forelimbs), especially in Marmotini evolving in soft to hard soil conditions. This study provides some clues to understand the evolutionary mechanisms shaping the skull of fossorial rodents, in relation to the time spent underground and to the nature of the soil. Our morphometric analyses permit to discuss the putative behavioral and environmental parameters shaping the skull of ground squirrels in relation to their digging activity. Our paper reports that these sciurid rodents do show convergent morphological traits on both cranium and mandible in relation to fossorial life. The different examples of skull shapes reported in ground squirrels are related to departure from tree life and to the strong constraints imposed by digging, especially in hard soils, and by the time spent underground.

1INTRODUCTIONFossorial species are known for their digging abilities to construct burrows mainly to protect from external conditions (e.g., predators and climate), but also for dispersion and foraging, especially when they spend most of their lifetime underground (e.g., Ellerman, 1956; Hildebrand, 1985; Nevo, 1979). Among mammals, rodents show numerous morphological specializations for digging. These adaptations are notably related to the use of claws for digging (i.e, scratch‐digging), but also to the use of ever‐growing incisors (i.e., chisel‐tooth digging), which is widespread in subterranean species, such as African and blind mole‐rats, pocket‐gophers, tuco‐tucos or cururos (e.g., Stein, 2000; Gomes Rodrigues et al. in press). More than 10 families of extant rodents show convergent adaptations to fossorial life, impacting their long bones and girdles, as well as their skull to various extents (e.g., Stein, 2000; Gomes Rodrigues et al. in press). Subterranean rodents generally show the most extreme cranial specializations due to their more intense activity of chisel‐tooth digging (e.g., procumbent incisors and massive masticatory muscles; Fournier et al., 2021; Gomes Rodrigues et al., 2016; Landry, 1957; Lessa & Patton, 1989; Marcy et al., 2016; McIntosh & Cox, 2016) and to the constraints related to life underground (e.g., reduced eyes and pinnae; Nevo, 1979; Scarpitti and Calede, 2022; Stein, 2000). The morphology of subterranean rodents has been studied, but a focus on less specialized fossorial taxa would have allowed a better comprehension of the evolutionary mechanisms underlying fossorial adaptations in rodents.Ground squirrels of the family Sciuridae are not strongly specialized fossorial rodents. They notably encompass marmots, prairie dogs, or susliks, and most of them construct burrows, mainly as nest and protection, but do not spend all their lifetime underground (i.e., they are not defined as subterranean; Koprowski et al., 2016; Nowak, 1999). These rodents use mainly their claws for digging and far less their incisors (Agrawal, 1967). Focusing on susliks and relatives from the genus Spermophilus, Lagaria and Youlatos said that “given that scratch digging is an integral part of their biology that may increase fitness, it is very likely that (these) ground squirrels exhibit associated morphological correlates.” This statement can be generalized to all fossorial ground squirrels since it has been demonstrated that they do have muscular, anatomical and histological adaptations for scratch‐digging revealed by investigations of both forelimbs and hindlimbs (Lagaria & Youlatos, 2006; Mielke et al., 2018; Scheidt et al., 2019; Thorington, Jr. et al., 1997; Wölfer & Nyakatura, 2019; Wölfer, Amson, et al., 2019). But, does the skull of ground squirrels similarly show fossorial specializations?In a study on extinct burrowing beavers including a comparison with many fossorial rodents, Samuels and Valkenburgh did not detect any cranial specialization for fossorial life in ground squirrels. Most studies on sciurid skull shape have highlighted the strong phylogenetic signal, the lesser influence of size and dietary habits on the mandible and thus pointed out the near lack of ecomorphological convergence (e.g., Cardini, 2003; Casanovas‐Vilar & Van Dam, 2013; Michaux et al., 2008; Zelditch et al., 2015, 2017). Nonetheless, some of them tend to show that ground squirrels set apart from tree squirrels (e.g., Lu et al., 2014; Michaux et al., 2008; Velhagen & Roth, 1997; Zelditch et al., 2015), in having morphologies clearly diverging from the ancestral arboreal condition of sciurids (Rocha et al., 2016; Steppan et al., 2004). One of these studies (Lu et al., 2014) also stressed putative fossorial traits on the skull of a few sciurids, but none of them mentioned morphological convergence in that respect. However, convergent evolution based on qualitative morphological similarities of the skull (i.e., flat and wide cranium, wide mandible) have been assumed between some ground squirrels and extinct and extant Aplodontiidae, a sister group of Sciuridae within Sciuromorpha, which has a stronger fossorial evolutionary history (Druzinsky, 2010; Hopkins, 2019).Here, our aim is to quantify and compare the skull shape of phylogenetically distant species of fossorial ground squirrels, involving Marmotini (e.g., marmots, prairie dogs, susliks, and chipmunks) and Xerini (e.g., unstriped ground squirrel, and long‐clawed ground squirrels; Figure 1). All these species are scratch diggers, but some works mentioned the occasional use of incisors for digging in Marmotini (e.g., Agrawal, 1967; Burns et al., 1989; Ramos‐Lara et al., 2014). Comparisons of fossorial species with some arboreal species are also drawn to quantify their morphological differences. Comparisons with the mountain beaver, which is a highly specialized fossorial species and the sole extant representative of Aplodontiidae, allow to evaluate the morphological similarities with fossorial sciurid species. Numerous studies on the mandible of sciurids have been published. Fewer studies have focused on the cranium (e.g., Cardini & O'Higgins, 2004; Roth, 1996), while the cranial shape of sciurids is assumed to be more importantly influenced by lifestyle than the mandible (Lu et al., 2014). Therefore, both parts of the skull are quantified here using three dimensional geometric morphometrics. We will consider to what extent the fossorial activities of sciurids impact their cranial and mandibular shapes compared with Aplodontiidae and arboreal species. We will also discuss these results in light of the fossorial adaptations observed in more specialized fossorial rodents (i.e., subterranean and chisel‐tooth digging species).Figure 1Phylogenetic relationships between the investigated Sciuromorpha (from Fabre et al., 2012; and Menéndez et al., 2021) and associated number of specimens. Specimens studied with their incisors intact are indicated in brackets. In bold and orange: fossorial taxa, in blue: arboreal/fossorial taxa, in green: arboreal taxa.2MATERIAL AND METHODS2.1Sample compositionSpecimens of Sciuromorpha are all from the collections of the Muséum National d'Histoire Naturelle in Paris (MNHN). We analyzed 59 mandibles and 60 crania representing one aplodontiid genus/species and 10 sciurid genera/species (Supporting Information: Table 1 in Supporting Information Online Material). Among them, one aplodontiid (Aplodontia) and five sciurids (Marmota, Cynomys, Spermophilus, Xerus, and Spermophilopsis) were here defined as fossorial because of their significant digging activity and the time they spent in burrows (Koprowski et al., 2016; Thorington, Jr. et al., 1997). Four other sciurids were defined as arboreal (Ratufa, Protoxerus, Sciurus Tamiasciurus), and one sciurid (Tamias) is defined as arboreal/fossorial because of its intermediate lifestyle (see Rocha et al., 2016; Steppan et al., 2004; and Mielke et al., 2018). These different categories of locomotor behavior also follow the nomenclature used in recent ecomorphological studies on Sciuromorpha (Mielke et al., 2018; Scheidt et al., 2019; Wölfer & Nyakatura, 2019; Figure 1). Numerous studies that focused on sciurid skull shape, especially on mandibles, have been performed on large comparative databases, which also permitted to reliably test for phylogenetic signal, dietary imprint on shape, as well as the allometric component (e.g., Calede et al., 2019; Casanovas‐Vilar & Van Dam, 2013; Lu et al., 2014; Michaux et al., 2008; Velhagen & Roth, 1997; Zelditch et al., 2015, 2017). We consider that the present sample, although smaller, is appropriate to evaluate the main morphological traits shared by the fossorial aplodontiid and the two targeted sciurid tribes encompassing iconic fossorial species (i.e., Marmotini and Xerini), and to assess the main potential differences between fossorial and arboreal sciurid species.2.2Geometric morphometric methodsMandibular and cranial forms were quantified using 11 and 62 anatomical landmarks, respectively (Figure 2; Supporting Information Online Material 2). This landmark data set was based on previous studies (Fournier et al., 2021; Gomes Rodrigues et al., 2016; Hautier et al., 2012) and was adapted to our sample. Landmarks #37 and #55 were not used in the analyses, because pterygoid processes are broken in some specimens, as was the tip of incisors. We nonetheless included additional landmarks (#70, #71, and #72) at the tip of upper incisors to obtain complementary information on a sub sample of crania for which eight specimens were removed due to damaged or extremely worn incisors (see Figure 1 and Supporting Information Online Material 1). Only the right hemi‐mandible was investigated here. Similar to the cranium, we also included an additional landmark (#73) at the tip of the right lower incisor on a sub sample of hemi‐mandibles for which five specimens were removed (see Figure 1 and Supporting Information Online Material 1). Digital data for most specimens were acquired using a Microscribe 3‐D digitizer (G2X, Immersion Corporation, measurement error: 0.0001 mm). The cranium and mandible of Cynomys ludovicianus (MNHN.ZM.MO1960‐3673) were scanned with a v|tome|x 240 L, Baker Hughes Digital Solutions, at the AST‐RX platform (MNHN, Paris) using a cubic voxel of 34.4 μm, to visualize virtual deformations within the data set. Landmarks were then digitized on this reconstructed mesh using the “LANDMARK editor” ( This protocol, involving digitization of both osteological and scanned specimens, was already tested in previous analyses in which measurement biases were negligible (e.g., Fournier et al., 2021; Gomes Rodrigues et al., 2016; Hautier et al., 2012).Figure 2Landmarks digitized on the cranium (a, ventral view, b, dorsal view; c, lateral view) and the mandible (d, occlusal view; e, lateral view) of Cynomys ludovicianus (MNHN.ZM.MO1960‐3673) imaged by using X‐ray conventional microtomographic 3D rendering. 3D, three dimensional.For the cranium, landmarks are mostly of type 1 (juxtaposition of tissues). Because the hemimandible of rodents is composed of a unique dentary bone of relatively simple shape, most of the landmarks taken on the dentary were of type 2 (e.g., maxima of curvature—Figure 2; Bookstein, 1991). All configurations (sets of landmarks) were superimposed using the Procrustes method of generalized least squares superimposition (GLS scaled, translated, and rotated configurations so that the intralandmark distances were minimized) following the method used by Rohlf and Slice , Bookstein , and Rohlf . Shape variation of the cranium and mandible was analyzed using principal components analyses (PCA; Figures 3 and 4, Supporting Information Online Material 3, 4, 5, and 6). Analysis and visualization of patterns of shape variation were performed with the interactive software package MORPHOTOOLS (Lebrun et al., 2010). We computed phylomorphospaces based on the mean coordinates of each genus from the first PC axes and on a simplified phylogeny of Sciuromorpha (Figure 1), and using the phylomorphospace function from the phytools R package (Revell, 2012; Figures 3 and 4). We calculated neighbor joining (NJ) trees to propose an overall representation of the morphological distance between each specimen and to evaluate the phenetic affinities of species (i.e., morphological similarity; Figure 5). We used the nj function from the APE R package (Paradis & Schliep, 2019) computed on a matrix of Euclidean distances, previously calculated on the first PCs representing more than 90% of the total variation. Multivariate analyses of variation (MANOVA) were also realized on these data (i.e., first PCs > 90%) to test for similarities between fossorial and arboreal categories. To do so, data were previously rank transformed data because they did not meet the assumptions required for such parametric tests (i.e., normality, homoscedasticity of variances; Conover et al., 1981). In parallel, these tests were performed on sciurids only, after removal of Aplodontia to test for morphological differences between fossorial and arboreal sciurids only. The arboreal/fossorial Tamias was not included in these tests, because of its small sample.3RESULTSBoth PCAs on crania and mandibles show a clear differentiation between most fossorial and arboreal genera on the first axis, especially concerning the mandibular analysis (Figures 3 and 4; Supporting Information Online Material 3 and 4). PC1, PC2, and PC3 account for 29.55%, 14.22%, and 8.01% of the total variation respectively for the cranium, and PC1, PC2, and PC3 account for 34.81%, 22.98%, and 9.49% of the total variation, respectively, for the mandible. Contributions on each axis as well as distributions of specimens are quite similar when coordinates for upper and lower incisors are included in the analyses (for crania—PC1: 30.56%, PC2: 14.49%, PC3: 8.89% of the total variation; for mandibles—PC1: 32.81%, PC2: 23.22, and PC3: 10.00% of the total variation; Supporting Information Online Material 5 and 6).The cranial analysis shows that Aplodontia plot on the negative side of PC1 (Figure 3a, Supporting Information Online Material 3a). Marmota and Cynomys also have negative values. They are closer to other fossorial taxa, Spermophilus, Xerus and Spermophilopsis, which are however located at the positive side of PC1, in the vicinity of arboreal taxa and of the arboreal/fossorial Tamias. The negative side of PC1 is characterized by a flat cranium showing an enlarged rostrum including elongated dental rows, but also reduced frontal bone areas and cranial vault, widened and posteriorly oriented zygomatic arches, a widened and anteriorly‐tilted occipital plate, and massive tympanic bullae. It can be mentioned that in the analysis involving incisors these specimens also present elongated and orthodont incisors (Supporting Information Online Material 5). At the positive side, the deformations are mostly represented by a higher, but slender cranium displaying enlarged cranial vault and frontal areas, a ventrally oriented foramen magnum, but a reduced rostrum combined with incisors reduced and curved backward. On PC2, most fossorial taxa plot on the negative side and are associated with a slender cranium including a reduced braincase, zygomatic plates posteriorly expanded and an elongated rostrum (Figure 3a, Supporting Information Online Material 3a). In contrast, Aplodontia and arboreal taxa are associated with a more massive, but shorter cranium on the positive side of PC2. On the negative side of PC3 (Figure 3b, Supporting Information Online Material 3b), Marmota, Cynomys and Ratufa present a virtual deformation with a widened rostrum, a reduced frontal area, combined with slightly proodont incisors (according to the additional analysis), conversely to Xerus plotting on the extreme part of the positive side of PC3.Figure 3Principal component analyses performed on crania of Sciuromorpha (mean specimens for each genus) with phylogenetic relationships and associated virtual deformation on the extreme sides of each axis. Yellow and violet code for increases and decreases in surface area, respectively. In orange: fossorial taxa, in blue: arboreal/fossorial taxa, in green: arboreal taxa.Concerning the PCA on mandibles, most fossorial taxa plot on the negative side of PC1, whereas arboreal taxa, the arboreal/fossorial Tamias and the fossorial Spermophilopsis plot on the positive side (Figure 4a, Supporting Information Online Material 4a). On the negative side, the virtual deformation is characterized by a short ramus including an enlarged and laterally projected angular process, and an enlarged body with an elongated dental row. The additional analysis including incisor also displays a mandibular shape with an elongated and anteriorly projected incisor (Supporting Information Online Material 6). Conversely, the virtual deformation on the positive side of PC1 shows a shorter mandible, but enlarged articular condyle and coronoid process. PC2 are mainly characterized by Xerus plotting on the negative side and Aplodontia on the positive side, while other taxa are located in between. If the negative side is characterized by a slender hemi‐mandible with a more developed condyle, on the positive side, the hemi‐mandible is more robust with an elongated coronoid process and a laterally projected angular process. The main information on PC3 relies on the plotting of Cynomys on the negative side with a virtual deformation showing a short but massive condyle close to the coronoid process, and an enlarged angular process (Figure 4b, Supporting Information Online Material 4b). On the additional analysis including the incisor, we observe slight differences on the plot with Marmota, Ratufa, and some Protoxerus plotting close to Cynomys on the negative side of PC3, and the associated virtual deformation also shows an elongated incisor.Figure 4Principal component analyses realized on hemi‐mandibles of Sciuromorpha (mean specimens for each genus) with phylogenetic relationships and associated virtual deformation on the extreme sides of each axis. Yellow and violet code for increases and decreases in surface area, respectively. In orange: fossorial taxa, in blue: arboreal/fossorial taxa, in green: arboreal taxa.Both NJ analyses show a clear clustering of fossorial genera on one side and of arboreal genera and Tamias on the other side (Figure 5). Intrageneric clustering is higher for the cranium than for the mandible for which Spermophilus and arboreal taxa are not strongly clustered. Among fossorial taxa, the cranial and mandibular shapes of Cynomys and Marmota are close to Aplodontia, which however shows more elongated branches. Spermophilopsis is the fossorial taxa showing the smallest cranial and mandibular morphological distances from arboreal taxa, in agreement with PCA analyses. These results are confirmed by the MANOVA showing that significant differences exist between fossorial and arboreal taxa for both the cranium (F = 48.7, p < .001) and the mandible (F = 57.73, p < .001), and even after removal of Aplodontia from the tests (for the cranium: F = 71.8, p < .001; for the mandible: F = 43, p < .001).Figure 5Trees illustrating morphological distances between Sciuromorpha resulting from neighbor joining analyses based on coordinates from the PCAs, and performed on crania (a) and hemi‐mandibles (b). Ap, Aplodontia; Cy, Cynomys; Ma, Marmota; Pr, Protoxerus; Ra, Ratufa; Sc, Sciurus; Sp, Spermophilus; Spi, Spermophilopsis; Tm, Tamias; Ts, Tamiasciurus; Xe, Xerus. In bold and orange: fossorial taxa, in blue: arboreal/fossorial taxa, in green: arboreal taxa.4DISCUSSION4.1Shared cranial and mandibular traits associated with fossorial lifeBoth analyses of cranial and mandible shapes show that the fossorial and arboreal Sciuromorpha species investigated here are clearly distinct. Aplodontiidae, Marmotini (except Tamias), and Xerini share cranial and mandibular traits (detailed below), even if they are less marked in this latter tribe. These results support previous analogous observations on the skull of aplodontiids and some ground squirrels (Druzinsky, 2010; Hopkins, 2019). They also concur with different analyses on mandible shape showing a similarity between some Marmotini and Xerini (Casanovas‐Vilar & Van Dam, 2013; Michaux et al., 2008; Zelditch et al., 2015). Tamias, the arboreal/fossorial species, which is an excellent climber and also digs burrows for nesting (Koprowski et al., 2016; Nowak, 1999), has a skull shape closer to arboreal species. This pattern probably highlights both inherited characters from the putative arboreal condition of the sciurid ancestor (Rocha et al., 2016; Steppan et al., 2004) and the fact that its fossorial activity does not have a significant imprint on its cranial morphology. In previous studies (Michaux et al., 2008; Zelditch et al., 2015), the definition of the skull shape of Tamias tends to be variable, but generally in‐between ground and arboreal squirrels.It could have been assumed that the clustering of phylogenetically distant fossorial sciurid species is simply related to relaxed selection on the skull because of a less constrained locomotion on the ground contrary to life on trees. These ground squirrels notably show: more posterior zygomatic arches and plates implying more posterior orbits limiting arboreal life; elongated rostrum and mandible, which preclude the consumption of hard tree seeds; and a less ventral foramen magnum similar to non‐fossorial ground squirrels not investigated here (e.g., Rhinosciurus, Menetes; Casanovas‐Vilar & Van Dam, 2013; Zelditch et al., 2015). However, the ground squirrels investigated here do show morphological characters associated with fossorial life. These characters are missing in nonfossorial ground squirrels, but are shared with Aplodontia, as suggested (yet not discussed) in Michaux et al. regarding clustering of mandible shapes. These taxa, especially Marmotini, notably present posteriorly oriented and widened zygomatic arches allowing insertion of massive masticatory muscles—as noticed by Lu et al. —coupled to more proodont incisors. These characters are observed in chisel‐tooth diggers, but in a markedly more pronounced way (e.g., Echeverría et al., 2017; Fournier et al., 2021; Gomes Rodrigues et al., 2016, in press; Marcy et al., 2016). They show a relatively wide and anteriorly tilted occipital plate for insertion of massive neck muscles necessary for removing earth out of the burrows (Stein, 2000), a distinctive character found in many extant and extinct fossorial rodents (Gomes Rodrigues et al. in press). As with most fossorial rodents, they also show a braincase relatively reduced contrary to arboreal species (Bertrand et al., 2021).The use of incisors in ground squirrels does not seem at first glance to be optimized for burrowing when looking at the mandible. Ground squirrels, mainly Marmotini, show a reduced condyle combined with an elongated dental row and a widened angular process allowing the insertion of a large superficial masseter. This configuration might improve the mastication of more fibrous and tough plants and roots (e.g., grasses and forbs; Koprowski et al., 2016) probably at the expense of incisor gnawing and biting force (Casanovas‐Vilar & Van Dam, 2013; Cox et al., 2012). Conversely, tree squirrels have a shorter mandible but an enlarged coronoid process for the insertion of a massive temporal muscle, and therefore a high output force at the incisors, in relation to the consumption of hard items (e.g., nuts and fruits; Ball & Roth, 1995; Casanovas‐Vilar & Van Dam, 2013; Cox et al., 2012; Freeman & Lemen, 2008; Velhagen & Roth, 1997). This latter configuration was described in many chisel‐tooth digging rodents, which have large temporal muscles to improve both jaw closing and the incisor output force for digging (e.g., Echeverria et al. 2017; Gomes Rodrigues et al., 2016, in press; Marcy et al., 2016). However, a wide mandible with short condyles, and proodont incisors is observed in fossorial Marmotini and in chisel‐tooth digging rodents, meaning that these characters might facilitate the occasional use of incisors for digging (e.g., bathyergids, ctenomyids, some octodontids, some cricetids, and some spalacids, Gomes Rodrigues et al., 2016; Kryštufek et al. 2016, Fournier et al., 2021). As a result, the mandible of ground squirrels, especially Marmotini, is likely impacted by their fossorial lifestyle, but to a lesser extent relative to the cranium, as suggested by Lu et al. .4.2From scratch digging to chisel‐tooth diggingMarmotini shows fossorial characters more distinctive than in Xerini (e.g., cranium with wide zygomatic arches and occipital plate, proodont incisors), and closer to the specialized morphology of Aplodontia (Calede & Hopkins, 2012; Druzinsky, 2010). It is interesting to note that these characters are only incipient in Spermophilopsis (see Casanovas‐Vilar & Van Dam, 2013 for similar observation on the mandible) showing the most elongated claws, which may improve its scratch digging activity in soft sandy soils (Ognev, 1966; Ružić, 1967), where the use of incisors is not necessary for burrowing. Wölfer and Nyakatura on femoral traits mentioned that Xerini are much closer to the arboreal condition than most Marmotini. This result might be based on diverse functional demands since that Xerini spend less time underground than most Marmotini, which can build burrows in soft to hard soils (e.g., Hoogland, 1996; Janderková et al., 2011; Ponomarenko, 2007). Digging abilities in hard soils are more important in chisel‐tooth diggers than in scratch diggers (Echeverria et al., 2017; Giannoni et al., 1996; Lessa & Thaeler, 1989; Marcy et al., 2016; Mora et al., 2003). The use of incisors for digging has often been observed in Marmota, Cynomys and Spermophilus (Agrawal, 1967; Burns et al., 1989; Ramos‐Lara et al., 2014), contrary to Xerus, Spermophilopsis, and the arboreal/fossorial Tamias, which mainly use their incisors for seed and fruit processing, like tree squirrels (Koprowski et al., 2016). This observation might also explain both the adaptability of fossorial Marmotini to dig in different types of soil and their more pronounced fossorial morphologies.Some rodent species, even if they are not highly fossorial or not adapted to dig with their incisors, can use them occasionally, as observed, for instance, in juveniles of laboratory rats to compensate the lower strength in their forelimbs (Gobetz, 2007). Fossorial ground squirrels do show some specializations for chisel‐tooth digging (e.g., wide cranium with widened occipital plate, wide mandible, and proodont incisors) even if they are not strongly adapted to do so. The use of incisors in addition to forelimbs to break the soil, especially in hard soil conditions, has been reported in other scratch‐digging species, such as degus (Octodon; Ebensperger & Bozinovic, 2000). To date, there are many other lines of evidence suggesting that the use of incisors for digging in rodents is primarily driven by the soil hardness, as noticed in some Marmotini. For instance, pocket‐gophers (Geomyidae) and tuco‐tucos (Ctenomyidae) show different modes of digging depending on the nature of the soil, and some species can use both modes alternately (e.g. Echeverría et al., 2017; Marcy et al., 2016). In African mole‐rats (Bathyergidae), the only scratch digger (Bathyergus) lives in sandy soil contrary to other South African chisel‐tooth digging genera occupying more consolidated soils (i.e., Georychus and Cryptomys; Cuthbert, 1975; Gomes Rodrigues et al., in press). It is likely the case in Rhizomyinae (Spalacidae), in which the African root‐rat (Tachyoryctes) uses only its incisors for digging and occupies more arid areas than its South Asian counterparts, bamboo rats (Rhizomys and Cannomys; Fournier et al., 2021).Consequently, if aridity and opening of environment generated the need to find shelter underground at the origin of the spread of fossorial habits (e.g., Jardine et al., 2012; Nevo, 1979), then frequent changes in the nature of the soil required a better efficacy to remove earth, and thus a more important use of incisors in rodents, as frequently observed in Marmotini. Conversely, the use of incisor for digging is not reported in Xerini, as mentioned above. They use to live in soft soils (Koprowski et al., 2016; Ognev, 1966), especially Spermophilopsis, but they do present cranial specializations, although minor, associated with fossorial life. It means that other behavioral or environmental parameters related to both burrowing and frequent life in burrows might impact their skull shape (e.g., brain size and auditory capacities), which does not extend to Tamias, which frequently climb trees. These different parameters related to the nature of the soil, the time spent underground, and the nature and length of the corresponding subterranean activities (e.g., digging, foraging, or dispersion) likely have a strong impact on both the degree of fossoriality (e.g., use of incisors or not for digging) and on the resulting skull shape, from scratch digging to chisel‐tooth digging rodents.The originality of ground squirrel compared to most fossorial rodents is definitely their morphology inherited from an arboreal condition. Despite their mosaic evolution, sciurids are generally viewed as constituting a morphologically conservative group, reinforced by their functionally versatile trophic morphology, which might limit ecomorphological convergences (see Zelditch et al., 2017). In the present case, the different examples of fossorial adaptations in sciurids (and aplodontiids) are exemplified by convergent skull traits related to departure from tree life and to the strong constraints on the whole skeleton imposed by digging, especially in hard soils (in addition to food foraging and processing). An accurate study of morphological variation of the skull combined with more data on the digging behavior in the different species of ground squirrels in relation to soil hardness will provide more information regarding their skull evolvability and adaptability compared to other fossorial rodents.AUTHOR CONTRIBUTIONS Helder Gomes Rodrigues: Conceptualization (lead); formal analysis (supporting); investigation (equal); methodology (lead); supervision (lead); validation (equal); visualization (equal); writing – original draft (lead). Mathilde Damette: Formal analysis (lead); investigation (equal); validation (equal); visualization (equal); writing – original draft (supporting).CONFLICT OF INTERESTThe authors declare no conflict of interest.Supporting informationSupporting information.Click here for additional data file.

PMC

International Journal of Circumpolar Health

38359161

PMC10877646

10.1080/22423982.2024.2314368

COVID-19 impacts in Northernmost Finland

Timlin Ulla, Rautio Arja

ABSTRACTThe COVID-19 pandemic challenged our lives during the years 2020–2022. Impacts could be seen in everyday life, both locally and nationally, through economic, mental and social elements. However, these effects varied depending on the life situation of individuals. This paper aims to gather information from the representatives and operators working in two Finnish municipalities, Inari and Utsjoki, to understand and learn about their experiences during the COVID-19 pandemic. The data (20 interviews) were collected between December 2021 and February 2022 and analysed following the principles of the qualitative content analysis. The results suggest that the effects of COVID-19 emerged through issues related to the national border between Finland and Norway, economic challenges, and the pressure that people experienced. However, despite challenges, people were supported by everyday life and a connection to nature, communality and close co-operation. Additionally, local needs were highlighted among participants. The results provide a deeper understanding about the public health impacts in these Northernmost municipalities and can therefore be utilised in future development work. They also provide relevant information on the experiences of Sámi people, and specific views related to Sámi people can be recognised.

IntroductionThe COVID-19 pandemic has challenged and impacted our lives, and the most intensive time was during the years 2020–2022. Around mid-March 2020, the WHO pronounced COVID-19 to be a pandemic [1,2]. After 6 months, it was found to be the cause of death for almost one million people worldwide . In Finland, COVID-19 started to spread during March 2020, even though the first case of infection was diagnosed from one tourist on the 29th of January [2,3] at a ski resort located in the municipality of Inari, Finland. Overall, during the pandemic, the spread of infection and number rates varied in Finland, depending on regions; most cases were identified in the Southern part . The COVID-19 pandemic started to spread in Finland late compared to other countries, which in turn, provided an opportunity to implement public health guidelines .Finland, together with many other countries, sets public health recommendations and restrictions, e.g. social distancing and testing. In cases of a positive infection, tracking, isolation, and treatment took place to control the spread of infection [2,4]. National regulations were set, which promoted remote working in organisations, businesses, and services, when possible, restaurants and public cultural and recreational services were closed, and public meetings were limited to up to 10 people [3,5]. Schools and educational organisations provided remote online studying for students over 9-years old [5–7], and it was also advised to provide home care for small children instead of daycare . Most regulations were released during the summer 2020, but restrictions related to hygiene standards, social distancing, and isolation when needed, remained, including control of public gatherings. New waves of the pandemic and regional variations demanded resetting of regulations during the pandemic when necessary – especially in autumn 2020 and spring 2021 [2,5].Starting from January 2021, COVID-19 vaccinations in Finland were provided to people , which helped to control the pandemic and protect the health of people . The pandemic was an economic challenge, resulting in dramatic financial problems for some , but it also burdened the capacity of the healthcare system and resources . Rantanen and colleagues found that these demands resulted in an experience of mental burden among healthcare workers, especially when workload was increased, there was a lack of time to recover from work, or protective COVID-19 instructions were insufficient. The new demands and changes were mentally challenging for working people , but also for family caregivers at home . In both studies, challenges were found to cause, e.g. anxiety, loneliness and stress. According to Koskela and colleagues , the pandemic caused extra worries and burden for parents due to remote studying. They were especially concerned about the use of online tools, control of everyday life and the overall learning and wellness of their children. Suddenly, parents found themselves in a situation where they had to deal with their own remote work, while taking care of their children and remote studying . The collaboration between educational organisations and families in supporting parents has been highlighted .At the beginning of the pandemic, Finland set travel bans, which included bans on international flights from high-risk areas and crossing the national borders between Nordic countries . As Northernmost Finland is the area for residents, including Sámi Indigenous Peoples, who are used to free movement between Nordic countries, this decision separated families and changed the everyday life of people living in these border municipalities . Tourism, reindeer herding, and fishing are the central livelihoods in the municipalities of Inari and Utsjoki, which have borders to Norway and Russia [14,15]. In fact, the municipality of Utsjoki is the only municipality in Finland that has a majority of Sámi residents .This study purposed to investigate the appearance and impacts of the COVID-19 pandemic in Northernmost Finland, keeping in mind the future recommendations related to the pandemic. The study aimed to gather information from the representatives and operators working in two municipalities, Inari and Utsjoki, to understand and learn about their experiences during the COVID-19 pandemic.Materials and methodsStudy participantsThis qualitative study interviewed adult study participants, who were representatives and operators working in the public and private sectors around the two Northernmost municipalities in Finland, Inari and Utsjoki. Both municipalities are in the Sámi homeland and have borders with Norway and Russia. Representatives and operators were working in different fields that were impacted by the COVID-19 pandemic, e.g. health and social work, management, education, culture, economics, business, tourism, communication, community, and volunteer work. They were both Finnish and Sámi; however, identity was not defined for the study. The determinative factor was to gather a wide variation of different representatives and operators working in the municipalities. In the individual interviews, there were nine men and ten women, and one group interview consisted of seven men and six women.Data collectionThe data were collected by a selection of researchers based on the structure of the municipality, using organisation charts and recognising central operators working in these municipalities. This process was finalised during the study as the goal was to achieve a comprehensive representation of representatives and operators working in the area. Researchers contacted study participants, informed about the study, and arranged interviews. This process resulted in 20 interviews. That number of interviews was enough as saturation was fulfilled. Interviews took place during December 2021 – February 2022, with an average duration of around 45–60 min. Interviews were conducted by the two authors of this study, whose backgrounds are in health sciences. Interviews were held at locations preferred by the study participants, usually their work offices. All interviews were completed in Finnish.Theme interviews followed the topics related to overall appearance, impacts and future recommendations. The specific topics, which were asked and discussed in the interviews, were as follows: appearance of the COVID-19 pandemic?negative/positive impactssupportive elements during the pandemicspecific elements related to Sámi identity or cultureimplications/recommendations for the futureData analysisData were analysed by inductive content analysis, which is a relevant analysis method in health research . The analysis process searched for answers to questions: In what ways has COVID-19 appeared/emerged in these municipalities?What has been important/supportive in solving the challenges of COVID-19?What kind of elements related to Sámi identity or culture needs to be considered?What should be considered/recommended for the future?The analysis started by reading the data through several times, to get familiar with it. Next, the open coding process started by coding the data. This was followed by creating coding sheets, and moreover, codes with a similar content were grouped together. These sub-categories were formulated throughout the data. Furthermore, sub-categories were grouped into categories, and the process continued by grouping categories into generic categories. Finally, the abstraction process was closed by creating two main categories (Figure 1). Codes that emerged from the data, which specifically answered the research question of Sámi identity and culture, were at first grouped into their own separate sub-categories. At the end of the analysis, they were merged to the whole data. The results integrate the responses of both Sámi and Finnish participants. Figure 1.Summary of results. From above: categories, generic categories, and main categories. includes especially Sámi peoples perspectives.EthicsFor the participants, all were voluntary, and they had a right to terminate the study at any time. Consent forms were signed, and oral information about the study was provided by the researchers. All participants agreed to recorded interviews. The raw data has been securely handled; only the authors had access. Individual interviews were given a specific research ID that supported the anonymity of participants and were used, while the research assistant transcribed interviews. The risk assessment/data protection impact assessment was completed, and all participants were given the privacy notice for scientific research participants. This study did not fulfill the criteria for an ethical review in human sciences by the University of Oulu, which are based on national guidelines of Finnish National Board on Research Integrity TENK nor the criteria for medical research.ResultsThe categories, generic categories and two main categories (COVID-19 in border municipalities and Internal power of the North) are presented in Figure 1 (see more detailed information in Table 1). The results are presented by the following five generic categories. Quotes can be found from the Tables 2–6. Table 1.Categories and sub-categories, divided by the main categories.COVID-19 IN BORDER MUNICIPALITIESINTERNAL POWER OF NORTHIntensive startImprint of COVID-19COVID-19 as a part of lifeHassle at the beginningStronger due to lessons learnedSomehow getting used to COVID-19Stopping of functions and actionsRemote working will remainGlimpse of normality Good preparedness and reactivityEnsuring the functionsInvesting to future actionsMeaning of nature and environmentNew functions to help community and peopleWorking environment moved onlineImportance of preparedness and co-operation for the futureInvesting in resourcesUncertainty of the futureMaintaining appropriate and meaningful functionsSpace to liveEmpowering natureCOVID-19 in border communityPaying attention to local needsForward togetherBurden from borderSudden separative border*Multidimensional border actions*Impacts on traditional lifeways*Extra burden on top of Covid-19*Importance of the right targeted and appropriate restrictions for the futureIssues related to culture and co-operation that need to be taken to account*Importance of co-operationPositive and multifaceted communalitySupporting Sámi identity during COVID-19COVID-19 in Sámi landSuccessful new solutionsSámi – how it is seen?*One Sámi land*Successful and converging remote workOther/alternative actions to maintain basic functionsBasic activities at riskCollapse and uncertainty of tourism businessEconomic challenges and problemsImpacts on public services*Mental burdenPsychological burden and concernsLiving apartDemands at homeAwakened memories*Uncertainty created by COVID-19Uncertain lifeConstant changes of COVID-19bold text categories• sub-categories.*especially Includes views from Sámi peopleTable 2.Quotes of the generic category COVID-19 meet northernmost Finland.QuotesCategory“World changed completely. On Monday we all worked together at the office and on a next day everyone worked remotely. That was a huge change”. (…) In the beginning it was a shock when we realized that all events will be cancelled. Nothing will happen. (25A)“In the beginning we all lived in total unawareness situation of what COVID-19 bill bring”. (38A)Intensive start (A)“We went outside to meet clients, and they could come to see us on stairs, or by the window. These happened that we could somehow help people”. (28D)Ensuring the functions (D)… during this summer we couldn’t go to Teno river for fishing. Everyday life started to get more difficult. We are used to it, that we can just go to Teno and access to other side and cross the bridge to Norwegian side. And now suddenly it was not obvious at all”. (34C)COVID-19 in border community (C)“We have a lot of families and relatives that live on both side of national borders”. (40B)COVID-19 in Sámi land (B)” But we have to remember that we legally need to provide all the services in three Sámi languages”. (23B) Table 3.Quotes of the generic category pressure caused by COVID-19.QuotesCategory“COVID-19 has hit extremely wide and intensely to whole livelihood and economy structure. First of all, livelihoods depend on business that happens on borders, tourism and reindeer herding. These are the main livelihoods here”. (32E)Basic activities in risk (E)” Loneliness, fear and anxiety seemed to be difficult. And when the tone changed in peoples’ talks, that we can’t cope anymore, because we have coped many weeks already and we feel frightened”. (28F)Mental burden (F)… when COVID-19 started. It was quite interesting, because we just received news about the Spanish flu, which raged here one hundred years ago. (…) And now we got this COVID-19, and we didn’t know in the beginning of what CODID-19 is, so elderly people thought immediately that what is this, is this something similar”. (22F)Mental burden (F)” People are more cautious to attend to events that are organized. (…) And then are thought that is it worth to organize anything, should things to be organized, or even planned, if new regulations will be given”. (31G)Uncertainty caused by COVID-19 (G)Table 4.Quotes of the generic category meaning of the locality.QuotesCategory” Perhaps that how useful these online and remote tools are, and how diverse use can be, to replace regular actions”. (…) Different video materials can be done, or stream something, or create discussions and meetings. Surely some of these will remain creating new practices”. (35H)” I guess COVID-19 impacted in a way that generally people are more prepared for the situations like this. (…) It came from nowhere on year 2020. Nobody was prepared. (…) And now at least we have all the equipment. And people don’t be so scared as year 2020. Everyone will understand a little, if we talk about infections, if the similar situation would happen”. (36H)“Generally, attitudes have changed, when there are more information and knowledge. (…) Surely these changes are lighter, at least according to the current knowledge. It has been a long way that we have complete in two years”. (38H)Imprint of COVID-19 (H)” But you can’t highlight enough the importance of preparedness”. (I29I)Investing to future actions (I)” Perhaps that, what local people have expressed here, is that somehow local views and perspective needs to be considered. It must be acknowledged that at least somehow Finland has recognized the meaning of the border community”. (32J)Paying attention to local needs (J)Table 5.Quotes of the generic category importance of everyday life.QuotesCategory” Surely, we had good readiness for this transfer. That we had had already remote studying and such activities. (…) That for example, teaching for children was, as far as I have understood, pretty much on different level compared to bigger cities”. (27K) ”But it seems that people used to it and adapted to it, to get along with”. (22K)COVID-19 as part of life (K)” But it, that helps the most, is the nature. Our nature is not closed, there are not restrictions. We have so much space to live, and when you go to the mountains”. (…) You can ski and walk out there. That is the thing which helps a lot. It may be, that many locals agree with this”. (37L)” Many elderly people are saying, that when we have space to live and move around, and people generally live fairly long in their own homes, and we have big gardens and forest and everything. That we don’t have that kind of tightness as in the cities. In that sense, it has been good”. (22L)Meaning of nature and environment (L)Table 6.Quotes of the generic category power of co-operation.QuotesCategory“On the other hand, it was nice to see that kind of communality here in the area of our municipality. People were ready to help each other, and those, whose life was not that strong. It was so nice to see. And COVID-19 fundraising was organized as well. People attended to it well”. (22M)” And then, shops donated. (…) Everyone was prepared that tourists will arrive, and then all these extra accessories. People were able to pick them up”. (28M)” These kind of remote discussions, unofficial, unformal discussions of what kind of remote working is, and what is difficult or creates anxiety or what is nice. At the beginning it felt, that it was really negative until people got used to it and we learned to guide it to the more positive direction. These are important to notice”. (30M)Forward together (M)“Yes, this has brought our municipality closer to the other world. Because now all the meetings and trainings can be completed online. It has become so established practice now, that it is so easy now, everybody can use it”. (31N)“By improvising all the projects were able to complete, at least somehow, and pretty well exactly”. (39N)Successful new solutions (N)COVID-19 in border municipalitiesCOVID-19 meets northernmost FinlandBased on the answers of the participants, the pandemic appeared to have an intensive start in Northern Lapland as the first diagnosed COVID-19 case in Finland was found from the municipality of Inari. National and international media were very interested in this case, and journalists travelled to Inari. During this time, the COVID-19 pandemic seemed chaotic and uncertain, and participants experienced that people felt confused and scared. They did not know how to act or what to do; there was uncertainty whether grocery stores would have enough food, so people stockpiled food and necessary items. The Social Insurance Institution of Finland became congested with new benefit applications due to the lack of incomes. People were afraid to meet elderly, and restrictions came into force. As a result, many locals went out into nature, which in turn, crowded popular forest areas. At the very beginning of the COVID-19 pandemic, there were still tourists from Asia and Southern Europe in municipalities, which caused confusion for locals as it was recommended not to travel and still tourists were around. This new situation with COVID-19 appeared to be serious: projects and development plans had to be put on hold, public and community events and activities were cancelled, people did not travel for work, and student exchanges were cancelled. Based on the answers, local schools and student dormitories were closed, when necessary, but also public premises, such as restaurants and libraries, had to be closed. People did not meet each other in person as usual.Participants felt that still, it was obvious that work and some activities had to be continued and carried out despite the COVID-19 pandemic – functions had to be ensured. That meant that the working mode and environment were modified by implementing new action models, such as completing minimal actions or with limited staff, and working online. They indicated that in-person or group meetings with customers or service users, e.g. health and social care and community work, were either on hold or had to be organised in a location without COVID-19 cases in order to carry on essential services. Furthermore, services were modified as well, for example medications and food were delivered to people, e.g. the elderly. Additionally, a free food service was started to help those in need, because people were lacking jobs and income. It was expressed that people met others, for example when picking up food, through windows or at the front of their homes. Student groups became smaller, and children’s summer camps were arranged as day camps. Moreover, traditional religious events had to be formulated in a new way, e.g. funerals and confirmation camps had to be postponed. This change to traditional funerals was distressing for some, as they were used to big funerals that were open to everyone; now due to the COVID-19 pandemic, this tradition seemed to be changing. Participants described that while in-person contact was reduced, telephone calls and messages and written letters returned to use when keeping in touch with customers, students, families, and elders. Also, newspapers were seen as an important element to pass on real-time information to locals, and their content was modified to include supportive and informative views during the pandemic. Online media related to COVID-19 was active, and news reached many locals. Overall, interviewees expressed that people wanted to receive information and had more time to read the news, so the media put an effort on that. Online working became real and familiar to all, with meetings, workshops and society clubs for children and youths, as well as different church services were held online.The COVID-19 pandemic was clearly felt by the border communities, especially those living close to the border between Finland and Norway – which is usually open. Participants indicated that people were not able to go to work, shop, attend hobbies, pick berries, or do normal, everyday activities. Border restrictions and closed borders caused extra problems, burden, and work for many officials, but also for all who lived in these border communities. The impacts on collaboration with Russia were noticed as well, but not as strongly as the border is normally controlled. Based on the interviews, the COVID-19 pandemic presented challenges to the Sámi by creating a real boundary between Finland and Norway, which heavily impacted their lives, and limited Indigenous rights. It limited and even stopped the normal interactions between families, people, and villages, but also had a negative impact on traditional culture. The Sámi people felt isolated and excluded, which created a heavy mental burden. Interviewees expressed concern that control over the closed national border was at times exaggerated, and interpretation of the rules was tight, but also extensive at times. The border guards defined family relationships differently compared to Sámi definitions, and there were experiences of misunderstanding when trying to cross the border due to the reasons of traditional lifeways – which was an acceptable reason. They pointed out that only reindeer herders were allowed to go to Norway when retrieving their reindeer.Study participants described that overall, the Sámi people felt unable to live a normal life, and no one supported them during the COVID-19 pandemic when they were not able to travel to Norway to meet their families. However, during this time, people were able to travel, e.g. from Southern Finland to the North to their villages. It felt unfair, especially when rates of cases were low in areas close to the national borders, but rates were high in the south of Finland. Participants indicated that traditional lifeways in Sámi culture, including reindeer herding and handicrafts, were impacted by the pandemic as well. Due to the closing of restaurants and decrease in the tourism business, the sale of reindeer meat decreased dramatically. This caused serious economic problems for reindeer herders and their families; for many it was the only source of income. Also, it was pointed out that border restrictions did not only have a negative impact on regular reindeer herding; it was also a problem that people were not able to buy equipment for handicrafts from Norway. At the same time as the COVID-19 pandemic, the Sámi people had to deal with extremely challenging winters, which caused the starvation and deaths of reindeer. Additionally, interviewees described that traditional salmon fishing became forbidden in the Teno river due to fishing regulations. All these changes, together with the pandemic, put people’s mental well-being and economic balance at risk. It was felt by the study participants felt that these were experienced even more strongly by Sámi people.Some participants assessed that the impacts of COVID-19 were stronger for Sámi people, especially on their community and culture, compared to Finnish people. For some participants, there were no specific elements or issues related to Sámi Indigenous people or culture that had to be considered during the pandemic, as it was normal and part of everyday life in these municipalities. Overall, Sámi Indigenous peoples and culture were seen among participants as important, essential, and valuable. Moreover, communication with three Sámi languages had to be remembered and respected in municipalities. Sámi culture and families were seen as one Sámi land that has no national borders. Families live in Finland and in Norway, which is normal, and the border is not a real border for the Sámi. Sámi culture and language appeared strong in this area, and everyday communication, collaboration, and traditional lifeways were a part of everyday life.Pressure caused by COVID-19Basic activities were at risk, which were especially noticed in the tourism business and public services, and these presented economic challenges. Study participants indicated that due to the pandemic, tourism was totally stopped, especially at the beginning of the pandemic. International flights were cancelled, and national travel was limited, too. The all-year-round tourism business was in crisis with no tourists, which had been an essential part of the economy of these municipalities. However, based on the interview responses, national tourism became busy especially in summer 2021, which was important for local companies and restaurants to fill the gap. Slowly, the tourism business started to revive, but it has remained vulnerable throughout the pandemic, and seasons varied a lot. Interviewees described that economic and livelihood impacts for municipalities and people were dramatic, not only in tourism but also in restaurant and commerce businesses, especially those close to national borders due to the lack of Norwegian customers. Many people faced layoffs, they did not have income, and suddenly they had to also feed their children during the daytime as schools were closed at the beginning of the pandemic. It was also indicated that media, e.g. newspapers, were economically impacted as well due to a lack of bought advertisements – suddenly, there were no events or such to advertise. Moreover, public services were at risk due to a pause in the co-operation with Norway in social and health care services. In addition, participants expressed concern that collaboration between libraries, schools, and child day care for border communities were on hold. These had impacts on services for Sámi as well, including mental health services and specific health care, e.g. giving birth in a Norwegian hospital (the closest hospital) was not possible. Additionally, based on the interviews, there were experiences that home caregivers had to cope at home without specific services.All these challenges related to COVID-19 caused mental burden for people. It was indicated that for some, changes in working life, such as remote work and online working, appeared to increase their workload. New systems and techniques demanded time, and people missed each other and in-person meetings. Overall, COVID-19 caused anxiety and fear. For some participants, it was challenging to keep updated and look for information about the pandemic situation. On the other hand, some experienced that being supportive to others was difficult or exhausting. They experienced that the pandemic felt endless, and being at home together with the whole family, cooking food, and remote working at the same time was demanding. The feeling of freedom was gone, and effectiveness at work was at risk. It was experienced that the whole community was mentally impacted, with different variations for children, youth, adults, and elders. These challenges were recognised in municipalities. For children, remote school caused extra challenges, and families did not necessarily have computers or other equipment. Alcohol use and domestic violence were recognised as possible problems, too. Participants described that a few weeks after the pandemic started, schools started to provide regular school lunches for children, which helped parents at home. However, they recognised that living alone and being isolated was demanding, especially for elders. People missed their family members and being together. Feelings of loneliness were strong. Regular family occasions, events, and celebrations were cancelled. Moreover, they expressed that it was difficult for people who had to travel long distances without their own car, for example, for vaccinations. On the other hand, as some of the interviewees described, the closed national borders during the COVID-19 pandemic brought up old, tragic memories, such as previous pandemics, e.g. Spanish influenza or World War II. For some, COVID-19 reminded them of life during the residential schools of Indigenous peoples.Participants recognised that the COVID-19 pandemic caused uncertainty for locals. People were unsure whether they could safely travel in Finland or abroad, despite the lack of restrictions to control that. This uncertainty was noticed in the tourism business and economically – the future seemed unsure and unbalanced. No one knew what would happen with the pandemic. They indicated it had resulted in the situation where it was difficult to plan anything, such as meeting people or conducting any events. Possible new restrictions were in interviewees’ minds, and they did not know whether all will be closed again. The COVID-19 pandemic appeared to be unpredictable and living with changes was challenging, especially in the tourism business, which excitedly waited for tourists to fully return. Even though there were good signs that tourism would recover, it felt too unsure to really believe it. Additionally, in the interviews it came out that the pandemic situations varied quickly and regionally. Therefore, people wished for a normal life and hoped that vaccinations would help in the control of the pandemic.Meaning of localityStudy participants felt that they had learned from the COVID-19 pandemic, and a certain imprint of COVID-19 was left. This experience and knowledge were important to recognise and utilise in the future in similar situations. Participants acknowledged that health promotion is important, decisions need to be made quickly, and crisis planning needs to be updated. They recognised how vulnerable health can be. The pandemic both educated and increased the awareness of locals. Additionally, online connections and working were believed to remain in the future, which participants also saw as an important way to reduce economic costs, e.g. travel costs. However, good interactions and supportive teamwork were highlighted when working remotely.When considering the future, interviewees pointed out that investing towards future actions were essential, for example investing in concrete preparedness and appropriate resources. Participants wished for specific action models for similar situations and highlighted the productive and fluent collaborations that aim to support and help individuals. Some felt that preparedness during the pandemic could have been better. Moreover, there was a lack of resources, both economic and human, which require investment, especially in the field of social and health care. Still, the uncertainty of the future was recognised by study participants, which can be shown globally through new viruses or inequality between people, e.g. access to vaccinations. For some, there is mistrust of officials or decision-makers. On the other hand, it is still unsure whether people really learned from this experience. Participants indicated that it is important to maintain appropriate functions in the future. Especially tourism was seen as essential for these municipalities, and it should be a target for investment. Economically, tourism is important, but so is business in general. A sense of community was important for participants as well.Local needs and paying attention to them were highlighted in the interviews by participants. This was especially important when considering and setting guidelines or restrictions to control the pandemic – the Northernmost situation and way of life should be recognised. Participants indicated that their knowledge and expertise are locally in the municipalities. Border restrictions should consider local needs and perceived double impacts, e.g. the restrictions for salmon fishing, should be avoided. Additionally, it was recognised that health is holistic, including not only physical health but also mental and social health. This needs to be considered when setting restrictions in future pandemics. For the future, closer collaboration should take place, for example with the Sámi Parliament, and reindeer herders should be more effectively included in decision-making. It was emphasised that Sámi should be seen as one who have their Indigenous rights, identity, and traditional culture, and this perspective should be included into future action models in similar situations. On the other hand, online actions and remote working or studying during the pandemic allowed Sámi people to stay at home more, which in turn, supported everyday life and traditional lifeways in their homeland. All these would increase adaptation to changes and support traditional life. As described by participants, overall, effective, and reflective interaction and collaboration between Sámi and Finnish peoples need to be remembered.Internal power of the northImportance of everyday lifeOverall, participants expressed a variety of supportive elements that helped them during the pandemic. In a way, COVID-19 became a part of their lives. They felt that after the intensive start and hassle, the situation calmed down a bit. The uncertainty was reduced, and the situation was brought under control, using the knowledge and means that they had at that time. It was felt that this pandemic will not go away, so we must just live with it. Participants felt that people managed, adapted, or learned to live this new way of life, and focused on their own health and protected themselves, and vaccinations were seen as an important solution that helped control the pandemic. On the other hand, participants experienced that they had managed the situation well; the pandemic had remained moderate in Northernmost Finland, the number of cases had been low, and therefore the impacts on lives had been lower compared to other parts in Finland. During the pandemic, people were able to live quite a normal life at times. Interviewees described that municipalities, organisations and working places were able to quickly react to changing pandemic situations, guidelines, and restrictions. This supported normal life, as well as working and school environments. Municipalities were seen as an effective operator. New ways of working helped, but it was also experienced that preparedness was important; for example, a variety of online activities were already available prior to the pandemic. Some organisations had good economic resources that helped them cope during the pandemic time. Participants expressed that they had a lot of space, environment, and nature to live in these municipalities compared to larger cities. People had their own houses and yards, there were fewer people, and they were able to go around in nature, which was an especially empowering and pacifying element for locals. Nature truly supported people to deal with the challenges that COVID-19 caused, and people met each other out in the nature.The power of co-operationParticipants expressed issues that described people going forwards together. Co-operation between them, communities, and different organisations was highlighted. People worked together and helped each other more intensively than before. COVID-19 was a joint enemy, a problem that had to be dealt with together. They indicated that some working places arranged remote discussion events or meetings in nature, since support of colleagues was important. Social and health services and different organisations or associations shared joint goals and strengthened their local collaboration. Moreover, it was noticed that people tried to support and encourage each other to do different things together. Fundraisings were organised, grocery stores donated food, and community people donated clothes for those who needed them. Volunteers packed and transported these further. Media published supportive news. Interviewees felt that a sense of community was very essential and positive, and it helped people a lot. All were in the same boat. It was described that some Sámi families met each other on the bridge between Finland and Norway and had coffee together without crossing the official border. They expressed elements that supported Sámi identity and culture during the pandemic. One central element was seen through the media. For example, radio, newspapers, and television provided important information and discussion in Sámi language, at times in all three Sámi languages. Moreover, online concerts were provided, and real, actual concerts were held outside when possible. The Sámi parliament attended different official meetings related to COVID-19 and collaborated with Sámi parliaments from Sweden and Norway. Based on the answers, Sámi associations provided support for locals. Overall, all these actions were seen as important.Participants expressed successful new solutions when handling the COVID-19 pandemic. They felt that online working, meetings, and studying were not only good and effective overall but it also brought people closer in a way. Locals had good skills for remote working; they were already used to it, and children talked and played with each other online. This was seen as an important way to connect with each other. They described that through online working, the world seemed to be closer now – not only for people living in Northernmost Finland, but it also brought these municipalities closer to people living in Southern Finland. Many felt that the pandemic taught other people to see this possibility, and they now received new invitations to meetings or collaboration networks, which did not happen before. It was seen that the pandemic strengthened the online skills of locals. Online working was not the only way to conduct work; participants also expressed other alternative ways to work during the pandemic. For example, online shops and home delivery were created, media put effort on online newspapers, e.g. completed interviews via telephone and took photos through windows. Libraries started to take orders via telephone too and deliver books to customers when libraries were closed. Interviewees pointed out that the tourism business put more effort on summer tourism, and some organisations renovated their premises as they now had time for this. For some employees, the pandemic offered the possibility to study new things, e.g. languages, or they organised their work in a new way, for example arranged groups and activities for customers outside. Overall, participants felt that people were creative.DiscussionThe pandemic created negative impacts on labour markets globally, causing uncertainty and resulting in higher unemployment rates [18,19]. In Finland, these effects hit especially new organisations and the service sector , such as tourism, restaurants and catering businesses [5,21], and businesses providing public events and happenings . Comparable findings were found in the present study, as participants expressed experiences related to economic challenges and unemployment, especially among service-sector providers. The tourism business struggled greatly due to the COVID-19 pandemic, which had further negative impacts on accommodation providers, restaurant businesses, and moreover on Sámi reindeer herders due to the decline in reindeer meat sales. Similar effects on the Indigenous traditional economy were found by the Arctic Council . A study conducted in Norway found that the pandemic had a minimal impact on daily work itself among reindeer herders in Norway, including Sámi herders. Still, negative consequences were noticed, such as economic impacts and closed national borders that limited normal life, e.g. work, family visits, and access to grocery stores . Changing guidelines and restrictions on international travel negatively affected travellers’ attitudes during the pandemic, and concerns related to health became after . Similarly, national border restrictions affected local peoples’ lives in this study, causing uncertainty, frustration and limiting their regular everyday life. Challenges related to the national border were recognised well among participants, and economic impacts were dramatic on businesses close to the border of Norway.Working life changed – not only the content or way of working but it also forced rearrangements in working places, e.g. services. For some, remote working was more of a natural shift, while for some this change appeared more concrete, requiring innovative solutions of how to conduct work. The pandemic was a challenge for part-time workers, women, and those who were not able to work remotely, especially in face-to-face services . Oksanen and colleagues have found that remote work seemed to be a softer change for those who had already used it as part of their work. However, overall, people working remotely experienced more anxiety, and the most challenging time was in the middle of the pandemic in Finland and employees who felt stress related to online environment, or had feelings of overwhelming or loneliness, experienced anxiety or concerns more. Specifically, being female or younger was connected to feelings of anxiety [11,26]. Perceived stress was found to be related to work ability, but also to a lack of ergonomics and support from the working organisation . Based on the results of our study, support and togetherness of colleagues were important for participants and helped while working from home.Changes during the pandemic, e.g. in economy, work and school, challenged life at home. They also led to the emergence of mental impacts, such as feelings of loneliness or being overwhelmed. For many, the pandemic separated families and brought new economic difficulties due to lack of income and increased living costs. According to Xiong and colleagues , younger age, being female or having a lower economic status or education were risk factors for mental symptoms during the pandemic. Similarly, females, younger parents, or parents with economic challenges or younger children seemed to have more challenges adapting to the situation during the first lockdown in Finland . Being at home together with family was found to be specifically demanding for mothers, especially during the first wave of the pandemic . The limited time and space had to be rearranged to complete remote studying and working, and often parents helped their children during the schoolday, adjusting their own working hours . Brooke and Jackson recognised that during similar situations as COVID-19, elderly people should be supported to maintain their social connections as well as possible, as we found in this study. Family caregivers of older adults experienced loneliness and worry about how COVID-19 may impact their health. Again, being female and having a higher experience of stress or low mood were related to feelings of loneliness and worry . Limited social interaction increased the feeling of loneliness [12,31], and lower physical health was connected to mental challenges during the pandemic [12,28]. These findings are in line with our present study. However, based on our results, we cannot make any conclusions related to gender. It has been found that compared to non-Sámi people, Sámi people were more concerned of getting infected by COVID-19, and this was more obvious among Sámi men. Moreover, even higher concern emerged among Sámi men and women towards others for getting sick, compared to non-Sámi participants. However, feelings of loneliness were less common among Sámi participants .For Indigenous Peoples, physical distancing put the natural interactions between extended families, traditional cultural happenings, and important activities, including spiritual and religious, at risk. Spending time with valuable elders, youth, connecting with the community, sharing food and practicing their own traditions are important for Indigenous Peoples. In Canada, COVID-19 resurfaced historical traumas related to colonialism – lockdown, being separate again, and given governmental guidelines . Similar experiences were found in our study, such as memories of the Spanish flu, but a more dramatic memory was related to the closed national border. This caused memories of World War II and residential schools to resurface. According to Heinikoski and Hyttinen , closed borders have been a great challenge to Sámi people; they have lived throughout history in one Sámi Land, without national borders. This in particular may have had a critical effect on their traditional lifeways, e.g. reindeer herding and family interactions.Often, Indigenous Peoples experience lower health and well-being compared to non-Indigenous, and COVID-19 challenged already vulnerable people [34–36]. This finding did not appear in our study; however, access to health care services in Norway was affected due to the pandemic and closed national border. According to Nilsson and colleagues , based on the study conducted in Sweden, there were some differences among Sámi and non-Sámi participants in seeking health care during the spring of 2021. Sámi men seemed to be less likely to avoid health care services compared to Swedish men, while Sámi women were a little more likely to avoid care compared to non-Sámi women. Despite the negative effect that the pandemic had on Sámi livelihoods and traditional practices, even out in nature, our results highlighted that nature and space around people were supportive elements for residents in these municipalities during the pandemic. Similarly, Sámi people in Sweden seemed to spend more time outside compared to Swedish participants . In the current literature, there is evidence supporting this finding; nature is indeed important for our health and well-being [37,38]. For Indigenous Peoples, being connected to nature is an essential part of everyday life and culture [39,40].Among nature, communality and togetherness helped people to deal with the pandemic and, in a way, brought people together. They helped each other, for example by sharing food and clothes, but people also reorganised their work to provide help and services. As found earlier, being able to interact with people in similar situations is important . Social relationships – being together in the same situation – support well-being and dealing with the pandemic . This was also highlighted among Finnish students and university staff during remote studying, although in-person meetings were missed . Similar results were found in this study, but participants also felt that online connections allowed them to be more active and connected with others – the world was closer now. Technology and online work provide the freedom to plan work, which in turn, supports work–life balance and work engagement . Organisational support and one’s positive attitude towards online work are essential, as they may help to maintain work engagement [44,45]. Additionally, media can support local people in challenging situations, as found in this study which investigated COVID-19 related news published in one local newspaper in Northernmost Finland. Despite the fact that Sámi perspectives were not in a focus, it can reflect, in a way, general feelings and atmosphere during the pandemic. Media’s role may be even solution seeker and empowering .For the future, public health actions should be focused on people with lack of social support and with economic difficulties . Preparedness of health care systems is essential, and social efforts need to be remembered during the pandemic, together with other public sectors, e.g. education and research . Knowledge exchange and equal communication were highlighted among Indigenous Peoples in Canada. It should be recognised between Indigenous peoples and national, governmental and regional leaders and professionals – people should work together. It is essential to acknowledge the local needs .Limitations and strengthsDuring the content analysis process, the researcher always checked the original data in case of unclarity to verify the codes and to correct the sub-category. This was done to ensure validation of the analysis and study results. It must be acknowledged that in the qualitative analysis process, the own experiences and views of the researcher may impact the interpretation and therefore the process of the abstraction. Keeping this risk in mind, the authors of the paper discussed constantly during the analysis process, reflecting on the decisions from coding to grouping main categories. The data include a variety of representatives and operators who worked in both public and private sectors, and in different fields. The data consist of different perspectives and experiences, providing wide views related to the impacts of the COVID-19 pandemic in the two Northernmost municipalities in Finland. Although the results cannot be generalised, they still provide a comprehensive description of the investigated area – and the information can help and support politicians and decision makers.ConclusionsThe main results of this study can be described in three parts. At first, the COVID-19 pandemic affected the lives of local people, including Sámi Indigenous Peoples. The effects were particularly seen through issues related to national borders and pressure that the pandemic caused – economically and mentally, but it also challenged service systems. Secondly, despite these challenging impacts, it seemed that everyday life with a connection to nature, togetherness, communality, and effective co-operation supported people to deal with challenges. Additionally, the pandemic situation remained better in Northernmost Finland compared to Southern Finland until early 2022. And thirdly, locality was seen as very important; it was highlighted among participants that future actions should acknowledge local needs. A deeper understanding of the overall public health impacts of the COVID-19 pandemic and recommended actions will provide valuable information for the development work and preparedness on the national and local levels, e.g. public health and social care in the future in similar situations. Additionally, it will provide relevant information on experiences in Sámi Land, and, therefore, has a special input to Sámi Indigenous Peoples, including their perspectives as well.

PMC

Genes & Diseases

PMC10308158

8-24-2022

10.1016/j.gendis.2022.08.004

Liver HMGCS2 is critical in the maintenance of liver lipid homeostasis during fasting

Zhou Yunfeng, Gu Tingting, Hu Shuyuan, Luo Zhaokang, Huang Ling, Yu Cong, Wang Jie, Liu Baohua, Zhang Xiaoyan, Guan Youfei

Fasting induces mammalian metabolic switch from glucose to fatty acid-derived ketones, resulting in a marked change in blood glucose, triglyceride, free fatty acid and β-hydroxybutyrate levels and a phenotype of transient hepatic steatosis. However, the underlying mechanism remains incompletely understood. This study aimed to investigate the role of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting enzyme in ketogenesis, in fasting-induced ketogenesis and hepatic lipid accumulation. Hepatocyte-specific HMGCS2 knockout (LKO) mice were generated and used to compare the difference in liver metabolic response to fasting between wild-type (WT) and LKO mice. Our findings demonstrate that HMGCS2 is essential in enhanced hepatic ketogenesis during fasting. Dysfunction of HMGCS2 leads to excessive hepatic triglyceride accumulation and severe liver injury possibly via increased liver fatty acid uptake by CD36. Therefore, liver HMGCS2 may represent a key enzyme in the maintenance of liver lipid homeostasis during fasting.After 24 h fasting, mouse body weight was significantly decreased (Fig. S1A). However, blood β-OH levels were increased ∼2-folds (Fig. S1B). The mRNA expression of HMGCS2 was markedly upregulated in the livers of the fasting mice (Fig. S1C). Consistently, Western blot assay revealed that the protein expression of hepatic HMGCS2 was also significantly elevated in the fasting mice (Fig. S1D). To determine the role of HMGCS2 in liver lipid homeostasis during fasting, we generated a liver-specific HMGCS2 gene knockout mouse line (L-HMGCS2−/−, LKO) by crossing an HMGCS2flox/flox mouse with an albumin gene promoter-driven Cre transgenic mouse (Alb-cre) (Fig. S2A, B). As expected, the expression of hepatic HMGCS2 was almost undetectable in the LKO mice as assessed by Western blot and immunohistochemistry assays (Fig. S2C, D). Consistently, hepatic HMGCS2 mRNA expression in the LKO mice was almost undetectable (Fig. S2E). However, the levels of other enzymes involved in hepatic ketogenesis including ACAT1, HMGCL and BDH1 were not altered in the LKO mice (Fig. S2F–H). Together, these results indicate that hepatocyte-specific HMGCS2 knockout mice were successfully generated.Under basal condition, blood β-OH levels were slightly lower in the LKO mice than those in wild-type (WT) mice. After 24-h fasting, WT mice exhibited a significant increase in blood β-OH levels. However, the LKO mice failed to develop hyperketonemia (Fig. S3A), suggesting that liver HMGCS2 is important for blood ketone levels in the fed-state, but is essential in the development of hyperketonemia during fasting. Consistently, it has been recently reported that although HMGCS2 is induced in both liver and kidney after starvation, the liver is the main ketogenic organ for circulating ketones during fasting.1 Similarly, 24-h fasting induced a significant elevation in blood triglyceride and free fatty acid levels in WT mice, which was almost completely abolished in the LKO mice (Fig. S3B, C). Little difference was found in blood triglyceride, free fatty acid, glucose and insulin levels between WT and LKO mice at the basal condition (Fig. S3B–E). Upon 24-h starvation, both genotypes exhibited a significant decrease in blood glucose levels, with slightly higher levels in the LKO mice than in WT mice (Fig. S3D). Although 24-h fasting resulted in a marked decrease in plasma insulin levels in both WT and LKO mice, but no difference was observed between two genotypes (Fig. S3E). Unlike other metabolic parameters, blood total cholesterol levels were similar between WT and LKO mice at both basal and fasting conditions (Fig. S3F). Together, these findings suggest that liver HMGCS2 deficiency mainly affects fatty acid and triglyceride metabolism with little effect on glucose and cholesterol homeostasis.Histological examination and functional analysis showed that the LKO mice exhibited more severe liver injury and function damage than that in WT mice after 24-h fasting (Fig. 1A–C). Consistent with a previous report,2 upon 24-h fasting WT mice showed normal liver enzymatic activity and a minor fatty liver phenotype characterized by a slight increase in the numbers and sizes of lipid droplets (LDs) and the contents of neutral lipids especially triglycerides as assessed by histological examination, Oil Red O staining and biochemical analysis (Fig. 1A–D). However, compared with WT mice, 24-h fasted LKO mice showed a significant increase in hepatic triglyceride and free fatty acid contents with a dramatic elevation in serum ALT and AST levels (Fig. 1A–D). To explore the underlying mechanisms, we used real-time PCR assay to determine the mRNA expression levels of genes involved in hepatic lipogenesis (ACC and FAS), lipid β-oxidation (CPT1α and AOX), lipid secretion (apoB and MTP), and fatty acid uptake (CD36 and LFABP) (Fig. 1E). The result showed that although no difference was found in the pathways of hepatic lipogenesis, lipid β-oxidation and VLDL excretion between two genotypes, CD36 expression in the LKO mice was ∼4-fold higher than that in WT mice. Consistently, Western blot analysis and immunohistochemistry study further confirmed a marked increase in hepatic CD36 protein expression in the LKO mice compared to WT mice (Fig. 1F, G). Moreover, in vitro results showed that the starvation medium (SM) (the HBBS medium with 5.5 mM glucose and without fetal bovine serum) treatment for 24 h significantly increased the protein levels of both HMGCS2 and CD36 and lipid accumulation in the BRL cells, a hepatocyte cell line (Fig. S4A–D). However, SM-induced CD36 expression and triglyceride contents were significantly increased by siRNA-mediated HMGCS2 knockdown (Fig. S4A–D). Together, these findings demonstrate that increased fatty acid uptake via CD36 is associated with worsened lipid accumulation observed in the livers of the LKO mice subject to 24 h fasting.Figure 1Liver-specific deletion of HMGCS2 worsens liver injury and hepatic lipid accumulation after 24 h fasting. (A) HE staining demonstrated significant hepatic injuries (arrows) in 24 h fasting LKO mice. Representative images: WT; WT-Fasting; LKO; 4) LKO-Fasting (Scale bar = 100 μm). (B) The LKO mice exhibited a significant increase in serum ALT and AST levels after fasting compared with WT mice. (C) Oil Red O staining showed a significant increase in 24 h fasting-induced lipid accumulation (arrows) in the livers of the LKO mice. (D) Biochemical assays showed that hepatic TG and FFA levels were significantly higher in 24 h fasting LKO mice than those in WT mice with fasting. (E) Quantitative RT-PCR analysis of mRNA levels of genes involved in hepatic lipid metabolism. (F) Changes of the protein levels of hepatic CD36 and HMGCS2 in fasting-induced WT and LKO mice. Representative Western blot are presented. Western blot assay showed that CD36 protein expression was induced in the livers of both genotypes after fasting. However, CD36 protein levels were markedly higher in the LKO mice than those in WT mice. (G) Representative immunostaining for CD36 by immunohistochemistry: WT; WT-Fasting; LKO; LKO-Fasting. Semi-quantification of CD36 protein immunoreactivity was performed. After fasting for 24 h, hepatic CD36 expression was markedly increased, which was further increased in the LKO mice. Data are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01 vs. WT; #P < 0.05, ##P < 0.01 vs. WT-Fs, n = 5–6. Note: Fs means 24 h fasting. Scale bar = 100 μm.Fig. 1The main finding of the present study was that the LKO mice had impaired adaptation to fasting-induced hepatic accumulation of LDs. The maladaptation to fasting-induced hepatic accumulation of LDs appears to result from enhanced hepatic uptake of free fatty acids, since hepatic expression of CD36 was markedly induced in WT mice and further increased in the LKO mice. CD36 (also known as scavenger receptor B2) is a multifunctional receptor that mediates cellular uptake of long-chain fatty acids and is ubiquitously present on the surface of many cell types.3 Therefore, induction of CD36 expression is very likely responsible for fasting-induced severe fatty liver in mice with liver-specific deletion of HMGCS2. In support, siRNA-mediated CD36 knockdown markedly decreased CD36 expression in the BRL cells (Fig. S5A, B). Moreover, CD36 knockdown abolished SM-induced lipid droplet and TG accumulation in SM-treated cells in the presence or absence of HMGCS2 siRNA treatment (Fig. S5C, D). These findings suggest that fasting-induced hepatocellular lipid accumulation is likely dependent on the upregulation of CD36 expression. However, the exact underlying mechanism by which hepatic HMGCS2 deficiency enhances liver CD36 expression remains unknown.HMGCS2 as a well-known rate-limiting enzyme in ketogenesis may be involved in both adaptive fatty liver during fasting and non-alcoholic fatty liver disease. Knockdown of mouse liver HMGCS2 with specific antisense oligonucleotides abolished high-fat diet (HFD)-induced hyperketonemia.4 Ketogenesis-insufficient mice exhibited worsened HFD-elicited fatty liver, which can be reversed by the supplementation of the precursors of ketone bodies, suggesting that insufficient ketogenesis contributes to HFD-induced fatty liver.4 It has also been reported that neonatal mice with global HMGCS2 gene knockout developed hepatosteatosis, suggesting that neonatal ketogenesis protects the energy-producing capacity of mitochondria possibly by preventing the hyperacetylation of mitochondrial proteins.5 The present study further provides direct evidence that liver-specific HMGCS2 ablation completely abolished fasting-induced hyperketonemia and markedly worsened adaptive fatty liver during fasting. Together, these findings highlight the important role of ketogenesis in preserving mitochondrial function and in the prevention of fatty liver with various etiologies. Thus, liver HMGCS2 might represent a potential therapeutic target for the treatment of fatty liver.In summary, we found that hepatic specific deletion of HMGCS2 completely abolished fasting-induced hyperketonemia and significantly attenuated fasting-induced elevation of blood triglyceride and free fatty acid levels. Liver-specific ablation of HMGCS2 markedly worsened fasting-elicited hepatic lipid accumulation and liver injury. The underlying mechanism may be due to upregulated expression of hepatic fatty acid translocase CD36. Together, our data demonstrate that hepatic HMGCS2-mediated ketogenesis is critical in maintaining liver fatty acid and triglyceride homeostasis during nutrient deprivation or fasting. Modulation of HMGCS2 activity may represent a potential therapeutic strategy for the treatment of fatty liver.Author contributionsY. Z. and Y. G. designed the study. Y. Z., T. G., L. Z., H. L., and Y. C. performed experiments; Y. Z., Z. L., W. J., and S. H. analyzed the data. Y. Z. drafted the manuscript. B. L., and X. Z. performed the critical review. Y. Z., and Y. G. edited the manuscript. All authors approved the final version of the manuscript.Conflict of interestsAuthors declare no conflict of interests.

PMC

ACS Omega

PMC10905584

02-16-2024

10.1021/acsomega.3c10031

PEG–PVP-Assisted Hydrothermal Synthesis and Electrochemical Performance of N-Doped MoS

Liu Wei, Yang Shenshen, Fan Dongsheng, Wu Yang, Zhang Jingbo, Lu Yaozong, Fu Linping

Molybdenum disulfide shows promise as an anode material for lithium-ion batteries. However, its commercial potential has been constrained due to the poor conductivity and significant volume expansion during the charge/discharge cycles. To address these issues, in this study, N-doped MoS2/C composites (NMC) were prepared via an enhanced hydrothermal method, using ammonium molybdate and thiourea as molybdenum and sulfur sources, respectively. Polyethylene glycol 400 (PEG400) and polyvinylpyrrolidone (PVP) were added in the hydrothermal procedure as soft template surfactants and nitrogen/carbon sources. The crystal structure, morphology, elemental composition, and surface valence state of the N-doped MoS2/C composites were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), high-resolution transmission electron microscopy (HRTEM), and X-ray photoelectron spectroscopy (XPS), respectively. The results indicate that the NMC prepared by this method are spherical particles with a nanoflower-like structure composed of MoS2 flakes, having an average particle size of about 500 nm. XPS analysis shows the existence of C and N elements in the samples as C–N, C–C, and pyrrolic N. As anodes for LIBs, the NMC without annealing deliver an initial discharge capacity of 548.2 mAh·g–1 at a current density of 500 mA·g–1. However, this capacity decays in the following cycles with a discharge capacity of 66.4 mAh·g–1 and a capacity retention rate of only 12% after 50 cycles. In contrast, the electrochemical properties of the counterparts are enhanced after annealing, which exhibits an initial discharge capacity of 575.9 mAh·g–1 and an ultimate discharge capacity of 669.2 mAh·g–1 after 70 cycles. The capacity retention rate decreases initially but later increases and elevated afterward to reach 116% at the 70th cycle, indicating an improvement in charge–discharge performance. The specimens after annealing have a smaller impedance, which indicates better charge transport and lithium-ion diffusion performance.

1IntroductionWith the rapid development of the global economy and population growth, the consumption rate of energy worldwide has greatly increased. The problem of energy exhaustion has become one of the major challenges faced by human society today as fossil energy is limited in stock and nonrenewable.1−3 Additionally,2 the combustion of fossil fuels will release a large number of harmful gases, which will cause serious harm to the ecology, such as the greenhouse effect. Therefore, the efficient conversion and full utilization of clean renewable energy have become a research hotspot. In recent years, the generation of renewable energy such as wind and solar energy has made great progress but is limited by the instability of its power source. Therefore, large-scale electrochemical energy storage devices with high capacity are crucial for the efficient utilization of clean energy.4−7 In addition, the rapid development of portable electronic devices has also put forward higher requirements for the performance of the secondary battery. Lithium-ion batteries (LIBs) have attracted much attention and good application prospects in the field of energy storage due to their advantages such as relatively high volumetric energy density, rapid charge/discharge capacity, long cycle life, and better safety performance.8−10 As an important component of LIBs, the anode material materials have become the key point to improve the electrochemical performances of LIBs.Since the commercialization of LIBs, graphite has consistently been the predominant selection for an anode material. Although it exhibits many advantages such as good safety and low price, its theoretical specific capacity is too low (∼372 mAh·g–1).11 High-power LIBs are currently limited by the cathode material that can meet the capacity demand, but with the breakthrough of cathode material, it is of significant necessity to exploit and research a new-type anode material with high specific capacity.As an alternative to the graphite anode, the molybdenum disulfide with a layered graphite-like structure has been extensively investigated because of its high theoretical specific capacity (∼670 mAh·g–1),12,13 low price, and abundant reservation. Nevertheless, there are some obstacles to its commercial applications,14−17 such as structural collapse resulting from volume expansion during charge/discharge processes and relatively poor conductivity as a type of semiconductor material.Thus, the primary objective of research aimed at improving the capacity of LIBs is to implement diverse strategies to enhance the charge–discharge performance, cycle stability, and conductivity of MoS2-based anode materials.Modulating the composition is an effective approach to increasing the conductivity of the material and achieving a higher capacity. Doping the nonmetallic N element effectively enhances the conductivity of MoS2, ensuring optimal electron transport. Additionally, it accelerates the oxidation-related multielectron transport process by promoting the relatively high valence state of the Mo element. According to the literature review, nitrogen is a favorable element for stable doping into MoS2 to increase its theoretical specific capacity by activating extra active sites and enhancing electrical conductivity.18−20 Nanoflower-like N-doped MoS2 was synthesized by Li et al.21 via a simple, one-step hydrothermal method and used as anode materials for lithium-ion batteries. The N-doped MoS2 anode material exhibits a specific capacity of 786 mAh·g–1 after 100 cycles at a high current density of 0.5C, indicating excellent cyclic stability compared to the pristine MoS2. This excellent cycling performance is attributed to the superior electronic conductivity of N-doped MoS2 anode materials, which provides a pathway for charge transport. Moreover, previous studies22−25 indicated that the incorporation of carbon-based materials with molybdenum disulfide can greatly improve the conductivity of materials due to the high conductivity and specific surface area of the former. Carbon-based materials, such as graphene, carbon nanotubes (CNTs), and porous carbon, are commonly combined with molybdenum disulfide.Therefore, it is hypothesized that the N element doping of the MoS2 material combined with carbon would result in a positive effect on the electrochemical performance. It has been found that the N-doped porous carbon layer in N-MoS2/C has both relatively high electrical conductivity and the ability to effectively retard the strain caused by the volume changes in the charge/discharge process. This layer also inhibits the accumulation or reaccumulation of MoS2 during the lithium storage process. Yan-Hong Shi et al.26 successfully integrate nanostructured MoS2 into N-doped porous carbon through a simple one-step hydrothermal reaction. MoS2/N–C microparticles greatly enhanced the electrical conductivity and electrochemical activity. Furthermore, the integration of nanoscale MoS2 particles into porous carbon yields a unique structural benefit that effectively combines the advantages of nanoscale MoS2 and micron-scale N-doped C. The particles exhibit excellent electrochemical behavior due to their increased surface area and improved conductivity. Consequently, the N-doped porous carbon materials serve as promising electrode materials for energy storage devices.In addition to composition modulation, morphology regulation is also a crucial method for material synthesis and modification. Polyethylpyrrolidine (PVP) intercalated molybdenum disulfide (PVP/MoS2) nanosheets with good interconnections were successfully prepared through an effective and versatile one-step hydrothermal strategy by Gu et al.27 The structural characterization indicates that the incorporation of PVP molecules into MoS2 leads to an enlarged interlayer spacing. The as-prepared PVP/MoS2 nanomaterials exhibit exceptional absorption properties, due to the enhanced specific surface area and functional groups of PVP/MoS2. Liu et al.28 produced molybdenum disulfide nanosheets with severe lattice distortion through solvothermal methods, employing PEG400 as both a surfactant and a reactant. The incorporation of oxygen-containing and carbon-containing groups into the molybdenum disulfide interlayer leads to heterogeneous coupling of Mo–O–C and Mo–O, which modifies the microstructure of molybdenum disulfide and expands its layer spacing to 0.97 nm, significantly larger than the 0.615 nm of pristine MoS2. This structure improves the electron transport rate and enhances the structural stability, allowing molybdenum disulfide to exhibit remarkable electrochemical properties without relying on carbon support or coating. As an anode material, it retains a capacity of 600 mAh·g–1 even after 1000 cycles at a current density of 0.5 A·g–1. Even at a high current density of 10 A·g–1, it maintains a capacity of 364 mAh·g–1, indicating an outstanding electrochemical performance.Herein, nonmetallic nitrogen elements and composite carbon-based materials were doped to the MoS2 matrix, to increase the number of charge carriers and inherent conductivity. Furthermore, surfactants were introduced to improve the structural stability by increasing the lattice spacing. The incorporation of these components will address the issues of high volume change rate, low conductivity, and suboptimal cycling performance found in pristine molybdenum disulfide when it is used as an anode material during the charging and discharging process. In this study, N-doped MoS2/C composites (NMC) were synthesized by an enhanced hydrothermal process, using ammonium molybdate and thiourea as sources of molybdenum and sulfur, respectively, and surfactants PEG400 and PVP to assist the process. The crystal structure, morphology, elemental composition, valence distribution, and electrochemical properties of the as-prepared composites were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), high-resolution transmission electron microscopy (HRTEM) and X-ray photoelectron spectroscopy (XPS), respectively. The effects of subsequent annealing on the properties of the powder were also investigated.2Experimental Section2.1Synthesis of N-MoS2/C CompositeAll chemical reagents used in the experiment were of analytical grade and used without further purification. In a typical synthesis process, 30 mL of polyethylene glycol 400 (PEG400, Tianjin Kermel Chemical Reagent Co., Ltd.) was first dissolved in 50 mL of deionized water, then 1.00 g sodium molybdate (Na2MoO4·2H2O, Tianjin Hengxin Chemical Co., Ltd.) and 1.84 g thiourea (CS(NH2)2, Tianjin Damao Chemical Reagent Co., Ltd.) were added in sequence during stirring. After being completely dissolved, 0.15 g of polyvinylpyrrolidone (PVP, Rhawn reagent Co., Ltd.) was added and stirred for 60 min to obtain a clear starting solution. Then, it was transferred to a magnetic stirring autoclave lined with polytetrafluoroethylene (PTFE) and reacted at 220 °C for 8h. After cooling to room temperature, the residue was centrifuged and washed with ethanol and deionized water alternately four times and then dried at 60 °C for 12 h. Subsequently, the obtained N-MoS2/C composite powder was annealed in a tubular furnace with argon (purity >99.999%) protection at 500 °C, 700, and 900 °C for 2 h at a heating rate of 5 °C/min and a flow rate of 250 mL/min. For comparison, MoS2 was synthesized under identical experimental conditions without any surfactants or with only one single type of PEG400 and PVP.2.2Characterization of N-MoS2/C CompositeThe phase analysis of the sample was carried out using a D8-Advanced X-ray diffractometer (XRD, Bruker, Germany). The morphology and microstructure were characterized by a JSM-IT800SHL field emission scanning electron microscope (FESEM, Nippon Electronics, Japan) and a JEM-2100 High-resolution transmission electron microscope (HRTEM, Nippon Electronics, Japan). ESCALAB 250Xi K-Alpha + X-ray photoelectron spectrometer (XPS, Thermo Fischer Scientific, USA) was used to analyze the elemental composition and valence states of the samples.2.3Electrochemical MeasurementsThe capacities and cycling properties of the as-prepared samples were performed by CR2032 buckle cells assembled in an argon-filled glovebox (Mikrouna (Shanghai) Ind. Int. Tech. Co., Ltd.), with a lithium sheet as the counter electrode. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) tests were carried out at the CHI660E Electrochemical Workstation (CH Instruments, USA). The charge–discharge cycle performance and rate performance were tested on a CT2001A (Wuhan Land Electronics Co., Ltd., China) button battery test system.3Results and Discussion3.1Crystal Structure, Morphology, and CompositionTo investigate the effect of different types of surfactants on the microscopic morphology of MoS2. MoS2 materials were added with 0.15 g of PVP and 30 mLPEG400, and both surfactants were prepared, respectively. A control experiment was performed with MoS2 without any added surfactant. As depicted in Figure 1, the FESEM images disclose the morphology of the obtained samples. The representative FESEM images of as-prepared samples are displayed in Figure 4a–d, revealing a porous nanosphere structure assembled from ultrathin MoS2 nanosheets. Such an open porous structure is in favor of electronic contact and rapid electron transfer during the discharge/charge process. Figure 1a shows MoS2 synthesized without any surfactant, which is composed of irregular spherical nanosheets. However, the average particle size is approximately 10 μm due to agglomeration. When PEG400 was added as the sole surfactant, it can be observed that the irregular surface of molybdenum disulfide transformed into small spheres, as shown in Figure 1b. The synthesized molybdenum disulfide yielded a microsphere with an average diameter of about 2–3 μm. Nevertheless, its dispersibility remained inadequate, as it clustered together to create large and irregularly shaped particles. When PVP was solely added, its refinement effect on the particle size was more prominent. Compared with the samples using PEG400, the MoS2 synthesized with PVP has a smaller spherical structure with an average particle size of 900 nm, as displayed in Figure 1c.Figure 1FESEM images of MoS2 and NMC synthesized using different surfactants (a, no surfactant; b, PEG400; c, PVP; d, PEG400+PVP).Additionally, the morphology images of MoS2 synthesized from both PEG400 and PVP are presented in Figure 1d. It can be observed from the figures that the particles are further refined with good dispersion and uniformity evident at low multiples. While at high multiples, the material maintains a nanoflower-like structure with an average particle size of around 500 nm. PEG400 is dispersed into the solution to form a spatial steric site resistance effect. The mutual contact between the molybdenum disulfide nuclei was prevented, which led to the morphology of MoS2 being transformed into microspheres. Therefore, the addition of PEG400 can refine the grain, but the dispersion is poor. When PVP was added to the molybdenum disulfide reaction system, it spontaneously assembled into a vesicular micelle with amide groups inside the outer alkyl backbone. It encapsulates the molybdenum disulfide nucleation core in solution to form a circular “microreactor”. Molybdenum disulfide nanosheets are oriented to grow in a specific region of the “microreactor” and the size of the material is controlled. Therefore, the addition of PVP both alleviated the agglomeration of molybdenum disulfide and improved the dispersion. When both surfactants were added at the same time, MoS2 showed uniformly dispersed fine particles.Figure 2 displays the XRD patterns of the NMC specimens synthesized with varying surfactants. As the sample with no surfactant, four diffraction peaks located at 14.1, 33.2, 39.8, and 58.9° can be indexed to , , , and crystal planes of 2H-MoS2 (JCPDS 37-1492), respectively. While the peak position of the patterns with surfactant is similar to that of the sample without surfactant. The results indicate that the crystalline structure of molybdenum disulfide is not affected by the introduction of surfactant. However, the intensity of all diffraction peaks was weakened after surfactants were added, with peak showing the greatest reduction, implying that the crystallization of molybdenum disulfide was reduced by the addition of surfactants. The intensity of the diffraction peak demonstrates a noteworthy decrease, suggesting that the addition of surfactants impedes the development of the molybdenum disulfide layer structure.23 The spacing between the adjacent crystal planes can be calculated by the Bragg eq (eq 1).1where d represents the distance between crystal planes, θ is the diffraction degree, n is the diffraction order, and λ is the X-ray wavelength (λ = 0.15406 nm for Cu target Ka radiation). As per the equation, a shift of the peak to a lower degree indicates an increase in the distance between the crystal planes. As shown in Figure 2, the peak position of MoS2 without surfactant is approximately 13.9°, which shifts to a lower degree with surfactant. With the addition of PEG400, the intensity of the peak decreased notably and shifted to roughly 13.6°. As for the PVP addition, the peak shifted further leftward to 12.5°. When both surfactants were added simultaneously, the peak shifted to around 12.2°, which is lower by approximately 1.7°, compared to the pristine MoS2. As a result, the introduction of surfactants enhances the interlayer spacing of MoS2.Figure 2XRD patterns of MoS2 and NMC synthesized with different surfactants.Molybdenum disulfide synthesized with PEG400 and PVP as synergistic surfactant was annealed at 500, 700, and 900 °C for 2 h, respectively. Figure 3 shows the FESEM images of the samples after annealing at different temperatures. The morphology of molybdenum disulfide does not change significantly before and after annealing, indicating that high-temperature treatment has little effect on the morphology of the specimens. Point scanning energy spectrum analysis was conducted at point m (unannealed) in Figure 3a and point n (annealed) in Figure 3c, and the EDS maps were obtained, as shown in Figure 4. It is evident from the figure that the main elements of the samples prior to and postannealing are C, O, S, and Mo. The atomic ratio of S to Mo before and after annealing is 18.23:9.26 and 28.61:14.81, respectively, which is approximately 2:1. Hence, it can be proven that the main phase of the as-prepared samples is MoS2, which is also identified by XRD analysis. The C element mainly originates from the residue of the surfactant. EDS analysis revealed a decline in the mass fraction of C and O elements in molybdenum disulfide after annealing at 700 °C for 2 h. This implies that some of the carbon is lost as gaseous oxides during the high-temperature treatment process. Nevertheless, not all carbon is eliminated since the residual PEG400 and PVP carbonizes to form amorphous carbon, which is uniformly distributed in molybdenum disulfide. The reduction of O content is caused by the decomposition of oxygen-containing functional groups on surfactants at high temperatures and the volatilization with carbon.Figure 3FESEM images of NMC before and after annealing (a, unannealed; b, 500 °C; c, 700 °C; d, 900 °C).Figure 4EDS maps of NMC before and after annealing.Figure 5 shows the XRD patterns of the NMC samples before and after annealing. Four diffraction peaks located at 2θ of 13.9, 33.2, 39.7, and 58.8° represent the , , , and crystal plane of 2H-MoS2 respectively (JCPDS 37-1492). As displayed in Figure 5, the intensity of the , , and diffraction peaks increased after high-temperature treatment, denoting the high crystallinity of the specimens. Interestingly, the intensity of the diffraction peak decreased at elevated temperatures, suggesting inhibited layered stacking of molybdenum disulfide. The reduced layered structure can be attributed to the superabundant defects in the structure caused by the high-temperature treatment.29 Nevertheless, the XRD patterns obtained after annealing at 700 and 900 °C exhibit very little disparity, indicating that the crystal structure of molybdenum disulfide will not be further affected when the annealing temperature exceeds 700 °C.Figure 5XRD patterns of N-doped MoS2/C composites before and after annealing.Figure 6 shows the XPS spectra of the NMC samples before and after annealing. The survey spectra (shown in Figure 6a) demonstrate that the samples both prior to and postannealing consist mainly of Mo, S, C, N, and O elements. The O element is mainly derived from both oxygen adsorption and oxygen-containing functional groups in surfactants. The C and N elements primarily originate from surfactant residues. Figure 6b,c displays Mo 3d spectra of NMC before and after annealing. As shown in Figure 6b, prior to annealing, the Mo 3d spectrum shows five distinct peaks, where the Mo4+ 3d5/2 and Mo4+ 3d3/4 peaks (at 229.37 and 232.47 eV, respectively) correspond to the molybdenum ion of 2H-MoS2. The Mo4+ 3d5/2 peak at 229.37 eV also displays a small peak toward the lower binding energy. This peak observed at 228.40 eV indicates the presence of a small quantity of 1T phase molybdenum disulfide in the synthesized material.30 Meanwhile, the peaks at 226.43 and 235.66 eV correspond to S 2s and Mo6+ 3d5/2, where the latter is derived from the M–O bond of MoO3. It is speculated that molybdenum oxide results from the incomplete reaction, leaving a small amount of MoO3 in the final product. Further, MoS2 is oxidized by residual oxygen molecules absorbed on the surface to generate molybdenum oxide during calcination.31Figure 6XPS spectra of NMC before and after annealing (a, survey spectra before and after annealing; b, Mo 3d before annealing; c, Mo 3d after annealing; d, S 2p before annealing; e, S 2p after annealing; f, C 1s before annealing; g, C 1s after annealing; h, N 1s before annealing; i, N 1s after annealing).Figure 6c shows the Mo 3d spectrum of NMC after roasting. The peaks at 229.82 and 232.95 eV correspond to Mo4+ 3d5/2 and Mo4+ 3D3/4, respectively. After annealing, the small peak at 229.37 eV present in the precalcined sample was absent. This indicates the complete conversion of metastable 1T-MoS2 to 2H-MoS2 during the annealing procedure. A considerable decrease was noted in the intensity and area of the Mo6+ 3d5/2 peak at 235.50 eV as contrasted to the values before roasting. The findings suggest that the majority of MoO3 has sublimed, leaving a small fraction behind during annealing. Consequently, the content of Mo6+ declined in the sample. Figure 6d,e illustrates the S 2P spectra, which depict the valence states of the molybdenum disulfide before and after annealing. Both samples exhibit two peaks, S2– 2p3/2 and S2– 2p1/2, which appear at 162.19 and 163.47 eV before annealing in Figure 6d, while at 162.24 and 163.84 eV after calcination in Figure 6e. Figure 6f displays the C 1s spectrum of the NMC prior to annealing. The peak at 284.65 eV corresponds to the C–C bond, which originates from the residual carbon chains of PEG400 and PVP. The peaks at 286.01 and 288.05 eV correspond to C–N and C=O bonds, respectively, and are derived from residual PVP. Figure 6g displays the C 1s spectrum of the specimen following a high-temperature treatment. The unheated sample exhibits three discernible peaks at 284.76, 286.1, and 288.57 eV, respectively, which corresponded to C–C, C–N, and C=O bonds, respectively.32 While the peak position relating to C–C bonds remained relatively stable, in contrast, those linked to C–N and C=O bonds experienced significant peak area reductions.According to the data presented in Table 1, the atomic ratio of C–N decreased from 14.48 to 10.77%, and C=O reduced from 6.06 to 2.30% after annealing. The surfactant within molybdenum disulfide was carbonized during roasting, resulting in the retention of most C–C bonds and the near-complete sublimation of pyrrole at 700 °C. Consequently, the C=O content was reduced to a minimum, while the N element attached to the carbon chain was partially preserved. Figure 6h,i shows the N 1s spectra of the as-prepared sample prior to and postannealing. It can be observed from Figure 6h that two peaks appear at 395.2 and 399.84 eV, corresponding to Mo 3p3/2 and Pyrrolic N, respectively.33 Pyrrole N is derived from polyvinylpyrrolidone (PVP). After annealing, the spectrum of N 1s reveals two peaks indicative of Mo 3p3/2 and Pyrrolic N at 395.69 and 399.64 eV, respectively, as shown in Figure 6i. Compared with samples prior to annealing, the pyrrole N content of samples after annealing is greatly reduced. Table 1 illustrates a 3.49% decrease in the atomic ratio of pyrrole N, declining from 10.12 to 6.63% after annealing, which is consistent with the reduction rate of the C–N bond. Thus, the experiment validates both the regulation of morphology and the achievement of N element doping into the MoS2/C composites. During the carbonization of the carbon chain in PVP, the N element distributes uniformly in molybdenum disulfide together with the carbon chain. N3– ions (0.171 nm) have a similar ionic radius to S2– ions (0.184 nm) but possess different valence states. Therefore, doping the N element into the lattice of MoS2 introduces defects and enhances the conductivity of MoS2.Table 1C 1s and N 1s Spectrogram Atomic Ratio of NMC Before and After Annealingtypebefore annealingafter annealingC–C17.78%15.87%C–N14.48%10.77%C=O6.06%2.30%Mo 3p3/224.58%30.01%Pyrrolic N10.15%6.63%Figure 7 depicts the HRTEM images for NMC samples prior to annealing in (a) and (b) and after annealing in (c) and (d). It can be observed that the morphology of NMC retains a flower-like structure composed of molybdenum disulfide nanosheets without any significant change after annealing. Figure 7b shows an HRTEM image (400,000×) of several few-layered ultrathin MoS2 nanosheets, each composed of only five or nine layers. The results show that the layer growth of the crystal plane is inhibited, which is consistent with the XRD analysis. Using Fourier transform and DigitalMicrograph software, the spacing of the crystal plane after annealing was determined to be 0.67 nm. After annealing, the crystal plane spacing increased by more than 0.64 nm. This was attributed to the carbonization of residual surfactants remaining in molybdenum disulfide. Consequently, molybdenum disulfide can fully contact the electrolyte. Figure 7d shows that the lattice fringe of the roasted molybdenum disulfide exhibits more bending and discontinuity compared to the unannealed samples. This suggests that a high-temperature environment has resulted in the formation of more defects,34 which in turn can provide more impingement sites for lithium ions and enhance the specific capacity. Furthermore, the enhanced spacing between layers can effectively buffer the expansion of the volume during the charging and discharging process, leading to an improvement in stability.Figure 7HRTEM images of NMC before and after annealing (a, before annealing, 50,000×; b, before annealing, 400,000×; c, after annealing at 700 °C, 50,000×; d, after annealing at 700 °C, 400,000×).3.2Analysis of Hydrothermal Synthesis MechanismAccording to characterization results in the previous sections, it is inferred that the reaction mechanism is as depicted in Figure 8. At the initial stage of the reaction, the molybdate ions in solution were attracted to the hydrophilic –O– bond on the carbon chain of PEG400 and bound to it as a “surface template” to determine their growth position.35 Subsequently, driven by a hydrophilic amide group and a hydrophobic alkyl backbone, PVP self-assembled spontaneously into vesicular micelles with amide groups outside and alkyl backbone chains inside, as reported by ref . The nucleation cores of MoS2 were then enclosed in the solution, forming a circular “microreactor”.37,38 The MoS2 nanosheets grew directionally in a specific region of the “microreactor” that serves as a “bottom template” to limit its growth space. In this manner, uniformly dispersed nanoflower-like MoS2 with an average grain size of approximately 500 nm was generated. Excessive PEG400 molecules were driven by hydrophilic and hydrophobic groups alternatively to form micellar aggregates,39 which were dispersed into the solution to prevent the MoS2 nuclei from aggregating contact due to the steric hindrance effect. Thus, the morphology of MoS2 was transformed into microspheres, and the agglomeration was reduced simultaneously. Then, the hydrothermal product was annealed for two h with a nitrogen atmosphere at 700 °C. After annealing, the surfactant remaining in molybdenum disulfide was carbonized, while the N element connected to the carbon chain via a C–N bond was retained. Finally, N-doped nanoflower-like MoS2/C composites were synthesized.Figure 8Illustration of the synthesis mechanism of NMC.3.3Electrochemical PerformanceFigure 9a displays the cyclic voltammetry (CV) curves of NMC samples after calcination, performed at a scan rate of 0.3 mV·s–1 in a potential range of 0.01–3 V. The figure shows two reduction peaks at 0.79 and 0.45 V during the first cathodic scan. The reduction peak at 0.79 V represents the intercalation process where Li+ is embedded in the MoS2 layer to form a compound, accompanied by a phase transition of MoS2 from 2H to 1T.40 The reaction equation for the intercalation of lithium ions with MoS2 is xLi+ + xe– + MoS2 → LixMoS2. However, the reduction peak at 0.45 V represents the transition of the intercalation compound LixMoS2 to the metallic molybdenum and Li2S. The reaction equation for this transition is LixMoS2 + (4 – x)Li+ + (4 – x)e– → Mo + 2Li2S. Furthermore, the reaction between lithium ions and electrolytes results in a solid electrolyte interface film (SEI film). The first anodic scan displays two oxidation peaks at 1.68 and 2.28 V. The oxidation peak at 1.68 V corresponds to the partial conversion of Mo to MoS2, as indicated by the following reaction equation: LixMoS2 → MoS2 + x Li+ + S + x e–. The oxidation peak at 2.28 V represents the conversion of Li2S to S, with the equation: Li2S-2e– → 2Li++S. The second cathodic scan in the subsequent cycle exhibits two reduction peaks, indicating that the reaction of the second cathodic scan was identical to that of the first scan. Specifically, it involved the transformation of MoS2 to metallic Mo nanoparticles and Li2S. Nonetheless, the position of the reduction peak differs from the first cycle, appearing at 1.92 and 1.06 V. This deviation is due to the consumption of a portion of lithium ions in the irreversible reaction that occurs during the SEI film formation in the first cycle.41,42 The comparable CV curves in the second and third cycles confirm that the material exhibits excellent stability and reversibility.Figure 9Electrochemical property of NMC (a, CV curves; b, charge and discharge curves of the first three turns; c, cycle charge and discharge curves; d, rate curve).Figure 9b shows the charge/discharge profiles of N-doped MoS2/C material after annealing at 700 °C for 2 h. The testing was performed at a current density of 500 mA·g–1, with a voltage range of 0.01–3 V for the first three cycles. The first discharge curve reveals two voltage platforms at 0.75 and 0.47 V. The voltage platform at 0.75 V denotes the insertion of Li+ ions between the MoS2 layers to form the intercalation compound LixMoS2. The voltage platform observed at 0.47 V suggests that the intercalation compound transforms into Mo nanoparticles and Li2S. In the first charge curve, a voltage platform appears between 2.18 and 2.4 V, representing the transformation of Li2S to S. The voltage platforms correspond distinctly with the REDOX peaks observed in the cyclic voltammetry (CV) curves. However, a slight discrepancy still exists and may be related to the battery manufacturing process. Nevertheless, the second and third charge/discharge curves display notable differences from the initial ones. This is attributed to the irreversible reaction of lithium ions with the electrolyte, resulting in the formation of the SEI film during the first discharge. Consequently, certain lithium ions are permanently lost, leading to inconsistencies between the first scan and the following two scans of the CV curves.The N-doped MoS2/C specimens after annealing at 500, 700, and 900 °C, respectively, for two h were assembled into button batteries. The charge–discharge cyclic performance of the material was tested at a current density of 500 mA·g–1, as shown in Figure 9c. It is apparent that the specific discharge capacity of the sample before roasting was 548.2 mA·g–1 for the first cycle. Although the capacity increased over the next five cycles, it dramatically declined after the fifth. By the 50th cycle, the specific discharge capacity had decreased to just 66.4 mA·g–1, with a capacity retention rate of only 12%. Annealing at elevated temperatures is a potential solution to enhance cycle capacity and stability. Samples annealed at 700 °C for 2 h demonstrated the best performance with an initial specific discharge capacity of 575.9 mA·g–1 and a gradual increase in the subsequent 70 cycles. After the 70th cycle, the specific discharge capacity reached 669.2 mA·g–1, while also maintaining a capacity retention rate of 116%. Therefore, annealing is essential for the cyclic performance of the as-prepared materials. The annealing treatment both benefits the crystallinity of the material and facilitates the carbonization of surfactant remaining in molybdenum disulfide. N-doped MoS2/C composites were synthesized by the doping of N elements, resulting in improved conductivity and enhanced electron transport. Moreover, the layer spacing of the plane was found to increase after annealing, leading to more contact sites for lithium ions and providing a buffer for volume expansion during the charge/discharge cycles. Consequently, the cycle performance of N-doped MoS2/C composites was significantly improved.During the first 30 cycles, the specific discharge capacity of the sample exhibited a substantial increase after roasting at 500 and 900 °C for 2 h. Nevertheless, the capacity retention rate was subpar, plummeting significantly after the 40th cycle. The sample treated at 500 °C demonstrated an initial specific discharge capacity of 488.7 mA·g–1, which dropped to 275.2 mA·g–1 with a capacity retention rate of 56% on the 70th cycle. The initial specific discharge capacity of the sample treated at 900 °C was 697.4 mA·g–1, which is the highest in the three samples. However, it decreased to 167.2 mA·g–1 in the following 70th cycle with a capacity retention rate of only 24%. In comparison to the samples treated at 700 °C, these values were notably lower. Thus, the optimal roasting temperature for the hydrothermal product was found to be 700 °C.The rate performance of the NMC samples annealed at 700 °C for 2 h was evaluated by cycling 10 times at each current density, as illustrated in Figure 9d. The discharge specific capacity of the NMC is 658.9, 634.3, 596.5, 557.5, and 479.7 mA·g–1 at current densities of 0.1, 0.2, 0.5, 1, and 2 A·g–1, respectively. The material exhibits excellent tolerance for changes in the current density. As the current density increases, there is only a slight decrease in the specific discharge capacity. Even at a high current density of 2 A·g–1, it still has a specific discharge capacity of 479.7 mA·g–1. When the current density drops to 0.1 A·g–1, the specific discharge capacity rises to 770.6 mA·g–1. Additionally, during the rate performance test, the specific discharge capacity exhibits a consistent upward trend. This is due to the fact that at lower current densities, electrons, and ions are transported more smoothly within the electrode material, resulting in increased reactivity. And as the current density decreases, the electrolyte ions have more time to redistribute in the electrode material, resulting in an increase in the specific discharge capacity. The discharge plateau is longer and smoother when the current density drops back to 0.1 A·g–1. This results in an increase in the specific discharge capacity. The initial discharge specific capacity was 786 mA·g–1, and the discharge specific capacity was 794.1 mA·g–1 at the end of the rate performance test. The capacity increases by 1% compared to the initial discharge specific capacity, which is consistent with the cyclic performance diagram shown in Figure 9c.The specimens before and after annealing were first cycled 10 times at a current density of 500 A·g–1 and subsequent take measurements of electrochemical impedance spectroscopy (EIS). The Nyquist diagram (Figure 10a) was obtained by analog circuit fitting in Zview software. As depicted in Figure 10a, each curve exhibits two distinct formations: a semicircle in the high-frequency region and a slash in the low-frequency region. The initial point of the high-frequency semicircle on the Z′ axis is not zero, indicating the presence of an electrolyte resistance Rs. The high-frequency semicircle represents the charge transfer resistance Rct between the electrode and the electrolyte. CPE is the constant phase element. The slanted line in the low-frequency region represents the Warburg impedance Zw, also recognized as the concentration polarization impedance, that occurs due to the impact of reactant concentration and diffusion on the electrode reaction.43 The equivalent circuit diagram is presented in Figure 10b. The Nyquist diagram in Figure 10a illustrates that the Rct of the pristine MoS2 material is 333.5 Ω, while the Rct of the N-doped MoS2/C material is 240.8 Ω. Remarkably, the Rct of NMC annealing at 700 °C for two h significantly drops to 108.6 Ω, which is the smallest value among the specimens. The results demonstrate that NMC after annealing have excellent charge transport performance and lithium ion diffusion performance. Specifically, MoS2 was doped with N and integrated with carbon, resulting in increased conductivity and a significantly enhanced electrochemical performance.Figure 10(a) Nyquist diagram of NMC before and after annealing and (b) equivalent circuit diagram.4ConclusionsNanocrystalline flower-like N-doped MoS2/C composites were synthesized by an enhanced hydrothermal method, utilizing sodium molybdate as a source of molybdenum and thiourea as a source of sulfur. Surfactants, including polyethylene glycol 400 (PEG400) and polyvinylpyrrolidone (PVP) were employed in the hydrothermal reaction as soft template surfactants and nitrogen sources. After annealing in an argon atmosphere at 700 °C for 2 h, the hydrothermal residue transformed into N-doped MoS2/C composites, as the residual surfactant was carbonized while retaining the N element during the annealing procedure. The as-prepared N-doped MoS2/C composites with an average particle size of approximately 500 nm were uniformly dispersed without agglomeration. No morphological changes were observed before and after annealing. The initial specific discharge capacity of the N-doped MoS2/C composite was 548.2 mA·g–1 without annealing. However, the specific discharge capacity dropped significantly to 66.4 mA·g–1 by the 50th cycle, exhibiting a capacity retention rate of only 12%. In contrast, the initial specific discharge capacity of the samples after annealing was 575.9 mA·g–1. Furthermore, there was a noticeable increase in the discharge capacity throughout the cycles, with a discharge specific capacity of 669.2 mA·g–1 by the 70th cycle. The capacity retention rate reached an impressive 116%, leading to a significantly improved cycle performance. Additionally, the impedance of the N-doped MoS2/C composites was reduced after annealing, resulting in a promoted electron/ion transfer at the electrode interface. This study manifests that the design of hybrid materials and carbonization at high temperatures can significantly modify the electrochemical performance of molybdenum disulfide as an anode material for lithium-ion batteries and also provides new research ideas for the application of other layered materials in energy storage devices.

PMC

International Journal of Molecular Medicine

37772381

PMC10558212

9-26-2023

10.3892/ijmm.2023.5308

[Corrigendum] Melanogenesis in uveal melanoma cells: Effect of argan oil

Caporarello Nunzia, Olivieri Melania, Cristaldi Martina, Rusciano Dario, Lupo Gabriella, Anfuso Carmelina Daniela

Int J Mol Med 40: 1277-1284, 2017; DOI: 10.3892/ijmm.2017.3104Following the publication of the above article, an interested reader drew to the authors' attention that, in Fig. 7 on p. 1282, a pair of the western blotting bands in the Akt blot positioned adjacent to each other looked strikingly similar. Although the authors considered that the data were correct as shown (and the Editorial Office were in agreement that it was not certain that the bands were identical), to avoid any possible confusion or suspicion, the authors requested that the figure be reprinted showing the Akt data obtained from one of the repeated experiments.The revised version of Fig. 7, containing the replacement data for the Akt western blotting data, is shown opposite. All the authors agree with the publication of this corrigendum, and are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this for the purposes of clarifying the presented data.

PMC

ACS Omega

PMC10975631

03-13-2024

10.1021/acsomega.4c00375

Integrative Analysis of Multi-Omic Data for the Characteristics of Endometrial Cancer

Li Tong, Ruan Zhijun, Song Chunli, Yin Feng, Zhang Tuanjie, Shi Liyun, Zuo Min, Lu Linlin, An Yuhao, Wang Rui, Ye Xiyang

Endometrial cancer (EC) is a frequently diagnosed gynecologic cancer. Identifying reliable prognostic genes for predicting EC onset is crucial for reducing patient morbidity and mortality. Here, a comprehensive strategy with transcriptomic and proteomic data was performed to measure EC’s characteristics. Based on the publicly available RNA-seq data, death-associated protein kinase 3, recombination signal-binding protein for the immunoglobulin kappa J region, and myosin light chain 9 were screened out as potential biomarkers that affect the EC patients’ prognosis. A linear model was further constructed by multivariate Cox regression for the prediction of the risk of being malignant. From further integrative analysis, exosomes were found to have a highly enriched role that might participate in EC occurrence. The findings were validated by qRT-polymerase chain reaction (PCR) and western blotting. Collectively, we constructed a prognostic-gene-based model for EC prediction and found that exosomes participate in EC incidents, revealing significantly promising support for the diagnosis of EC.

IntroductionEndometrial cancer (EC) is a commonly diagnosed gynecologic malignancy among women.1 The precursor lesion for endometrioid adenocarcinoma of the endometrium, which accounts for the majority of endometrial carcinomas, is endometrial hyperplasia (EH). EH is a noninvasive, abnormal proliferation of the endometrial lining of the uterus and is associated with a significant risk of concurrent EC or progression to EC.2 EH could be further classified into two subtypes during its development: endometrial hyperplasia without atypia (EHA)3 and endometrial atypical hyperplasia (EAH). EHA is a benign disease without significant somatic genetic changes, along with a significant risk of transforming EC and persistent EH.4 EAH is regarded as a precancerous condition leading to EC,5 whose pathological progression is complex and exhibits a multiplicity over time and spatial distribution.6 It is widely recognized that women with EAH have a higher risk of progressing to EC compared to those with EHA. However, the magnitude of this risk is uncertain.7The rising incidence of EC and its growing population of new diagnoses underscore the severe challenge to women’s health. To combat this condition, it is essential to identify reliable prognostic biomarker genes to assist in the risk assessment of malignancy and to inform clinical treatment decisions for EC. Nowadays, high-throughput technologies have made it possible to obtain large-scale information at multidimensional biological expression levels. An integrated analysis that encompasses multiple biological layers, such as transcripts, proteins, or metabolites, provides a comprehensive approach to gain a more detailed understanding of the molecular mechanisms underlying public health and disease.8In this study, we collected the publicly available ribonucleic acid (RNA)-seq data, including 572 tissues (normal = 35, EC = 537). Death-associated protein kinase-3 (DAPK3) and recombination signal binding protein for immunoglobulin kappa J region (RBPJ) were identified as the potential prognostic genes to affect the patients’ prognosis. Based on the above two genes, a linear model was constructed to evaluate the risk of being malignant in EC patients. Meanwhile, the biopsies (EHA = 3, EAH = 4, EH = 6, and EC = 7) were collected for proteomic data generation by liquid-chromatogram tandem mass spectrometry (LC-MS/MS). In prognostic gene validation, the DAPK3 protein was found in our proteomics analysis. Through protein–protein interaction analysis, a DAPK3 binding partner called myosin light chain 9 (MYL9) was found to significantly affect the EC patients’ prognosis, making us speculate that MYL9 might be closely related to EC. Four of the six genes were further validated by qRT-polymerase chain reaction (PCR) and western blotting, including three exosome-related genes (HSPE1, RAB14, and SEC31A) and one prognostic gene (MYL9). To investigate the relationships at both transcriptomic and proteomic levels, we also compared their shared genes and the biological annotations from the significantly changed genes during EC incidents, finding that most exosomal proteins might participate in tumorigenesis. EHA and EAH have similar patterns of feature proteins involved in exosomes, indicating they might carry a higher risk compared with EH. Altogether, the utilization of integrative analysis based on a multiomics strategy can be used to discover the prognostic genes for the clinical diagnosis of EC.Experimental ProceduresSample Preparation for MSAfter washing with ice-cold phosphate-buffered saline, approximately 50–100 mg of uterine tumors and normal tissues were cut into small pieces. Samples were then homogenized by a homogenizer (Bertin Precellys, France) at 4 °C and processed with the following settings: 8 × 20 s at 5500 rpm, break 30 s. The freeze-crushed tissue samples were precipitated with ice-cold acetone at −20 °C overnight. The protein precipitant was centrifuged at 14,000g for 30 min. The tissue was washed separately with 1 mL of acetone and then air-dried on ice. Tissues were softly homogenized separately in 500 μL of lysis buffer (8 M urea, 100 mM Tris, pH 8.0, 1:100 v/v MCE protease inhibitor cocktail). Lysates were precleared by centrifugation at 20,000g for 30 min at 4 °C and protein concentrations were determined by a Bradford assay (Thermo Fisher Scientific). Proteins were reduced with 10 mM dithiothreitol for 30 min at 37 °C and subsequently alkylated with 55 mM iodoacetamide for 45 min in the dark. Before digestion, samples were loaded into the Microcon Ultracel YM-30 filtration devices (Millipore, Billerica, MA, USA) and centrifuged at 14,000g for 20 min. After three washes in 50 mM ABC, trypsin (Pierce, Thermo Fisher Scientific) solution was added to the filter (enzyme-to-protein ratio 1:100 w/w), and samples were incubated at 37 °C overnight. Peptides were collected by centrifugation, followed by an additional wash with 50 mM ABC. The digestion was stopped by acidifying the solution to a final concentration of 1% (v/v) formic acid. Tryptic peptides were desalted on a C18 SPE and dried for LC-MS/MS analysis.LC-MS/MS AnalysesAll analyses were performed using an EASY-nLC 1200 system (Thermo Fisher Scientific) on an Orbitrap 480. After reconstituting peptides in 20 μL of 0.1% FA, 1 μg of the peptide mixture was injected and loaded directly onto a C18 column (25 cm/75 μm, 2 μm beads, Thermo Fisher Scientific) and separated with a 90 min gradient from 4 to 40% B at 300 nL/min in typically. Parameters are as follows in Full MS/data dependent—MS2 TopN mode: mass analyzer over m/z range of 350–1500 with a mass resolution of 60,000 (at m/z = 200) in a data-dependent mode, 1.6 m/z isolation window. Twenty of the most intense ions are selected for MS/MS analysis at a resolution of 15,000 using the collision mode of HCD.Data AcquisitionTandem MS data were queried against a human database using Proteome Discoverer version 2.4 software (Thermo Fisher Scientific). The normalized abundance of a given protein was calculated from the average area of the three most intense peptide signals. For this software, proteins for which area intensities were below the minimum range or were not detected were assigned an area of zero. For the proteins that were identified by multiple UniProt9 IDs (release 2023/12/26).The transcriptome profiling (RNA-seq), which was preprocessed by fragments per kilobase of an exon model per million mapped fragments (FPKM), was from the EC project of the Cancer Genome Atlas (TCGA)10 database, including 537 tumor samples and 35 normal samples. The analyses were based on the genes expressed in all samples using a cutoff value of FPKM ≥1.Computational AnalysesSoftware tools used for this study were available as open source packages under the R v4.2.2 environment, including: “tidyverse”11 v2.0.0 for basic data operations, such as data cleaning; “missForest”12 v1.5 for empty value filling; “limma”13 v3.54.1 for differential expression analysis; “pheatmap”14 v1.0.12 for plotting all samples’ abundance and classification; the DAVID15 database for terms enrichment, such as KEGG16 and GO term;17 “Boruta”18 v8.0.0 for feature selection; “survival”19 v3.5-5 and “survminer”20 v0.4.9 for drawing survival curve and Cox regression; “leaps”21 v3.1 for best subset regression (BSR); “glmnet”22 v4.1-7 for lasso regression with 10-fold cross validation; “pathview”23 v1.40.0 for alterations specific pathways demonstration.Quantitative Reverse Transcription Polymerase Chain ReactionTransfer the accurately weighed RNA extraction sample to a liquid nitrogen precooled mortar and grind the tissue with grinding (liquid nitrogen needs to be continuously added to the mortar during the grinding process) until it is ground into powder. Then, an appropriate amount of RNA extraction reagent was added to the powder and mixed well. The above mixture was transferred to a centrifuge tube and thoroughly mixed by repeatedly pipetting and blowing. Let it stand at room temperature for 5 min, then centrifuge at 12,000 rpm at 4 °C for 5 min. Carefully aspirate the supernatant and transfer it into a new centrifuge tube before proceeding with subsequent RNA extraction operations. The process of RNA extraction was done using the Trizol reagent (Accurate Biotechnology, Changsha, China) according to the manufacturer’s protocol. Total RNA was quantified by a spectrophotometer (Nano-Drop ND-2000), and 2 μg of total RNA was reverse transcribed to complementary DNA using the reverse transcriptase kit (Accurate Biotechnology, Changsha, China) according to the manufacturer’s instructions. The messenger RNA (mRNA) levels of the pS2 gene were detected by RT-PCR using the SYBR Green Premix qPCR Kit (Accurate Biotechnology, Changsha, China) in the Bio-Rad CFX Connect PCR system.The primer of HSPE1-forward (5′-3′) was CAACAGTAGTCGCTGTTGGA, and that of HSPE1-reverse (5′-3′) was CCTCCATATTCTGGGAGAAGAAC.The primer of MYL9-forward was (5′-3′) GTCCCAGATCCAGGAGTTTAAG, and that of MYL9-reverse (5′-3′) was CATCATGCCCTCCAGGTATT.The primer of SEC31A-forward was (5′-3′) GAAGTTGTGATTGCCCAGAATG, and that of SEC31A-reverse (5′-3′) was GCACCAGAAGCTACCAGATTAG.The primer of MSN-forward was (5′-3′) CCACCTGGCTGAAACTCAATAA, and that of MSN-reverse (5′-3′) was GGACACATCCTCAGGGTAGAA.The primer of RAB14-forward was (5′-3′) GGAGCGATTTAGGGCTGTTA, and that of RAB14-reverse (5′-3′) was ACCAGCTGCTTAAGTGGTTAT.The primer of DAPK3-forward was (5′-3′) CGTTCACTACCTGCACTCTAAGC, and that of DAPK3-reverse (5′-3′) was CCGAAGTCGATGAGCTTGAT.Western Blotting Analysis30 μg of EVP proteins were boiled using 4 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer at 95 °C for 10 min, run on 15% SDS-PAGE gels (polyacrylamide gel electrophoresis), and transferred onto poly(vinylidene difluoride) membranes (Millipore, USA). Membranes were sequentially blocked with 5% nonfat milk (w/v) and incubated with primary antibodies, including an Exosome Panel Kit (Abcam, ab275018) and Anti-GAPDH (TransGen Biotech, HC301-01), overnight at 4 °C. After washing three times with 1× TBST, membranes were incubated with antimouse (Cell Signaling, 7076S) or antirabbit (Cell Signaling, 7074S) secondary antibodies for 1 h and washed again to remove unbound antibodies. Bound antibody complexes were imaged by a ChemiDoc Imager (Bio-Rad).Results and DiscussionData Processing and Analysis StrategiesThe whole flowchart of this study is shown in Figure 1. We first collected the RNA-seq data of EC from the TCGA10 database and prepared the expression data and clinical information for the further associated analysis.24−26 To provide enough data for the model validation, we also divided the whole RNA-seq data set into two equal subsets: the training and the testing data set. The differentially expressed genes (DEGs) between the normal and cancer samples were combined with the prognostic genes screened out by univariate Cox regression for the selection of the genes whose expression is positively correlated with the hazard ratio by the least absolute shrinkage and selection operator (LASSO) regression for biomarker discovery. BSR was performed to reduce the count of independent variables for model simplification. Finally, a linear model was constructed for the risk score prediction and validated by the whole RNA-seq data set and the testing data set.Figure 1Flowchart of the whole study.At the proteomic level, four types of biopsies were collected and calculated by two strategies: one was that the EC and non-EC (EHA/EAH/EH) samples were compared to get the significantly changed proteins along the tumorigenesis; another strategy was that the feature proteins of the three non-EC samples were measured by the random forest algorithm. After biological annotations, some hints were found to indicate the possible conversion from nontumor to tumor, and exosomes were finally found to possibly participate in the conversion of nontumor to tumor.Discovery of Prognosis-Related GenesData mining based on publicly available data plays a vital role in addressing clinical issues. In this study, we collected RNA-seq data from 35 normal individuals and 537 EC patients from the TCGA10 database. 4932 genes with more than 1 FPKM were identified that were expressed in all samples. To optimize model generalization during the training process, the size of the test set was made equal to that of the training set. According to the screening methods and criteria discussed above, 346 prognosis-related genes (hazard ratio ≠1) were found in the training set (n = 266). To link survival information and genes’ abundance, the prognosis-related genes were intersected with the DEGs. 27 genes with positive correlations between abundance and hazard ratio were screened out, including 3 up-regulated DEGs with HR >1 and 24 down-regulated DEGs with HR <1. LASSO regression with 10-fold cross-validation was performed based on the 27 genes to get the optimal lambda value that came from the minimum partial likelihood deviance (Lambda min = 0.015), which was related to 13 potential biomarker candidates (Figure 2A,B). For further analysis by BSR, the optimal three prognostic genes were selected according to the lowest value of the Bayesian information criterion in the evaluation process of model performance, namely DAPK3, RBPJ, and SLC40A1 (Figure 2C). Through Kaplan–Meier analysis, it was found that the expression of genes significantly impacted the patients’ prognosis (Figure 2D). The forest plots (Figure 2E) and boxplots (Figure 2F) provided more details about the three prognostic genes. To ascertain the existence of a significant prognostic value for each gene for EC patients, we conducted a multivariate Cox proportional hazards regression analysis and constructed a linear model. After optimizing the model, the two gene-based models for risk score evaluation were established with respective Cox coefficients: risk score = −0.009767733 × Exp (DAPK3) – 0.021571099 × Exp (RBPJ). The risk score of each patient in the testing set was calculated to plot the survival curve, showing that patients with high scores had poorer life conditions compared to those with low scores. The result suggests that the model holds a predictive value in assessing the risk of malignancy (Figure 2G). To validate the performance of the model, both the testing set (n = 266) and the entire data set (n = 532) were used for evaluation. This comprehensive approach ensures a more accurate and reliable assessment of the model’s predictive capabilities. Time-dependent ROC analysis showed that the AUC for the testing and the entire set were 0.622 and 0.665, respectively (Figure 2H). Indeed, the model was constructed using only two genes, highlighting the significant role of these two prognostic genes in predicting overall survival among EC patients. Collectively, these genes could potentially be valuable biomarkers for prognosis and might even be targeted for therapeutic interventions.Figure 2(A) LASSO coefficient profiles of training set genes. (B) LASSO regression with 10-fold cross-validation obtained 13 prognostic genes by minimum lambda value. (C) Best three biomarker candidates from BSR. (D) Kaplan–Meier survival analysis of DAPK3, RBPJ, and SLC40A1. (E) Forest plot of three prognostic genes from the multivariate Cox regression analysis. (F) Expression of the three genes in normal and EC tissues. (G) Kaplan–Meier survival analysis of the risk score model. (H) Model evaluation.Feature Protein Selection by Machine LearningGene expression has been defined as the “production of an observable phenotype by a gene—usually by directing the synthesis of a protein”.27 To investigate the protein characteristics during tumorigenesis, EC and three types of non-EC biopsies were collected for proteomic data generation using LC-MS/MS (Figure 3A), which is a central analytical technique for protein research and for the study of biomolecules in general. Feature selection is a fundamental step in many machine learning pipelines, which aims to simplify the problem by removing useless features that would introduce unnecessary noise. The genes were treated as features used for the recognition of different types of samples in this study. 61 and 42 feature proteins were identified to distinguish between EC and non-EC types and to discern the intragroup differences among the three types of non-EC, respectively (Figure 3B,C). Interestingly, in the abnormal samples, such as EHA, EAH, and EH, EHA and EAH showed similar patterns compared with those of EH (Figure 3C). As biological annotation showed, the up-regulated proteins in EC were involved in extracellular exosomes and cytoplasm, containing the same functions as the annotations of non-EC feature proteins (Figure 3D). Additionally, the overexpressed proteins in EHA and EAH were the same terms as the EC feature selection, indicating that both of them might carry a higher risk of being malignant than that of EH.Figure 3(A) Workflow of proteomic data generation. (B) Patterns of non-EC and EC feature proteins screened out by a random forest algorithm. (C) Patterns of three types of non-EC samples. (D) Biological annotations of the feature proteins.Alterations in Target Pathways during ECGenetic alterations as well as various cell-signaling pathways have been implicated in EC development and progression, including the MAPK/ERK pathway and Wnt/β-catenin signaling cascades (together with APC/β-catenin signaling). Endometrial tumor tissues have been shown to contain mutations in these signaling pathways, which are generally regarded as the primary drivers of carcinogenesis.28 MAPK/ERK expression is critical for development, and their hyperactivation plays a well-known role in cancer development and progression.29 The Wnt/β-catenin signaling pathway is an extremely conserved pathway that is involved in a variety of cellular processes in the female genital system, including development, cell proliferation, cell survival, adhesion, and motility, as well as the regulation of the menstrual cycle.30,31 Based on the above findings, we measured the gene products at different expression levels. Interestingly, the genes associated with these two pathways have reverse expression patterns (Figure S1). Along with EC occurrence, the proteins were highly expressed, but the RNAs were down-regulated. Although it is widely recognized that gene expression can exhibit inconsistent trends, the inverse expression in specific pathways, which is strongly associated with cancer development, still warrants careful consideration.Integrative Differential Expression AnalysisThe advent of omics technologies has generated an ever-growing number of omics datasets that allow researchers to study gene expression at various levels. Multiomics analysis in biomedical research could help explain the complex relationships between molecular layers, improving disease prevention, early detection, and prediction. To investigate the connections between transcriptomic and proteomic levels, we measured the significantly changed genes in tumorigenesis. The biological annotations of DEGs (RNAs = 1322, proteins = 668) were integrated (Figure 4A). The biological annotations were related to cell proliferation, growth, communication, and immigration, indicating that the DEGs have a closely tight connection with cancer occurrence (Figure 4B).Figure 4(A) Distributions of DEG products. (B) Biological annotations of DEGs at the transcriptomic and proteomic levels.The comparison of normal and EC from RNA-seq data showed similar counts of up- and down-regulated genes. However, at the protein level, overexpressed proteins make up a significant proportion of the differentially expressed proteins shared with the abnormal tissues (Figure 5A,B), which means there were many variations up-regulated along with EC incidents even though compared with the benign non-EC samples. The biological annotations (Figure 5C) were consistent with the cellular component annotations (Figure 4B), demonstrating that exosomes were critical for EC development. There were 116 DEGs shared and expressed at both the protein and RNA levels (Figure 5D). Based on the proteomic data, DAPK3, and its binding partner MYL9 were found. MYL9 had a tight connection with DAPK3 (confidence = 0.926907) and had been identified as a fibroblast-specific biomarker of a poorer prognosis for colorectal cancer.32 Low MYL9 abundance also was reported a significant association with the development of nonsmall-cell lung cancer.33 Thus, we speculated that MYL9 could also be a biomarker candidate for EC.Figure 5(A) Patterns of shared DEG products. (B) Biological annotations of protein-RNA shared DEGs. (C) Biological annotations of protein-RNA shared DEGs. (D) Expression trend of the shared 116 DEG products at two levels.Validation of Exosome-Related and Prognostic GenesTo substantiate the insights resulting from omics data analysis, it is essential to furnish solid evidence. We selected six genes associated with exosomes and prognosis, including RAB14, SEC31A, HSPE1, MSN, DAPK3, and MYL9, and measured their expression by qRT-PCR and western blotting.Heat shock protein family E (Hsp10) member 1 (HSPE1) is usually used together with the cochaperonin heat shock protein 60 (Hsp60) to maintain protein homeostasis.34 Hsp60 is well-known as being related to exosomes, so we speculated that HSPE1 might participate in exosome activity and cooperate with Hsp60. Moesin (MSN) is part of the ezrin, radixin, and moesin protein families. These proteins play crucial roles in linking the plasma membrane to the actin cytoskeleton. MSN has been reported as a potential exosomal protein biomarker for bladder cancer.35 Ras-related protein Rab-14 (RAB14) is a small GTPase involved in the regulation of intracellular membrane trafficking and vesicle transport, which plays a crucial role in vesicular transport within cells, especially during vesicular transport from the Golgi apparatus to the cell surface.36 There were reports showing RAB14 protein expression was positively correlated with increased tumor size,37 and its activity could regulate exosome secretions and cell growth.38 Protein transport protein Sec31A (SEC31A) is a core component of the COPII coat complex, which is involved in the formation of transport vesicles from the endoplasmic reticulum (ER), contributing to the efficient transport of proteins from the ER to the Golgi apparatus. This gene is essential for maintaining proper protein trafficking and secretion within the cell.39 DAPK3 was a potential prognostic gene found in RNA-seq data mining, which is a serine/threonine kinase that belongs to the DAP kinase family. Members of this family serve as crucial regulators of cell apoptosis. DAPK3 plays a pivotal role in diverse biological processes such as cell apoptosis, autophagy, cytoskeleton remodeling, and immune response.40−42 As a DAPK3 binding partner, MYL9 was widely reported to be overexpressed in several colorectal cancer cell lines, promoting cell proliferation, invasion, migration, and angiogenesis, while silencing MYL9 exerted the opposite effects.43In Table 1, the four genes related to exosomes have the same expression trend in RNA-seq data and qRT-PCR, except for MSN. Table 2 and Figure 6A show the results at the RNA level are consistent. High expression of MYL9 mRNA was associated with worse survival status in EC patients (Figure 6B). MYL9 was validated at the protein level by western blotting (Figure 6C). The result of univariate Cox regression was not significant (HR = 1, p-value = 0.2), suggesting that additional factors may synergize with MYL9 to worsen the prognosis of EC patients. Overall, the experiments supported our findings based on the omics data, even though the comparisons were normal versus EC and EH versus EC.Table 1Fold Change Values of Exosome-Related Genes From Transcriptomic DataTable 2Fold Change Values of Potential Biomarkers at Different LevelsFigure 6(A) Expression of DAPK3 and MYL9 in omic data. (B) Kaplan–Meier survival analysis of MYL9. (C) Western blotting of two potential biomarkers.ConclusionsIn summary of our study, DAPK3, RBPJ, and MYL9 have been identified as potential prognostic biomarkers for EC, and a linear model has been developed based on these findings to predict the patients’ prognosis. To describe the characteristics of EC at different levels, differential expression analysis and machine learning algorithms were adopted, finding that extracellular exosomes might be associated with EC development. The biological pathways named the “MAPK signaling pathway” and “WNT signaling pathway” are well-known and involved in tumorigenesis, and the DEG products in the two pathways have inverse trends in this study, demonstrating that there are potential regulation networks that exist among multibiolayers depending on this way. Three exosome-related genes (HSPE1, RAB14, and SEC31A) and one prognostic gene (MYL9) were validated by wet experiments, supporting the omic findings resulting above. Altogether, based on the approach of integrating multiomics data from the transcriptome and proteome, we provided a landscape for a better understanding of EC incidents.

PMC

Asian Pacific Journal of Cancer Prevention : APJCP

37116133

PMC10352758

Jan-01-2023

10.31557/APJCP.2023.24.4.1131

The Potential of Expression of Cyclin-D1 on Neoadjuvant Chemotherapy in Invasive Breast Carcinoma

Rustamadji Primariadewi, Wiyarta Elvan, Anggreani Ineke

Background:Patients undergoing neoadjuvant chemotherapy (NC) for invasive breast cancer (IBC) need indicators to track their progress during treatment. The goal of this research is to learn how cyclin D1 works in conjunction with taxane and non-taxane therapy for people with IBC. Methods:There were 31 examples divided into two groups, based on: those using a different type of NC (taxane- or non-taxane-based), and NC administration time (before or after). Tumor grade, age, PR, ER, Ki-67, HER2, and Cyclin D1 expression were among the factors considered. Using immunohistochemical labeling, we were able to categorize cyclin D1 levels according to a threshold value, and we supplemented this with data we found in our databases. To analyze the data, we used a modified linear model.Results:The expression of Cyclin D1 decreased after NC delivery (p=0.086). Cyclin D1 expression was reduced in the taxane group (p=0.792). The non-taxane group also saw no differences in outcomes (p = 0.065). There was a larger decrease in Cyclin D1 expression in the non-taxane group compared to the taxane group, but the difference was not statistically significant (p=0.200). Conclusion:Cyclin D1 expression, even if the differences are not statistically significant, may be a prognostic indicator of NC reaction in IBC. The involvement of Cyclin D1 in NC warrants more research with bigger IBC sample sizes.

IntroductionCancer deaths from breast cancer are the leading cause of mortality among women. According to Global Burden of Cancer (GLOBOCAN) statistics, women will be diagnosed with breast cancer at 11.7% of all cases and 6.9% of all deaths linked with the disease in 2020 (GLOBOCAN, 2020). Breast cancer can be classified as invasive (IBC) or non-invasive (nIBC) (GLOBOCAN, 2020). There are several subtypes of IBC, which is the most frequent form of breast cancer. Because of the high variation of IBC, the treatment must also be done early and aggressively (Rustamadji et al., 2021; Rustamadji et al., 2021; Sharma et al., 2010).Neoadjuvant chemotherapy (NC), which is administered before surgery, is a crucial part of modern IBC care (Lee et al., 2011). Currently, NC is the gold standard for patients with locally progressed breast cancer and is the therapy of choice for early-stage, possibly treatable diseases (Lee et al., 2011). There are two primary categories of NC: those founded on taxanes and those that do not (Zhang et al., 2019). In breast cancer therapy, the taxane is one of the most influential and extensively used systemic therapies. Resistance to NC, on the other hand, impacts breast cancer treatment (Zhang et al., 2019).A mechanism of self-protection has been devised by cancer cells in order to fight the effects of NC, for example, the NFkB activation pathway (Biliran et al., 2005). One of the NFkB protein complex’s most important functions is regulating gene expression(Biliran et al., 2005). By controlling many anti-apoptotic genes, NFkB may help cancer cells withstand chemotherapy(Biliran et al., 2005). Survival factors like Cyclin D1 are among them (Biliran et al., 2005; Garg et al., 2016; Pacifico et al., 2006). The involvement of cyclin D1 in the NC resistance pathway makes it a candidate for use as a prognostic indicator (Mohammadizadeh et al., 2013). Predictive indicators are ultimately used in therapy to improve total mortality after NC administration in IBC. Both taxane-based and non-taxane-based NC for IBC will be compared for their effects on cyclin-D1 expression before and after treatment. We hypothesized that cyclin-D1 expression is associated with NC delivery in IBC patients.Materials and Methods Data Collection and Design of Study This research was carried out between January and May of 2022 in the Pathological Anatomy Laboratory of the University of Indonesia’s Faculty of Medicine. The Universitas Indonesia Institutional Review Board approved the 21-11-1252 testing protocols in November 2021. Each participant gave a written agreement and understood the goal of the research. The research adheres to Declaration of Helsinki (Rickham, 1964). The data collection period ran from January 2014 through June 2016, and the five-year monitoring phase will run from January 2019 through May 2021. Data were collected, including tumor grade, age, tumor size, HER2 status, Ki-67, underarm lymph node spread, lymphovascular penetration, and NC type (taxane-based or non-taxane-based). Quantitative analysis of the IHC labeling findings on the paraffin sample was also performed to collect data on cyclin D1 expression. Samples Primary tumor paraffin samples were taken from female breast surgery patients who had initially been identified with IBC histopathologically. Specimens from individuals with non-IBC diseases, systemic illnesses, and damaged paraffin blocks are discarded. Both the NC treatment status (before or after) and the type of NC used (non-taxane or taxane) were used to categorize the data. The selected group represents the largest representative selection from the available records in the department. To avoid any potential for prejudice, only one researcher (E.W.) had access to the final groupings. Researchers didn’t have access to the studies’ classifications until after the analysis was done. Preparation of Samples Kusmardi et al., Wiyarta et al., and Primariadewi et al. all use this staining method . In xylol (Brataco.inc, Bogor, Indonesia), we deparaffinized the paraffin block, then rehydrated it in 96%, 70%, and purified water for 5 minutes, as per protocol. Heat-induced antigen recovery in pH 9.0 Tris EDTA (Merk, Jakarta, Indonesia). was performed in a 96°C chamber for 20 minutes. There was a 15-minute period of peroxidase block (Merk, Jakarta, Indonesia) after antigen retrieval, then followed by 15 minutes of PBS pH 7.4 (Brataco Inc., Bogor, Indonesia) rinses. Post-primary and Novolink polymer incubations were conducted after anti-cyclin D1 antibodies (ab134175, Abcam, Cambridge, UK) were incubated for one hour. Hematoxylin and 5 percent lithium carbonate were used to counterstain the tissue slices before they were examined under a microscope for DAB staining (Abcam, Jakarta, Indonesia). Quantification of Cyclin D1 Expression Histopathology experts P.R. and I.A. assessed the immunohistochemistry stainings. A Leica DM750 microscope with a 400x total magnification was used to examine each specimen. Five sites were chosen at random, each containing 500 tumor cells, and their Cyclin D1 expression was analyzed. Each location had at least one hundred malignant cells. Cyclin D1 expression was detected by a dark smear in the tumor cytoplasm (Fusté et al., 2016). Cell counter were used to assess the brown hues and to classify the staining intensity as negative , weak (1+), moderate (2+), or strong (3+)(O’Brien et al., 2016). In order to measure the expression of cyclin D1, Peurala et al. developed a quantification formula (Ortiz et al., 2017). The percentage values of the very positive, positive, and low positive categories are combined to quantify the cyclin D1 expression (Ortiz et al., 2017). In addition, the cyclin D1 expression group was separated in to high and low expressions based on the prior quantification values. Samples with values higher than 0.4 were classified as having strong cyclin D1 expression (Ortiz et al., 2017). Two raters independently collected all of the results for the group. Until the entire sample has been examined, the results of the calculations that have already been performed are merged and sent to additional scholars (E.W.). The combined rating from the two raters will serve as the basis for further study. Statistical Analysis All data was been tabulated in Microsoft Excel (Microsoft Corp, Redmond, WA, USA) before research. Using SPSS (Statistical Package for the Social Sciences) 20, we examined and portrayed the collected data (IBM Corp, Armonk, NY, USA). To categorize cyclin D1 expression, the number 0.4 was used (Ortiz et al., 2017). The combined ratings from the two raters were then compared to the threshold to determine the overall rating (high or low). Each sample’s Cyclin D1 expression level is represented these overall rating.ResultsAll thirty-one samples were stained for cyclin D1 by immunohistochemistry. Before and after administration of NC, each specimen exhibits the clinicopathologic features outlined in Tables 1 and 2. Figure 1 depicts the results of exemplary IHC staining. Negative, low positive, positive, and high positive samples of tumor cells are shown in each image, respectively. The images are composites of different parts of a single sample. These samples are analyzed further after the strength of the brown color has been measured and assigned a number. All 31 samples were assessed separately by two experts (I.A and P.R.).Following NC therapy, as shown in Figure 2 and Table 3, Cyclin D1 expression dropped, albeit not significantly (p=0.086). Both Table 4 and Figure 3 demonstrate the data’s separation into taxane and non-taxane group. Cyclin D1 was downregulated in the taxane-treated cohort. However, the numbers were too low to be considered significant (p=0.792). Similar results were observed in the control sample (p=0.065) that did not include taxanes. The level of cyclin D1 was also compared between the two groups. Cyclin D1 levels were significantly lower in the non-taxane group than in the taxane group. The difference between the two groups was not statistically significant (p=0.200).Figure 1IHC Staining for cyclin-D1 Expression in IBC Tumor Cells at 400x Magnification before and after NC Administration. The scale bar represents 50 μm for all imagesTable 1Clinicopathological Characteristics before Neoadjuvant Chemotherapy AdministrationVariablesCategoryCyclin-D1 Expressionp-valueHigh (%)Low (%)Age≥50 y.o.14 (83.9%)2 (12.5%)0.654<50 y.o.12 (80.0%)3 (20.0%)Tumor grade38 (88.9%)1 (11.1%)0.581215 (78.9%)4 (21.1%)13 (100.0%)0 (00.0%)ER statusPositive14 (93.3%)1 (6.7%)0.165Negative12 (75.0%)4 (25.0%)PR statusPositive12 (85.7%)2 (14.3%)0.8Negative14 (82.4%)3 (17.6%)HER2 statusPositive11 (78.6%)3 (21.4%)0.467Negative15 (88.2%)2 (11.8%)Ki67 statusPositive4 (80.0%)1 (20.0%)0.797Negative22 (84.6%)4 (15.4%)TaxaneWith9 (90.0%)1 (10.0%)0.522Without17 (81.0%)4 (19.0%)ER, estrogen receptor; PR, progesterone receptor, HER2, human epidermal growth factor receptor 2; Univariate analysis was performed using the chi-square test with continuity correlation;* p-value less than 0.05 is considered statistically significantTable 2Clinicopathological Characteristics after Neoadjuvant Chemotherapy AdministrationVariablesCategoryCyclin-D1 Expressionp-valueHigh (%)Low (%)Age≥50 y.o.14 (87.5%)2 (12.5%)0.57<50 y.o.12 (80.0%)3 (20.0%)Tumor grade310 (90.9%)1 (9.1%)0.732212 (80.0%)3 (20.0%)14 (80.0%)1 (20.0%)ER statusPositive14 (93.3%)1 (6.7%)0.165Negative12 (75.0%)4 (25.0%)PR statusPositive12 (85.7%)2 (14.3%)0.8Negative14 (82.4%)3 (17.6%)HER2 statusPositive11 (78.6%)3 (21.4%)0.467Negative15 (88.2%)2 (11.8%)Ki67 statusPositive4 (80.0%)1 (20.0%)0.797Negative22 (84.6%)4 (15.4%)ALNMYes12 (80.0%)3 (20.0%)0.57No14 (87.5%)2 (12.5%)LVIYes12 (75.0%)4 (25.0%)0.165No14 (93.3%)1 (6.7%)TaxaneWith9 (90.0%)1 (10.0%)0.522Without17 (81.0%)4 (19.0%)ALNM, Axillary lymph node metastasis; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; LVI, Lymphovascular invasion PR, progesterone receptor; Univariate analysis was performed using the chi-square test with continuity correlation; * p-value less than 0.05 is considered statistically significantFigure 2Individual before-after Line Showing Overall Changes in Cyclin-D1 Expression before and after Administration of Neoadjuvant ChemotherapyTable 3Overall Changes in Cyclin-D1 Expression before and after Administration of NeoadjuvantCategorynCyclin-D1 ExpressionP valueBefore310.700 ± 0.2380.086After310.578 ± 0.308Statistical analysis was performed using the generalized linear model Figure 3Individual before-after Line Showing Changes in Cyclin-D1 Expression before and after Administration of Neoadjuvant Chemotherapy in Taxane-based vs. non-Taxane-based GroupTable 4Changes in Cyclin-D1 Expression before and after Administration of Neoadjuvant in Taxane vs non-Taxane GroupGroupCategorynCyclin-D1 ExpressionP valueCyclin-D1 Expression Mean DifferenceP valueTaxaneBefore100.735 ± 0.2310.7920.17 ± 0.290.2After100.706 ± 0.253Non-TaxaneBefore210.683 ± 0.2450.0650.03 ± 0.19After210.517 ± 0.318Statistical analysis was performed using the generalized linear modelDiscussionCyclin D1 expression was significantly changed in individuals with IBC who received NC. These alterations are detectable on an individual and collective level. Pre-NC administration, cyclin D1 expression was often higher than after administration of NC. This provides evidence for a causal relationship between NC and a reduction in cyclin D1 in IBC cells. Possible explanations for these observations involve the role of Cyclin D1 in drug resistance and the impact of it on the efficacy of therapy (Pysz et al., 2014). Consequently, several kinds of NC were created to target this biomarker (Villegas et al., 2018). Numerous investigations have shown that some anticancer drugs function by reducing cyclin D1 synthesis (Grillo et al., 2006). This is consistent with Grillo et al., , which demonstrate that siRNA-mediated suppression of cyclin D1 in MCF-7 breast cancer cells has anticancer drug target potential. Overexpression of cyclin D1 was also linked to resistance to NC. Overexpression of cyclin D1 increases tumor cell proliferation and imparts resistance to cisplatin-mediated apoptosis, as reported by Biliran et al., . All of these factors explain the involvement of NC in cyclin D1 expression modification.Pre- and post-NC groups and taxane and non-taxane groups showed differences in cyclin D1 expression. This research found that the non-taxane group experienced a greater shift in cyclin D1 than the taxane group. Although these findings are not statistically significant, they suggest a possibly more prominent decreasing tendency in the group using non-taxane-based NC. Some research may account for this discovery. Overexpression of cyclin D1 was also linked to IBC in patients who underwent surgery followed by anthracycline-based treatment, as was previously reported by Reis-Filho et al., . In a separate investigation, cyclin-dependent kinase was linked with a more accurate prognostic model and lower pathological complete response rates (Wachter et al., 2013). This observation may be because anthracyclines (such as epirubicin and pirarubicin) interact with topoisomerase II and inhibit DNA transcription (Fisher et al., 1997; Li et al., 2011). In the context of neoadjuvant treatment, combining these two drugs is one of the most often reported chemotherapy regimens (Li et al., 2011; Tiezzi et al., 2007).Even though participants were recruited from an IBC referral center, the study’s sample size was insufficient. It’s possible that this is the case because NC has not been extensively adopted in the area where the research is taking place. Consequently, the expression of cyclin D1 was very minimally changed. Despite the need to consider clinical and laboratory importance, cyclin D1 expression is decreasing across all categories. As part of this initiative, more research on cyclin D1’s role in IBC and NC will need to be done.In conclusion, in IBC, cyclin D1 expression may be a prognostic indicator of NC response and clinical outcome, despite the fact that the differences are not statistically significant, as determined by an analysis of clinical and biochemical data. The involvement of cyclin D1 in NC warrants more research with bigger IBC sample sizes.Author Contribution StatementConceptualisation, P.R., E.W., I.A.; methodology, P.R., E.W.; software, E.W.; validation, P.R., I.A.; formal analysis, P.R., E.W.; investigation, E.W., I.A.; resources, P.R.; data curation, P.R., E.W.; writing—original draft preparation, P.R., E.W.; writing—review and editing, all authors; visualization, E.W.; supervision, P.R.; project administration, E.W.; funding acquisition, P.R. The published version of the work has been reviewed and approved by all authors.

PMC

Indian Journal of Ophthalmology

37322666

PMC10417997

6-01-2023

10.4103/ijo.IJO_3068_21

Outcomes of preloaded toric intraocular lens implantation in eyes undergoing phacoemulsification

Nagpal Ritu, Shakkarwal Chetan, Ahsan Saima, Maharana Prafulla Kumar, Goel Madhav, Sharma Namrata

Purpose:To evaluate the clinical outcomes of preloaded toric intraocular lens (IOLs) implantation in eyes undergoing phacoemulsification.Methods:This prospective study included 51 eyes of 51 patients with visually significant cataracts and corneal astigmatism ranging between 0.75 and 5.50 D. All patients underwent phacoemulsification with SupraPhob toric intraocular lens implantation under topical anesthesia. The main outcome measures were uncorrected distance visual acuity (UDVA), residual refractive cylinder, spherical equivalent, and IOL stability at 3 months follow-up.Results:At 3 months, 49% (25/51) of patients had UDVA equal to or better than 20/25 with 100% of eyes achieving better than 20/40. Mean logMAR UDVA improved from 1.02 ± 0.39, preoperatively to 0.11 ± 0.10 at 3 months follow-up (P < 0.001, Wilcoxon signed-rank test). The mean refractive cylinder improved from − 1.56 ± 1.25 D preoperatively to − 0.12 ± 0.31 D at 3 months follow-up (P < 0.001) while the mean spherical equivalent value changed from − 1.93 ± 3.71D preoperatively to − 0.16 ± 0.27D (P = 0.0013). The mean root mean square value for higher order aberrations was 0.30 ± 0.18 μm while the average contrast sensitivity value (Pelli-Robson chart) was 1.56 ± 0.10 log unit, at the final follow-up. The mean IOL rotation at 3 weeks was 1.7 ± 1.61 degrees, which did not change significantly at 3 months (P = 0.988) follow-up. There were no intraoperative or postoperative complications.Conclusion:SupraPhob toric IOL implantation is an effective method for addressing preexisting corneal astigmatism in eyes undergoing phacoemulsification with good rotational stability.

Addressing preexisting corneal astigmatism in patients undergoing cataract surgery is a key factor to achieve spectacle independence and optimal quality of vision. Various methods have been described in the literature to address this preexisting corneal astigmatism, each having its own benefits, limitations, and complications. Cataract surgeons all over the world moved from incisional refractive procedures (selective positioning of phacoemulsification incision, opposite clear corneal incisions, astigmatic keratotomy, limbal relaxing incisions) to excimer laser based procedures, (laser in situ keratomileusis and photorefractive keratectomy) and finally to the current preferred mode of correction by toric intraocular lens (IOL) implantation.[7-9] These IOLs allow the cataract surgeons to address astigmatism correction at the time of surgery itself, without any added risks of corneal epithelial injury or biomechanical changes.Toric IOLs have been in use since 1994 when the first silicone toric IOL was implanted with promising visual outcomes. A large proportion of these eyes however had significant postoperative IOL rotation (more than 10 degrees). Various advancements have been made since then pertaining to the material and design of IOLs, to improve the rotational stability, and hence the postoperative outcomes. A wide range of toric IOLs currently exists that offers correction of cylindrical power over a variable range. Other than primary cataract surgery, these IOLs have also been used to address the higher degree of astigmatism such as developing following keratoplasty.This study looked into the outcomes of a preloaded toric IOL (SupraPhob toric IOL; Appaswamy Associates, Tamil Nadu, India) in eyes undergoing routine cataract surgery.MethodsStudy designThis prospective study included 51 eyes of 51 patients, presenting in the outpatient department of the institute, between November 2014 and December 2019. Patients which were planned for phacoemulsification with SupraPhob toric IOL (Appaswamy Associates, Tamil Nadu, India) implantation were enrolled. Written and informed consent was obtained from all patients after explaining the nature and consequences of the surgical procedure. The study adhered to the tenets of the Declaration of Helsinki. Ethics approval was obtained from the ethics committee of the institute (IEC/NP-349/08-10-2014).Patient selectionInclusion criteria for study enrollment were the presence of visually significant cataract and presence of regular corneal astigmatism ranging from 0.75 to 5.5 D. Patients who were planned for phacoemulsification with SupraPhob toric IOL implantation were enrolled. The IOLs were provided free of cost to all patients as a part of the study by the manufacturer. Exclusion criteria for the study included the presence of irregular astigmatism, corneal scar, mature cataract, presence of other ocular comorbidities such as glaucoma, retinal pathology, anterior or posterior segment inflammation, history of a prior surgical procedure, and ocular trauma.Preoperative evaluationPreoperatively, all patients underwent a detailed evaluation involving assessment of uncorrected (UDVA) and corrected distance (CDVA) as well as near visual acuity, manifest refraction, intraocular pressure, anterior chamber depth, iris details, grade of cataract, and posterior segment status. Visual acuity was assessed using the Snellen acuity chart at all follow-up visits. B scan ultrasonography was done in case the posterior segment was not visible clinically. Axial length, keratometry, anterior chamber depth, and lens thickness were determined using a swept source biometry; IOL master (Zeiss; Jena, Germany). Patients recruited in the study before October 2018 (n = 21), were evaluated using the IOL Master 500 while those enrolled after that period (n = 31) were evaluated using the IOL Master 700.Corneal topography was performed in all cases, using the Oculus Pentacam (HR Typ70900; Wetzlar, Germany) to ensure the regularity of the corneal surface.SupraPhob toric intraocular lensSupraPhob toric intraocular lens is made up of a premium quality biocompatible acrylic material with a refractive index of 1.497. The overall length of the IOL is 13.00 mm and the optic diameter is 6.00 mm. Each curvature of the IOL has an aspheric design with markings on the anterior surface, corresponding to the flat meridian of the lens. The non-touch preloaded system allows for its convenient insertion.The IOL is available from + 10.00 dioptre (D) to +30.00D spherical power in 0.5D increments and from 1.50 to 6D cylindrical power at the IOL plane and 1.03 to 4.11 D cylindrical power at the corneal plane. The IOL constant was optimized based on the results of a pilot study conducted on 10 patients wherein the IOL was initially implanted. An optimized A constant of 118.8 was then used for the current study. Table 1 shows various available models of SupraPhob toric IOL with their respective powers in the corneal and IOL planes. The toric IOL power calculation was done using an online calculator provided by the manufacturer at om/toric.php. The Appaswamy Toric IOL calculator takes into account the magnitude and axis of flat and steep keratometry, magnitude of surgically induced astigmatism, IOL spherical power, and the incision location. IOL power calculations were done taking into account the surgical induced astigmatism (SIA) value of 0.5 and incision location as 180 degree for the right eye and 0 degree for the left eye.Table 1Available models of SupraPhob Toric Intraocular lensCylindrical powerCylindrical power at corneal planeSP TORICT31.501.03SP TORICT42.251.55SP TORICT53.002.06SP TORICT63.752.57SP TORICT74.503.08SP TORICT85.253.60SP TORICT96.004.11SP=SupraPhobSurgical techniqueAll surgeries were performed by the same surgeon under topical anesthesia using 0.5% proparacaine hydrochloride (Paracaine; Sunways India Pvt Ltd, Mumbai, India). A temporal approach was used for all cases with an initial clear corneal incision of 2.8 mm. The exact location of the incision, size, and location of the capsulorhexis and the final IOL placement axis were determined using a computer-assisted cataract surgery system; ZEISS CALLISTO eye (Carl Zeiss Meditec AG, Jena, Germany) image-guided system. The reference image captured preoperatively by the IOL Master and the keratometry data are transferred to the Callisto eye before starting the surgery. The reference image is used for intraoperative matching with the live eye image, thereby assisting in the precise localization of incisions, capsulorhexis size and location, and the final toric IOL axis placement. The use of intraoperative image-guided systems obviates the need for preoperative axis marking.A temporal approach was used for all cases with an initial clear corneal incision using a 2.75 mm keratome (slit knife; Alcon Laboratories, Inc, Fort Worth, Texas, USA). After injecting viscoelastic (Healon, 1% sodium hyaluronate; Abbott Medical Optics, Sweden) capsulorhexis of diameter 5.0–5.50 mm was made to ensure an adequate overlap with the IOL optic. After phacoemulsification, the preloaded SupraPhob toric IOL was inserted into the capsular bag. After the complete removal of viscoelastic from the capsular bag was ensured, the IOL was aligned with the axis as indicated by the image-guided system. The corneal wound was hydrated and no sutures were applied.Postoperative evaluationPostoperatively, all patients received topical 0.5% moxifloxacin solution (Vigamox; Alcon Laboratories, Inc, Fort Worth, Texas, USA) and 1% prednisolone acetate ophthalmic suspension (PredForte; Allergan, Dublin, Ireland) in tapering doses for 6 weeks. Patients were evaluated on day 1, 1-week, 3-weeks, and at 3-months follow-up visit. Investigations included assessment of UDVA and CDVA, manifest refraction, corneal status, anterior segment details, intraocular lens position and stability, and posterior segment details. Two observers assessed the IOL position in the postoperative period, with the patient seated on a slit lamp under retro illumination. Axis measurement was done using a thin coaxial slit, which was rotated until it overlapped with the marks over the IOL. The corresponding value on the slit lamp was noted and the difference between the actual IOL placement axis and the measured value was taken as the amount of rotation.In addition, in 20 patients, visual quality was assessed at 3-months follow-up visit. iTrace ray-tracing aberrometry (Tracey™ Technologies, Texas, USA) was performed using the HOYA iTrace™ Surgical Workstation (HOYA surgical optics, Singapore) to assess the magnitude of postoperative higher-order aberrations. Contrast sensitivity was measured using the Pelli-Robson chart.Statistical analysisStatistical analysis was done using STATA version 12.1. For the purpose of analysis, the visual acuity recorded on a Snellen acuity chart was converted to logMAR visual acuity. Mean values, standard deviations, and frequency (%) distributions were calculated for each parameter. Intergroup comparisons were done using the Wilcoxon signed rank test. Results were considered statistically significant if P value was less than 0.05.ResultsFifty-one eyes of 51 patients underwent SupraPhob toric IOL implantation including 21 males and 30 females. All patients were followed up till the required study follow-up time, with no loss. The mean age of patients was 60.94 ± 11.04 years (range: 38–82). The mean keratometry at baseline was 44.44 ± 2.04 D and the keratometric cylinder was 1.85 ± 2.41 D (range: 0.91–5.35). The demographic and other preoperative variables have been summarized in Table 2.Table 2Demographic and clinical parameters of patients which underwent SupraPhob toric intraocular lens implantationParametersvaluesA. Demographic variables Number of patients51 Number of eyes51 Age (years) Mean s.d60.94 1.04 Gender distribution (M/F)41%/59% Laterality (OD/OS)43%/57%B. Visual acuity logMAR UDVA (mean s.d)1.02 0.39 logMAR CDVA (mean s.d)0.81 0.29C. Keratometric cylinder (D) (mean s.d)1.85 2.41D. Manifest refraction (mean±s.d) Sphere (D)−1.04 2.92 Cylinder (D)−1.56 1.25 Spherical equivalent (D)−1.93 3.71E. IOL power (mean±s.d) Spherical (D)21.53 2.28 Cylindrical (D)2.42 0.78F. Anterior chamber depth (mm) (means.d)3.07 0.18G. Lens thickness (mm) (mean s.d)4.27 0.24H. Axial length (mm) (means.d)23.130.86UDVA=Uncorrected distance visual acuity, CDVA=Corrected distance visual acuity, D=Dioptre, IOL=Intraocular lensVisual and refractive outcomeMean logMar UDVA at presentation was 1.02 ± 0.39 which improved to 0.18 ± 0.20 logMAR at 3-weeks follow-up, 0.13 ± 0.12 at 6-weeks follow-up, and 0.11 ± 0.10 at 3-months follow-up visit [Table 3]. The change from preoperative value to the final follow-up visit was statistically significant.Table 3Postoperative outcomes at various time follow-up visits, following SupraPhob toric intraocular lens implantation3 weeks follow-up6 weeks follow-up3 months follow-uplogMAR UDVA (mean s.d)0.18 0.200.13 0.120.11 0.10logMAR CDVA (mean s.d)0.06 0.060.040.03Manifest refraction (mean s.d) Sphere (D)−1.32 0.32−0.11 0.27−0.11 0.27 Cylinder (D)−1.86 0.37−0.12 0.32−0.12 0.31 Spherical equivalent (D)−0.21 0.39−0.24 0.74−0.16 0.27Toric IOL rotation (degree) (mean s.d)1.7 1.371.74 1.331.71 1.36UDVA=Uncorrected distance visual acuity, CDVA=Corrected distance visual acuity, D=Dioptre, IOL=Intraocular lensFurthermore, 49% (25/51) of patients had UDVA equal to or better than 20/25 and 100% of eyes had UDVA equal to or better than 20/40 at the last follow-up. A total of 100% of eyes had CDVA equal to or better than 20/32 at 3 months postoperatively.The mean refractive cylinder improved from a preoperative value of − 1.56 ± 1.25 D to − 0.12 ± 0.31 at 3 months follow-up (P = 0.0000) while the mean spherical equivalent value changed from − 1.93 ± 3.71 preoperatively to 0.16 ± 0.27 at final follow-up (P = 0.0013) [Table 4].Table 4Comparison of preoperative and 3 months postoperative variables following SupraPhob toric intraocular lens implantationPreoperative3 months postoperative P logMAR UDVA (mean s.d)1.02 0.390.11 0.100.0000logMAR CDVA (mean s.d)0.81 0.290.030.0000Manifest refraction (mean s.d) Sphere (D)−1.04 2.92−0.11 0.270.0386 Cylinder (D)−1.56 1.25−0.12 0.310.0000 Spherical equivalent (D)−1.93 3.71−0.16 0.270.0013UDVA=Uncorrected distance visual acuity, CDVA=Corrected distance visual acuity, D=DioptreA total of 64% of eyes had residual refractive cylinder (33/51) less than 0.5 D. In addition, 94% of eyes (48/51) had spherical equivalent equal to or better than 0.5D.Visual qualityThe mean root means square value for higher order aberrations at 5 mm pupillary zone was 0.30 ± 0.18 mm. Mean contrast sensitivity value as measured with the Pelli-Robson chart 1.56 ± 0.10 log unit at 3 months follow-up.IOL rotationThe mean IOL rotation at 3 weeks was 1.7 ± 1.37 degrees at 3 weeks follow-up, which did not change significantly at 6 weeks and at 3 months follow-up.There were no intraoperative or postoperative complications.Analysis of change in astigmatismThe change in astigmatism was analyzed based on preoperative keratometry, predicted postoperative refraction, and the manifest refraction obtained at 3 months follow-up. Fig. 1a and 1B show cumulative histograms showing the distribution of the magnitude of preoperative keratometric and postoperative refractive astigmatism at the spectacle plane [Fig. 1a] as well as at the corneal plane [Fig. 1b]. A total of 100% of eyes had preoperative corneal astigmatism ranging between 1D and 2D, whereas postoperatively 92% of eyes had refractive astigmatism less than equal to 0.5D at the spectacle plane.Figure 1(a) Cumulative histogram showing the distribution of the magnitude of preoperative keratometric and postoperative refractive astigmatism at the spectacle plane. (b) Cumulative histogram showing the distribution of the magnitude of preoperative keratometric and postoperative refractive astigmatism at the corneal planeFigs. 2 and 3 show double-angle plots showing preoperative corneal and postoperative refractive astigmatism at the spectacle plane [Fig. 2a] and at the corneal plane [Fig. 2b] showing mean and standard deviation values and the 95% confidence ellipse of the centroid as well as of the dataset. Preoperatively, the majority of values lay outside the 95% confidence ellipse of the centroid. This is in contrast to the postoperative plots where most values were limited to the area within the 95% confidence ellipse of the centroid. A significant reduction in both the mean as well as the standard deviation of the magnitude of astigmatism was noted both at the spectacle plane as well as the corneal plane. Fig. 3 shows double angle plots, representing the prediction error of postoperative refractive astigmatism at the spectacle plane [Fig. 3a] and at the corneal plane [Fig. 3b].Figure 2(a) Double angle plot showing preoperative corneal and postoperative refractive astigmatism at the spectacle plane. (b) Double angle plots showing preoperative corneal and postoperative refractive astigmatism at the corneal planeFigure 3(a) Double angle plot representing the prediction error of postoperative refractive astigmatism at the spectacle plane. (b) Double angle plots, representing the prediction error of postoperative refractive astigmatism at the corneal planeDiscussionThe study evaluated the outcomes of a preloaded toric IOL (SupraPhob) implantation in eyes undergoing phacoemulsification with a wide range of preexisting corneal astigmatism (0.75–5.50 D) at 3 months follow-up. As mentioned earlier in the methods part, the IOL comes in a ready-to-use preloaded IOL, which comes in a ready-to-use non-touch preloaded system. Using a preloaded delivery system for IOL insertion is advantageous as it helps to streamline the process of lens preparation as well as delivery. It also ensures reliable delivery of IOL within the capsular bag, helping to retain the integrity of the IOL. Using a preloaded system also minimizes the chances of IOL damage occurring during the loading process as well as the risk of microbial contamination. Apart from the ease of surgical procedure, an important concern while planning cataract surgery in patients with significant corneal astigmatism is the cost of surgery. Toric IOLs are an expensive means for addressing corneal astigmatism when compared with other methods such as incisional procedures, (LRI, AK) but still continue to be the surgeon’s preferred choice since they do not hamper the biomechanical stability of the cornea. A cost-effective toric IOL which reliably addresses astigmatism with minimal complications is therefore the need of the hour. Table 5 enlists and compares various types of toric IOLs in terms of their cost and the insertion technique.Table 5Comparison of various toric IOLs available in the market, in terms of their loading system and the cost borne by the patientModelPreloaded (√/×)Cost in USDAcrySof IQ Toric SN6AT3 (Alcon)×204.36Tecnis monofocal toric (ZCT150-600) (Johnson & Johnson Vision)×201.67AT TORBI 709M×174.78AT TORBI 709MP√(ZEISS)enVista®0 toric MX60PT (Bausch & Lomb)√ (SimplifEYE delivery system)121SupraPhob toric (Appaswamy Associates)√121IOL=Intraocular lens, USD=United States DollarThe amount of preoperative corneal astigmatism corrected in various randomized trials performed with different models of toric IOLs ranges from 0.75 to 3D with favorable visual and refractive outcomes. Postoperative rotation is the most common complication which has been reported following the implantation of toric IOLs. With increased use of imaging systems such as the Callisto eye and intraoperative aberrometers, guiding accurate intraoperative placement, the incidence of this complication has lessened, but it is not completely eliminated and postoperative refractive surprise is a possibility. IOL stability, therefore, is an important factor determining postoperative refraction, spectacle independence, quality of vision, and overall patient satisfaction.Miyake et al. reported 2-year outcomes of implantation of Acrysof toric IOL in a large series of 378 eyes. A total of 94.7% of patients achieved a UDVA of 20/25 or better with mean refractive cylinder of −0.59 ± 0.62 D. Mean IOL misalignment was 4.1 ± 3.0 degrees at 2 years follow-up. The authors observed a higher amount of IOL rotation (more than 20 degrees) in the immediate postoperative period (within 10 days) in eyes that had a longer axial length (more than 25.0 mm). In another comparative study by Holland et al., 256 eyes underwent Acrysof toric IOL implantation and the results were compared with a similar set of 261 patients who underwent Acrysof spherical IOL. Furthermore, 40.7% of eyes achieved a UDVA of 20/20 or better with a residual refractive cylinder of 0.59D. The mean IOL rotation was 3.8 degrees at 1-year follow-up. Waltz et al. reported the outcomes of tecnis toric IOL implantation in 172 eyes. The mean absolute change in axis orientation between visits was less than 3 degrees for all visit intervals. Five eyes had an axis shift of 10 degrees or more between day 1 and 6 months, requiring realignment. In our series, the mean change in orientation between various visits was 0.04 degrees, with none of the patient requiring IOL realignment.The average contrast sensitivity value of our cases was 1.56 ± 0.10 log unit, at the final follow-up. The study by Yang et al. evaluated and compared the outcomes of Acrysof toric IOL with tecnis toric IOLs and found comparable results in terms of postoperative contrast sensitivity. Our results compare well with their reported ones. The mean root mean square value of higher order aberrations (5 mm pupil zone) at the last follow-up visit was 0.30 ± 0.18 μm. In a previous study by the same authors, the outcomes of single-stage Acrysof toric IOL implantation were randomly compared with sequential monofocal IOL implantation followed by PRK for astigmatism correction in eyes undergoing phacoemulsification. The median value for root mean square value of higher order aberrations (5 mm pupil zone) after 6 months of Acrysof toric IOL implantation was 0.395 mm and following 6 months of PRK was 0.63 mm, which is higher compared with what we observed in this study, following 3 months of SupraPhob toric IOL implantation.Our results with SupraPhob toric IOL implantation are comparable with the above-reported ones, both in terms of efficacy as well as safety even in eyes with a relatively higher amount of preexiting corneal astigmatism. The mean axial length of patients in our study was 23.13 ± 0.86 mm. The mean change in axis orientation between various visits was 0.04 degrees, with none of the patient requiring IOL realignment. In terms of visual quality, the amount of high-order aberrations and the contrast sensitivity values were within the acceptable range. Another advantage of SupraPhob IOL is the reduced cost, compared with other models of toric IOLs available in the market.ConclusionTo our knowledge, this is the first study reporting the outcomes of Supraphob toric IOL, a preloaded IOL being widely used by cataract surgeons. The initial results have been encouraging in terms of efficacy and postoperative complications. The study is, however, limited by its short follow-up time and the lack of a control group. Randomized studies with head-to-head comparison of SupraPhob toric IOL with other toric IOLs in a large number of eyes, will be more informative and should be undertaken as the next step.Financial support and sponsorshipNil.Conflicts of interestThere are no conflicts of interest.

PMC

Heliyon

38192832

PMC10772616

12-16-2023

10.1016/j.heliyon.2023.e23730

Increased serum visfatin level is associated with fat deposition of the lumbar spine in ankylosing spondylitis patients

Shen Jie, Tao Sha-Sha, Wang Rui-Yuan, Shi Shi-Kui, Jiang Chao, Mei Yong-Jun

ObjectivesTo assess the serum visfatin levels in patients with ankylosing spondylitis (AS), as well as its correlation with fat deposition of the lumbar spine.MethodsSerum visfatin levels were detected by enzyme-linked immunosorbent assay (ELISA) in 50 AS patients and 75 sex-and age-matched healthy controls. The clinical and laboratory indexes of AS patients were recorded, and the lumbar spine magnetic resonance scan was performed to evaluate the lumbar spine fat deposition in AS patients. The level of serum visfatin and its correlation with lumbar fat deposition were analyzed, and the risk factors of AS lumbar MRI fat deposition were evaluated by Logistic regression.ResultsSerum visfatin levels in AS patients were elevated compared with that in healthy controls (p < 0.001), and were more significant in patients with fat deposition and syndesmophyte formation (p = 0.017 and p = 0.014, respectively). Serum visfatin levels were positively correlated with CRP, BASDAI, mSASSS and fat deposition (all p < 0.05). Age (OR = 1.085, 95% CI: 1.005–1.173, p = 0.038), disease duration (OR = 1.267, 95% CI: 1.017–1.578, p = 0.035), and visfatin (OR = 1.846, 95% CI: 1.004–3.393, p = 0.048) were risk factors for fat deposition in AS patients.ConclusionsThe level of serum visfatin in AS patients is significantly increased, which is associated with fat deposition on lumbar MRI. Elevated visfatin level is an independent risk factor for AS lumbar fat deposition.

1IntroductionAnkylosing spondylitis (AS) is a chronic progressive inflammatory disease, which is more common in men and mainly affects the axial joint. Sacroiliac joint involvement is usually considered a sign of the disease. AS is insidious and the most common and characteristic symptoms are low back pain and back stiffness . It is characterized by new bone formation during disease progression and can cause spinal ankylosis and ligamentous calcification in advanced stages, manifesting as hunchback or bamboo-like changes. Many studies have investigated the radiological progression of AS by imaging methods, such as radiographs and magnetic resonance imaging (MRI), and therefore imaging has an important role in the diagnosis and prognosis assessment of the disease.Adipokines are highly biologically active factors secreted by adipose tissue, mainly including visfatin, adiponectin, leptin, and resistin, which can affect different tissues and cells systemically and locally, contributing to immune regulation and bone reconstitution mechanisms . It has been found that visfatin, a novel adipokine, leads to the release of pro-inflammatory factors, plays an important role in various acute and chronic inflammatory responses, and plays a role in bone homeostasis by stimulating osteoblast proliferation and inhibiting osteoclast production . In this study, the level of visfatin in the serum of AS patients was detected, and the difference between AS patients and the control group was observed. Combined with imaging data, the correlation between visfatin and lumbar MRI fat deposition in AS patients was analyzed, and the effect of serum visfatin level on AS lumbar fat deposition was evaluated.2Materials and methods2.1Study subjectsA total of 50 AS patients (42 males, 8 females, aged 16–62 years, with an average age of 34.40 ± 12.64 years) who were admitted to the Department of Rheumatology and Immunology of the First Affiliated Hospital of Bengbu Medical College from October 2020 to December 2021 were selected as the AS group, all patients met the New York criteria for AS 1984 revision. Seventy-five healthy controls (62 males and 13 females, range from 20 to 60 years, mean age 35.67 ± 10.09 years) were collected during the same period. The enrolled patients were excluded from infections, tumors, and other rheumatic diseases. The age, disease duration, body mass index (BMI), HLA-B27, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), platelet count (PLT), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and Bath Ankylosing Spondylitis Function Index (BASFI) were recorded for all AS patients. Normal reference range of erythrocyte sedimentation rate: male ≤15 mm/h, female ≤20 mm/h; the normal reference range of CRP was ≤6 mg/L. The study was approved by the Ethics Committee of the First Affiliated Hospital of Bengbu Medical College. Written informed consent was obtained from all individuals before the initiation of the study.2.2Laboratory analysisBlood samples of 5 ml were collected from all the individuals, and centrifuged at 1000 r/min for 20 min after natural clotting and the serum samples were stored at −80 °C until analysis. Visfatin levels were detected by enzyme-linked immunosorbent assay (ELISA). The reagents were purchased from Wuxi Donglin Sci & Tech Development Co., Ltd. The detection was performed according to the manufacturers’ instructions.2.3ImagingAll AS patients underwent MRI scanning of the lumbar spine in sagittal position with T1, T2 (Fig. 1a) and short-tau inversion recovery (STIR) phases (Fig. 1b), mainly to observe fat deposition in the vertebral horn (Fig. 1), and to record the number of fat-deposited vertebrae. Frontal and lateral radiographs of the cervical, thoracic and lumbar spine were performed, and the modified Stoke ankylosing spondylitis spine score (mSASSS) was used to assess structural damage of the spine. The examination reports were obtained by two radiologists who reviewed the films individually.Fig. 1(1a) Lumbar MRI T1 sequence, fat deposition as shown in the arrow; (1b) STIR sequence images of the same lumbar spine.Fig. 12.4Statistical analysisStatistical analyses were performed with Statistical Package for the Social Sciences (SPSS) statistical software for Windows, version 21.0 (SPSS Inc., IL, USA). Normal distribution data were presented as mean ± SD, skewed data were presented as median (interquartile range, IQR). Independent samples t-test or rank sum test was used to compare differences between groups, and chi-square test was used for comparison of categorical variables. Spearman correlation coefficients were calculated to determine the association between visfatin levels and other variables, and risk factors for fat deposition in patients with AS were analyzed using Logistic regression analysis, p values less than 0.05 were considered statistically significant.3Results3.1Characteristics of patientsThe patients’ demographic and clinical characteristics are summarized in Table 1. We examined two groups of subjects: 50 AS patients and 75 healthy controls. Their mean age (±SD) was 34.40 ± 12.64 and 35.67 ± 10.09 years, respectively. Males accounted for 84% of the AS group and 82.7% of the healthy controls. According to the Goutellier criteria, which measures the fat content of muscle tissue by imaging techniques such as CT or MRI, and divides the fat deposits into 5 grades from 0 to 4, we classified fat deposits of AS patients on the basis of fat infiltration of paravertebral muscles. AS patients were classified into 0–4 grade with 22, 17, 5, 2, and 4 patients, accounting for 44%, 34%, 10%, 4%, and 8%, respectively. Which corresponded to 22, 17, 5, 2, and 4 patients with AS, with the proportions of 44%, 34%, 10%, 4%, and 8%, respectively.Table 1The general features of study subjects.Table 1ParametersAS (n = 50)HCs (n = 75)p valueAge (years)34.40 ± 12.6435.67 ± 10.090.536Gender (male/female)42/862/130.845Visfatin (pg/ml)5.57 ± 2.201.41 ± 0.82 0.05) (Table 2).Fig. 3Serum visfatin levels in AS patients were positively correlated with CRP (a), BASDAI (b), mSASSS (c) and fat deposition (d) (p < 0.05).Fig. 3Table 2Correlation between visfatin level and clinical indexes of AS.Table 2ParametersvisfatinrpAge0.0290.844Disease duration0.0900.536ESR0.0180.900CRP0.2930.039*PLT−0.1490.301BASDAI0.3290.019*BASFI0.0690.636mSASSS0.3110.028*Fat deposition0.3450.014*Note: *p < 0.05.3.4The difference of fat deposition rate in AS patients between different general index groupsThe difference of lumbar fat deposition rate between different age and course groups was statistically significant (x2 = 8.179, p = 0.004; x2 = 9.149, p = 0.002). There was no significant difference in lumbar fat deposition rate between different gender, BMI and HLA-B27 groups (x2 = 0.628, p = 0.428; x2 = 2.710, p = 0.100; x2 = 0.968, p = 0.325) (Table 3).Table 3Comparison of lumbar fat deposition rate between different general index groups of AS patients.Table 3ParametersGroupNFat depositionn%Age<40351542.9*≥40151386.7Gendermale422252.4female8675.0BMI<24231043.5≥24271866.7Disease duration<5291137.9*≥5211781.0HLA-B27+33100.0_472553.2*p < 0.05.3.5Analysis of risk factors associated with fat depositionAge, sex, disease duration, BMI, visfatin, and mSASSS were used as independent variables, and the presence or absence of fat deposition in lumbar MRI (0 = no fat deposition, 1 = fat deposition) was used as a response variable. The risk factors affecting fat deposition in the lumbar spine of patients with AS were analyzed by logistic regression. The results showed that age, disease duration, and visfatin were risk factors for fat deposition in patients with AS (Fig. 4).Fig. 4Baseline characteristics were used for analysisAge, course of disease, visfatin were risk factors for fat deposition in AS patients. The non-effective line was OR = 1.Fig. 44DiscussionVisfatin is an adipocytokine that causes inflammation by activating human white blood cells to release Proinflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor-α . Studies have found that visfatin is associated with numerous diseases such as acute lung injury, sepsis, atherosclerosis, and rheumatoid arthritis, its serum level is elevated in a variety of acute and chronic inflammatory diseases and plays an important role in the pathogenesis of these diseases. Comparing the serum of AS patients with that of healthy controls, we found that serum visfatin levels were significantly higher in AS patients than in the normal population, this proves that the expression level of serum visfatin in Chinese Han AS population is consistent with that in German and Czech [5,6], suggesting that visfatin plays a role in the course of AS.Inflammation (bone marrow edema), fat deposition, and syndesmophyte formation as a trilogy of bone structure progression in AS can lead to spinal stiffness and ankylosis in patients with AS in the later stages of the disease. Rademacher et al. demonstrated that visfatin levels were significantly associated with imaging progression in patients with AS. MRI can show the fat deposition of joint bone marrow in the early stage by its sensitivity , reflecting metabolic changes in the marrow tissue noninvasively at the molecular level. Studies have shown that the fatty lesions in the anterior horn of the spinal vertebral on MRI predict the formation of new bone . T1 weighted sequences can show spinal structural damage (such as fatty lesions, erosion, and stiffness). After the inflammation in the erosion subsides, it fills the cavities in the eroded bone through fat metaplasia and evolves into bridging ankylosis . MRI links the inflammatory lesions of the spine with new bone formation and becomes a bridge to observe the progression of AS. To further investigate the role of visfatin in the progression of AS patients, we observed lumbar spine MRI fat deposition in AS patients, combined with the level of visfatin detected, we found that AS patients with elevated serum visfatin levels had a higher frequency of fat deposition in the lumbar spine and were more likely to have syndesmophyte formation. In the histological analysis of the joints of AS patients, it was found that the joint remodeling process of AS has many similarities with osteoarthritis (OA) patients [11,12]. Studies have found high levels of visfatin production in the joint tissues of patients with osteoarthritis, especially in synovial and adipose tissues . Therefore, we believe that visfatin may be involved in the new bone formation of AS.Elevated CRP and ESR can be used the reference index of AS disease activity, and BASDAI is a common indicator to evaluate the activity of ankylosing spondylitis. Tsiklauri et al. found that visfatin participated in the bone remodeling of adipose tissue/bone interface by inducing pro-inflammatory factors and imbalanced MMP/TIMP during the differentiation of mesenchymal stem cells, and changed the balance between bone resorption and bone formation. In a study of 64 patients with axSpA and 61 age-and sex-matched healthy controls, Hulejová et al. not only found elevated serum visfatin levels in patients with axSpA, moreover, visfatin level was positively correlated with mSASSS score. Therefore, we used Sperman to analyze the correlation between serum visfatin and clinical indicators in AS patients. The results showed that there was a correlation between serum visfatin level and CRP, BASDAI, mSASSS and fat deposition in AS patients, and there was a positive correlation between them, which indicated that visfatin could reflect the activity of AS to a certain extent. However, this correlation was not found in ESR, which could explain the majority of AS patients in this study being repeat patients, and the difference in study results could be related to the control of inflammatory markers between individual AS patients after treatment and the bias of the small sample size. In addition, we did not observe a correlation between visfatin levels and age, disease duration, PLT or BASFI.In rheumatoid arthritis (RA), visfatin is expressed in joint sites of arthrocyte destruction and in periarticular adipose tissue . In the present study, it was found that the increase in visfatin level had an important effect on the fat deposition in the vertebral angle of patients with AS. In addition, advanced age and long disease duration are also factors that affect fat deposition in AS patients. However other factors may also contribute to the fat deposition at older age group, and what is the correlation between age and disease duration, we have not investigated deeply, which needs further study and discussion. Several studies have found that fat deposition is associated with new bone formation in AS, and Machado et al. concluded that there is a sequential link between vertebral corner inflammation (VCI), vertebral corner fat deposition (VCFD) on MRI, and radiologic of the same vertebral body, and the results revealed that both VCI and VCFD promoted new bone formation in AS, especially when VCI occurred before VCFD, confirming the relationship between fat deposition and new bone formation. Hartl et al. showed a positive correlation between visfatin and radiological spine progression. Similar results were also verified in the study of Syrbe et al., who proposed that elevated serum visfatin level was an independent predictor of radiological progress and intervertebral process formation/progress in AS patients. Therefore, visfatin plays an integral role in the disease progression of AS and is an important indicator of fat deposition and new bone formation in AS. In patients with AS who have developed fat deposition, single anti-inflammatory therapy is limited in inhibiting new bone formation and delaying imaging progression. At present, various targeted drugs have emerged in clinical practice, and the prognosis of AS patients has been continuously improved. In the future treatment of patients with AS, the goal of our therapy is to pursue anti-inflammation while delaying disease progression and preventing structural damage. Visfatin not only plays a role in the inflammation of AS but also is a key mediator affecting bone remodeling, providing us with new possible therapeutic research directions for the treatment of AS in the future. In this study, we found that serum visfatin levels in patients with active AS was increased, and the serum visfatin level was associated with fat deposition on lumbar MRI in AS patients. These results indicate that AS patients are more likely to experience spinal imaging progression. In our daily clinical practice, elevated visfatin may be used as a biomarker of increased disease activity and new bone formation in AS patients. Patients with elevated visfatin levels should be given timely attention and treatment. Visfatin, as one of the possible therapeutic targets for AS, the development of its inhibitor needs further study.Based on the fact that our study is a cross-sectional study, some limitations are unavoidable, on the one hand, the inability to dynamically observe changes in serum visfatin levels before and after fat deposition in patients with AS, on the other hand, the observation of fat deposition could not be assessed using fat quantification techniques due to the limitations of the conditions, and there is a slight limitation to the accurate calculation of fat deposition objectively. In the future, it may be necessary to increase the longitudinal research direction and adopt more accurate methods to evaluate the level of fat deposition.5ConclusionIn conclusion, increased serum visfatin level is associated with fat deposition of the lumbar spine in ankylosing spondylitis patients, suggesting that it may play an important role in the pathophysiological process of new bone formation in AS. In addition, the increased serum visfatin level is an independent risk factor for lumbar spine fat deposition in AS patients. This finding reveals the relationship between visfatin and fat deposition, which helps us to further explore the pathogenesis of new bone formation in AS and provides new ideas for preventing spinal stiffness and delaying disease progression.Ethics statementThe studies involving human participants were reviewed and approved by the ethics committee of the First Affiliated Hospital of Bengbu Medical College ([2020 No. 134]). The participants provided their written informed consent to participate in this study. Among them, the written informed consent was obtained from the patient of which images were included in this study for the publication of his images.Data availability statementThe datasets generated for this study are available on request to the corresponding author.CRediT authorship contribution statementJie Shen: Writing - original draft, Formal analysis. Sha-Sha Tao: Writing - review & editing, Writing - original draft. Rui-Yuan Wang: Investigation. Shi-Kui Shi: Investigation. Jiang Chao: Visualization. Yong-Jun Mei: Conceptualization.Declaration of competing interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Plant Biology (Stuttgart, Germany)

34651391

PMC10078684

12-01-2022

10.1111/plb.13349

Summer temperatures reach the thermal tolerance threshold of photosynthetic decline in temperate conifers

Kunert N., Hajek P., Hietz P., Morris H., Rosner S., Tholen D.

Abstract Climate change‐related environmental stress has been recognized as a driving force in accelerating forest mortality over the last decades in Central Europe. Here, we aim to elucidate the thermal sensitivity of three native conifer species, namely Norway spruce (Picea abies), Scots pine (Pinus sylvestris) and silver fir (Abies alba), and three non‐native species, namely Austrian pine (Pinus nigra), Douglas fir (Pseudotsuga menziesii) and Atlas cedar (Cedrus atlantica).Thermal sensitivity, defined here as a decline of the maximum quantum yield of photosystem II (Fv/Fm) with increasing temperature, was measured under varying levels of heat stress and compared with the turgor loss point (πtlp) as a drought resistance trait. We calculated three different leaf thermotolerance traits: the temperature at the onset (5%) of the Fv/Fm decline (T5), the temperature at which Fv/Fm was half the maximum value (T50) and the temperature at which only 5% Fv/Fm remained (T95).T5 ranged from 38.5 ± 0.8 °C to 43.1 ± 0.6 °C across all species, while T50 values were at least 9 to 11 degrees above the maximum air temperatures on record for all species. Only Austrian pine had a notably higher T5 value than recorded maximum air temperatures. Species with higher T5 values were characterized by a less negative πtlp compared to species with lower T5.The six species could be divided into ‘drought‐tolerant heat‐sensitive’ and ‘drought‐sensitive heat‐tolerant’ groups. Exposure to short‐term high temperatures thus exhibits a considerable threat to conifer species in Central European forest production systems. Temperatures recorded during heat waves in Central Europe exceed the thermal tolerance threshold of native and non‐native conifer species.

INTRODUCTIONForest disturbance is a natural part of forest ecosystem dynamics (Seidl et al. 2017), but increasing climate‐induced tree mortality has been observed in a wide range of forest ecosystems worldwide (Allen et al. 2010; Cobb et al. 2017). In European temperate forests, mortality of canopy trees has doubled over the last three decades (Senf et al. 2018) and has been primarily linked to climate‐induced drought (Senf et al. 2020). Tree mortality results in changes to ecological communities, shifts in ecosystem functions with a reduction in ecosystem services, together with unexpected land–climate feedbacks (Anderegg et al. 2013). A mechanistic understanding of tree mortality is crucial to lessen economic and cultural harm (Allen et al. 2010) and sustain forest production systems. The predicted increases in frequency and intensity of temperature extremes (heatwaves) are unequivocally linked to climate change and occur independently of drought events (IPCC, 2019). However, heatwaves can cause drought stress due to high potential evapotranspiration in combination with little or no rainfall. The duration and intensity of heatwaves can seriously impact the physiological performance of trees (Billon et al. 2020), some of which are important timber trees.Over centuries, Norway spruce and Scots pine have been the most important timber species for the forestry sector in Central Europe. Large‐scale wind disturbance and associated bark beetle outbreaks have been historically major disturbances in forest production systems consisting mainly of Norway spruce (Eriksson et al. 2007). However, the increasing frequency and longer duration of heatwaves has favoured bark beetle infestation and replaced windthrow as the main cause of mortality (Hentschel et al. 2014). Although Scots pine has been considered relatively drought‐tolerant compared to e.g., Norway spruce, an accelerated mortality of Scots pine has been observed in recent years. Besides Norway spruce and Scots pine, other species in Europe are expected to be impacted by the expected rise in drought and heatwaves in future climate change scenarios (irrespective of the actual cause of tree mortality) (Hartmann et al. 2018).Several advantages of conifers over broadleaved tree species, e.g., optimal mechanical wood properties, potentially faster growth rates, larger carbon sink per area and increased economic yield, means forest owners would, in the future, preferably plant a drought‐adapted conifer species. Consequently, the native silver fir has in recent years been planted in increasing numbers across Central Europe, aiming to achieve higher drought resistance in forest stands (Muck et al. 2008). Likewise, the substitution of former Norway spruce and Scots pine forests by non‐native drought‐tolerant conifer species or the intermixing of these with native species became common practice (Bolte et al. 2009). The three non‐native species, Douglas fir, Austrian pine or Atlas cedar, have been considered as potentially suitable alternatives in forest production systems under a future climate (De Avila & Albrecht 2018). However, criticism arises on admixing non‐native species due to their potentially invasive character and potential detrimental impact on forest ecosystems (Pötzelsberger et al. 2020). Also, it remains unknown how such species will perform under a changing climate.The actual drivers and mechanisms causing tree mortality are still under debate and are most probably a result of complex interdependencies of mutually inclusive mechanics (Hajek et al. 2020). Most studies on tree mortality focus predominantly on drought stress as the initial trigger of tree mortality (e.g., Pretzsch et al. 2020), neglecting heat‐induced changes in morphological, physiological and biochemical processes, although these do affect the overall performance, growth and ultimately the survival of plants (Song et al. 2014). However, there is growing evidence that heat‐induced tree mortality can cause abrupt changes in forest biomass stocks (Chaste et al. 2019; Breshears et al. 2021), which suggests a limited ability of trees to locally acclimate to higher temperatures (Konôpková et al. 2018). Drought‐induced effects on forest performance could potentially be mitigated by the application of silvicultural practices, such as stand density reduction (Sohn et al. 2013), which in turn reduces the canopy cover, negatively affecting the forest understorey during warm periods (von Arx et al. 2012). Thus, short‐term management options to increase forest resistance to heat events are even more limited than forest management options to increase forest drought resistance (Burschel & Huss 2003). Therefore, a better understanding of temperature thresholds that trigger forest decline and mortality is necessary for choosing suitable species for future forest production systems.Photosystem II (PSII) is a pigment–protein complex located in the thylakoid membranes of the chloroplasts and is considered the most heat‐sensitive component of photosynthesis (Ashraf & Harris 2013), with temperatures above a critical value leading to irreversible damage to the photochemistry (Tiwari et al, 2021; Slot et al. 2021). One approach to quantify thermotolerance is to analyse the effect of extreme temperatures on the photochemistry of the leaves. A quantitative measure of the photochemical efficiency of PSII is the ratio between variable and maximum chlorophyll fluorescence (Fv/Fm), which is a good indicator of drought and heat stress (Yu & Guy 2004). The temperature at which the quantum efficiency declines by 50% (T50) has been used as a proxy for the critical temperature at which irreversible damage to the photochemistry occurs (Krause et al. 2010; Tiwari et al. 2021). Data on T50 values are predominantly available from subtropical and tropical regions (Sastry et al. 2017; Leon‐Garcia & Lasso 2019; Perez & Feeley 2020), where trees have adapted to elevated temperatures. However, tree species from mid‐latitudes are expected to have a very narrow thermal tolerance range and thus may be more susceptible to heatwaves (O'Sullivan et al. 2017).In this study, we used the aforementioned temperature‐induced changes in Fv/Fm to assess thermal tolerance for three conifer species native to Central Europe and three that are potentially or increasingly being planted for future forest production systems. We aimed to address the following questions: (i) will current summer temperatures exceed the thermal thresholds of conifer species, and (ii) how will their thermal tolerance be related to hydraulic leaf properties, namely turgor loss point? The overall goal was to provide a first assessment on thermo‐tolerance of the economically most important conifer species and their suitability in forest production systems under a changing climate—in particular under the threat of the increasing frequency of heatwaves in Central Europe.MATERIAL AND METHODSMaterial from native and non‐native conifers was collected in the surroundings of Sichersdorf, in the rural district of Fürth in Middle Franconia, Germany (49°24'04.4" N 10°56'01.3" E). The district is characterized by very patchy forest distribution, with approximately 30% of the area stocked with forest. Scots pine (Pinus sylvestris L.) has been the main species cultivated in the area, followed by Norway spruce (Picea abies Karst.) and European oak (Quercus robur L.). A few autochthonous silver fir (Abies alba L.) are scattered sparsely throughout those forests. The region has been affected by high tree mortality in recent years. The mortality increased after two very dry summers combined with heatwaves in 2015 and 2016, and accelerated during the following dry and very hot conditions in 2019 and 2020. The non‐native conifer species selected for this study, i.e., Atlas cedar (Cedrus atlantica ‘Glauca group’ (Endl.) G. Manetti ex Carrière) and Douglas fir (Pseudotsuga menziesii (Mirbel) Franco), are commonly grown in urban areas in the district and the botanical material was collected in the adjacent township. Austrian pine (Pinus nigra sensu lato J.F. Arnold) was collected from a forest stand on a former military training ground planted by the US military in the 1970s (49°25'16.6" N, 10°59'47.9" E). For each species, one sun‐exposed branch was sampled from five individuals in early spring 2021. In the forests, we targeted dominant canopy trees, and a minimum diameter at breast height (DBH) of 25 cm. Per individual, needles from 1‐year‐old shoots were collected. Needles were dark‐acclimated for 30 min and tested for initial maximum photosynthetic efficiency (Fv/Fm) to ensure leaf health (Fv/Fm between 0.83 and 0.75) with a chlorophyll fluorometer (MINI‐PAM; Walz, Effeltrich, Germany). Thermal dependence of Fv/Fm was assessed following the protocol of Krause et al. . Therefore, needles were placed in Microcloth, separated by cloth layers to prevent anoxic conditions, and transferred into watertight Whirlpack bags. The bags were bathed under water in a precision cooker at varying temperatures for 15 min. The exact temperature was monitored with a digital thermometer (TFA; Dostmann, Wertheim, Germany). The mean temperature during the vegetation period in the region is 15° C (DWD 2021), hence we used 15° C as starting temperature. Temperature treatments covered the range between 15 °C and 59 °C. We started with 5‐degree steps between treatments and lower steps of 2 degrees within the critical temperature range, starting at 30 °C. Per individual, about eight needles were selected and each needle was randomly assigned to a temperature treatment. Overall, 40 needles were used to establish one curve. Heat‐treated needles were incubated under controlled conditions (15 °C, ~20 μmol m−2 s−1 light) in Petri dishes containing a thin film of water (Tiwari et al. 2021). The next day, the recovery of Fv/Fm was measured after a 30‐min dark adaptation period. We used the data on turgor loss point collected by Kunert & Tomsakova to compare the thermal tolerance traits with hydraulic properties. Thermal and hydraulic traits were sampled on the same tree individuals. We used air temperature data available on the data platform of the Deutscher Wetterdienst (station number 3668; DWD, 2021) to compare the increase in frequency of hot days.After correcting for a typographical error, we used the same log‐logistic curve for the Fv/Fm response as described by Tiwari et al. : Fv/Fm=c+d‐c1+Exp[bLog[T/T50]] where T represents the temperature, c is the Fv/Fm of the lower plateau, d is the higher plateau, T50 is the temperature at which Fv/Fm reached 50% of the total decrease, b is the slope of the curve at T = T50. Because our initial results showed clearly asymmetric curves for some species, we also used an extension of Equation proposed by Ricketts & Head allowing for a difference in curvature before and after T = T50: Fv/Fm=c+d‐c1+fExp[b1Log[T/T50]]+(1‐f)Exp[b2Log[T/T50]]withf=(1+Exp[b1b2b1+b2Log[T/T50]])‐1 where b1 and b2 now represent a possibly different curvature before and after T50. Both response curves were fitted using the ‘modelFit’ function of the ‘drc’ package using the LL.4 (Equation ) and baro5 (Equation ) methods (Ritz et al. 2016) in R. Akaike’s information criterion was used to decide between Equation and Equation . Half of the species’ thermal Fv/Fm decline could be described by the four‐parameter logistic curve (Equation ; Scots pine, Austrian pine and Douglas fir), whereas the best fitting model for the other half was a five‐parameter asymmetric logistic (Equation ) function (silver fir, Norway spruce and Atlas cedar) (see Figs 1 and 2). Five different measures were extracted from the fitted curves to compare different species. The first three measures (T5, T50, T95) correspond to temperatures at which the change in Fv/Fm is 5, 50 or 95% of the maximum change (c–d). We fitted a simpler model to species pairs to compare the deviated from the species‐specific dose–response curves with an approximate F‐test (Ritz et al. 2016). In cases where the null hypothesis was rejected, the ‘compParm’ function was used to identify species differences in T5, T50 and T95. Analogous to Tiwari et al. , we also defined two decline widths for the temperature difference between different Fv/Fm levels: before the turning point (T50): T50–T5, and after the turning point: T95–T50. These decline widths are inversely related to the slope parameters b, b1 and b2 , with smaller values indicating more rapid changes in Fv/Fm. All presented means are ± SE. Data analysis was performed using the R program, version 4.0.4 (R Core Team, 2021).Fig. 1Temperature response of PSII efficiency (Fv/Fm) to 15‐min duration heat treatment of needles of three native conifer species. Top panel: Silver fir (Abies alba); middle panel: Norway spruce (Picea abies); bottom panel: Scots pine (Pinus sylvestris). Five individuals per species were measured to establish the thermal vulnerability curve.Fig. 2Temperature response of PSII efficiency (Fv/Fm) to 15‐min duration heat treatment of needles of the three exotic conifer species. Top panel: Atlas cedar (Cedrus atlantica); middle panel: Austrian pine (Pinus nigra); bottom panel Douglas fir (Pseudotsuga menziesii). Five individuals per species were measured to establish the thermal vulnerability curve.RESULTSThe T5 was on average at 40.3 ± 0.7 °C across all six conifer species. Austrian pine had the highest T5 (43.1 ± 0.6 °C) and Norway spruce the lowest T5 (38.5 ± 0.8 °C). T5 of Norway spruce was reached by the maximum air temperature recorded in the area (Fig. 3). T50 was on average 50.0 ± 0.9 °C, and silver fir had the highest T50 (52.3 ± 0.2 °C) of all species, followed by Atlas cedar (51.8 ± 0.4 °C). The two pine species had both very low T50 values of 47.8 ± 0.3 °C. T95 averaged 55.2 ± 0.6 °C across all species and was highest in Austrian pine (57.5 ± 1.4 °C) and lowest in Norway spruce and Douglas fir (both 53.9 °C; for ±SE, see Table 1).Fig. 3Thermal tolerance of conifer needles for six species represented by a boxplot diagram (note: boxplot y‐ordinate positioning indicative and not absolute, the logistic curve indicates combined fit for all six species). Thermal tolerance was measured as Fv/Fm temperature response to 15‐min duration heat treatment. The histogram highlights the warming trend in daily maximum air temperature relative to the two 10‐year periods between 1990–1999 and 2011–2020. Black bars are difference in frequency between the two periods. Maximum temperature represents the temperature record observed in the region as recorded in 2020.Table 1Summary of the measured thermal tolerance for the six European conifer species.Common nameLatin nameNative/introducedT5T50T95°C ± SE°C ± SE°C ± SESilver fir Abies alba Native39.5 ± 1.0a 52.3 ± 0.2a 54.6 ± 0.7a Norway spruce Picea abies Native38.5 ± 0.8a 51.6 ± 0.2a 53.9 ± 0.2a Scots pine Pinus sylvestris Native41.9 ± 0.7b 47.8 ± 0.3b 54.6 ± 1.0b Douglas fir Pseudotsuga menziesii Non‐native39.8 ± 1.0a 48.3 ± 0.3a 53.9 ± 0.6a Austrian pine Pinus nigra Non‐native* 43.1 ± 0.6b 47.8 ± 0.3b 57.5 ± 1.4b Atlas cedar Cedrus atlantica Non‐native39.0 ± 1.6a 51.8 ± 0.4a 56.5 ± 1.2a Mean40.3 ± 0.750.0 ± 0.955.2 ± 0.6Breaking point temperature at which PSII efficiency declines 5% (T5), temperature at which efficiency is at 50% (T50) of the maximum, and temperature at which only 5% of the maximum efficiency remains (T95). Superscript letters indicate significant differences between species (z‐test).*Austrian pine is native to Central Europe, but does not naturally occur in the study area.John Wiley & Sons, LtdThe T5 and decline width (T95–T5) were not significantly related (r2 = 0.385. P = 0.188; Fig. 4a). However, T5 correlated significantly with the decline width before the turning point (T50–T5) and after the turning point (T95–T50) (see Fig. 4b and c). Species with lower T5 were characterized by a wider decline width from T50–T5. Species—namely the two pine species—characterized by a high breakpoint temperature show sudden and steeper Fv/Fm decline over a narrow decline width.Fig. 4Relationships between PSII maximum quantum yield (Fv/Fm) breakpoint temperature, T5, and decline width. The temperature windows are shown when Fv/Fm declines from (a) 95% to 5% of the maximum Fv/Fm level (T95–T5), (b), the Fv/Fm decline from 5% to 50% of the maximum Fv/Fm level, and (c) Fv/Fm declines from 50% to 95% of the maximum Fv/Fm level.The T5 values of the six species correlated significantly with the turgor loss point (πtlp; Fig. 5a). Species with a higher T5 were characterized by a less negative πtlp, and species with a lower T5 by a more negative πtlp. There was no significant relationship between T50 and πtlp, nor T95 and πtlp (Fig. 5b and c).Fig. 5Relationship between thermal tolerance (T5, T50 and T95) and leaf turgor loss point (πtlp). πtlp data from Kunert & Tomaskova , except Cedrus atlantica (Kunert, unpublished data).DISCUSSIONWe report the first temperature‐dependent Fv/Fm measurements made on foliage of mature conifers from a temperate region. The most important finding is that the breakpoint temperature T5, the temperature when Fv/Fm starts to decline, lies on average at 40.3 ± 0.7 °C. For all six species, T5 is similar or marginally above the maximum summer air temperature recorded in the study area, and T50 values that were at least 9 to 11 degrees above the maximum summer air temperatures on record. Further, T5 correlates with leaf turgor loss point, indicating two diverging mechanistic strategies of species to deal with heat and drought stress. Species are classified as either ‘drought‐tolerant heat‐sensitive’ or ‘drought‐sensitive heat‐tolerant’.Summer temperatures reach thermal threshold of photosynthetic declineWe found clear evidence that summer temperatures measured during heatwaves exceed the thermal threshold, when the functioning of PSII of the investigated conifer species is significantly affected. The most sensitive species was Norway spruce, in which the maximum temperature measured during a heatwave in 2021 in the region (38.5 °C; see Fig. 2) will already result in initial PSII damage (T5 = 38.5 ± 0.8 °C). Ideally, the thermal tolerance thresholds should be related to leaf rather than air temperature; however, during heatwaves, limited access to soil water commonly results in stomatal closure. Stomatal closure, in turn. results in a rise of leaf temperatures to a level that even exceeds the ambient air temperature (Krause et al. 2010). In the course of a heatwave, trees in full sunlight are exposed to critical temperatures and high light intensities that can significantly change physiological and biochemical processes (Tiwari et al. 2021). The latter changes would likely affect the overall performance, growth and, ultimately, the survival of Norway spruce. Hence, thermal stress may contribute to the accelerated mortality of Norway spruce. We expect two possible consequences when the thermal threshold is surpassed, which might have a direct lethal outcome or induce long‐term physiological disorders in Norway spruce. First, thermal stress often triggers leaf senescence (Way, 2013). In the case of severe damage to the foliage, the canopy will be defoliated, leading to irreversible loss of carbon reserves as this species is unable to resprout new leaves within the same season (Galiano et al. 2011). Second, less severe damage to PSII can reduce electron transport rates that may decrease photosynthesis. A reduction in photosynthesis rate may weaken the defence mechanisms of Norway spruce and increase its susceptibility to bark beetle attacks (Huang et al. 2020) in the subsequent vegetation period.Air temperatures above 40 °C have been observed on a regular basis at different climate stations throughout Central Europe in the last few years (e.g., Herold & Schappert, 2019). Only Scots pine and Austrian pine had a substantially higher T5 (41.9 ± 0.7 °C and 43.2 ± 0.6 °C, respectively), whereas T5 values of silver fir, Douglas fir and Atlas cedar were only marginally above the recorded temperatures. Temperature extremes, in particular more frequent and hotter heatwaves, have significantly increased on a regional and global scale over the last few decades (Perkins‐Kirkpatrick & Lewis 2020). Increasing temperature extremes have also been observed in our study area (Fig. 3). The latter trend is predicted to intensify in the near future, with forests likely to experience increasing levels of thermal stress resulting in tree mortality events, potentially amplifying positive climate–carbon cycle feedbacks.Heat tolerant or sensitiveTiwari et al. describe a continuous spectrum of strategies found in six tropical broadleaved tree species. Our results suggest that the investigated six temperate conifer species can be classified as either ‘heat‐sensitive’ or ‘heat‐tolerant’: Douglas fir, silver fir, Norway spruce and Atlas cedar represent the heat‐sensitive species, characterized by an early decline in Fv/Fm and wide decline width in particular between T5 and T50. Fv/Fm in these mentioned four species declined rapidly after T50 (narrow decline width from T50 to T95). Both pine species, namely Scots pine and Austrian pine, can be classified as heat‐tolerant species, with a higher T5 and a narrow decline width between T5 and T50. The decline width of the pines between T50 and T95 was wider than in the other conifers, indicating a broader tolerance to high temperatures. Differences among species in their tolerance to high temperatures supports the Tiwari et al. hypothesis of classifying species into ‘heat‐sensitive’ and ‘heat‐tolerant’; however, the total decline width between T5 and T95 was found to be a rather weak indicator of thermal sensitivity (r2 = 0.385, P = 0.188), whereas the decline widths T50–T5 (r2 = 0.888, P = 0.005) and T95–T50 (r2 = 0.822, P = 0.013) were good predictors of heat tolerance strategies in the investigated conifers.Trade‐off between hydraulic and thermal vulnerability in leaves?Heat sensitivity is intrinsically linked to drought avoidance; plants can either cool through transpiration, thus risking drought stress, or speedily close the stomata to avoid drought stress while risking heat damage (Konôpková et al. 2018). In our case, the first group comprising the two pine species has a less negative turgor loss point (−2.24 MPa and −2.39 MPa; Scots pine and Austrian pine, respectively) than the other four species (all between −2.70 MPa and −3.02 MPa; see Kunert & Tomsakova 2020). The second group might keep stomata open for longer durations under more water‐limiting conditions while pines trigger early stomatal closure in response to a water deficit. Turgor loss point is a ‘robust proxy of a species' degree of anisohydry’ (Meinzer et al. 2017) and, accordingly, the two pine species could be classified as being more drought‐sensitive than the other four conifer species, which are classified as drought‐tolerant. The drought‐tolerant species may follow another strategy by using transpirational cooling of the leaf to avoid or reduce heat stress instead of adopting a more heat‐tolerant photochemistry, as found in drought‐sensitive species. In our study, the conifer species with high drought tolerance tend to be more heat sensitive (i.e., have a lower breaking point temperature), but might compensate for this through maintaining transpirational cooling under drought and/or heat stress. The latter strategy could be described as ‘drought‐tolerant heat‐sensitive’ while, conversely, ‘drought‐sensitive heat‐tolerant’ species (e.g., Scots pine, Austrian pine, etc.) are more sensitive to a water deficit but can tolerate high temperatures. The latter confirms the findings of Kunert that pine mortality is triggered predominantly by a complex of interdependencies related to drought stress. Further, spruce mortality is more likely the result of heat stress. However, the results also show that tree species can follow different strategies to occupy the same ecological niche.CONCLUSIONSWe found considerable variation in the thermal tolerance of the investigated temperate conifer species. During heatwaves, all six species were already operating very close to their thermal tolerance threshold. Species follow different strategies and either adapt their photochemistry to function under higher temperatures or adapt their osmotic potential to enable more transpirational cooling. Further research is required to understand how the interaction between heat stress and water limitation is affecting forest ecosystems. In the meantime, we suggest that the temperature of incipient PSII damage (T5) could be used to parameterize forest ecosystem models, enabling us to predict the effects of a future climate with increasing heatwave frequency and intensity.

PMC

JACC Case Reports

PMC10313485

5-17-2023

10.1016/j.jaccas.2023.101874

A Stepwise Approach for Transcatheter Edge-to-Edge Repair in Very Advanced Tricuspid Regurgitation

Ivannikova Maria, Rudolph Tanja K., Friedrichs Kai, Gummert Jan, Rudolph Volker, Omran Hazem

Transcatheter edge-to-edge repair (TEER) is the most widely used approach for tricuspid regurgitation in patients with prohibitive surgical risk. However, TEER might not be feasible in advanced tricuspid regurgitation. In such cases, a stepwise approach with initial annuloplasty and subsequent TEER can be a worthwhile alternative, which is reported in this series. (Level of Difficulty: Intermediate.) Central Illustration

Case 1An 82-year-old woman with history of permanent atrial fibrillation and arterial hypertension presented to our clinic with progressive dyspnea on exertion (NYHA functional class III). She had no clinical signs of congestion on a stable dose of diuretic agents (torsemide 10 mg). Transthoracic echocardiography (TTE) showed a “torrential” tricuspid regurgitation (TR) (VC biplane 14 mm, effective regurgitant orifice area [EROA] proximal isovelocity surface area 1.3 cm2, regurgitant volume [RVol] 95 mL) with a large coaptation gap with good systolic left and right ventricular function, moderate aortic regurgitation, and no other relevant abnormalities.Learning Objectives•To understand the role of thorough echocardiographic assessment for tailoring treatment options in TR.•To highlight the possibility of a stepwise approach for the treatment of advanced TR.Right heart catheterization showed isolated postcapillary pulmonal hypertension (mean pulmonary artery pressure 23 mm Hg, pulmonary capillary wedge pressure 18 mm Hg, and pulmonary vascular resistance 1.5 WU), most likely caused by heart failure with preserved ejection fraction secondary to hypertensive heart disease.Transesophageal echocardiography confirmed torrential functional TR (grade 5 of 5) with a large gap of 8 mm and a restrictive septal leaflet unfavorable for tricuspid transcatheter edge-to-edge repair (TEER) (Figure 1A, Video 1).Figure 1Periprocedural Images of Case 1(A) Initial transesophageal echocardiography (TEE) showing a coaptation gap with a restriction of the septal leaflet and mild tenting. (B) Postprocedural TEE (annuloplasty) showing a reduction of gap with a restrictive septal leaflet. (C) TEE after transcatheter edge-to-edge repair showing a good result, with only mild residual tricuspid regurgitation.Because the patient was at high surgical risk (TRI-SCORE 4 of 12, 8%), our heart team decided to go for a stepwise approach with an initial percutaneous annuloplasty with the Cardioband system (Edwards Lifesciences) and the option of subsequent TEER (Supplemental Figure 1, Video 2). A reduction in TR was achieved from torrential (5 of 5) to severe (3 of 5) (Video 3) and the big gap was almost closed (Figure 1B, Supplemental Figure 2).We evaluated the patient after 5 months. Although she reported better exercise tolerance, she remained symptomatic with onset of leg edema so that the dose of diuretic agents had to be increased. Follow-up TTE revealed severe residual TR (vena contracta [VC] biplane 13 mm, EROA 0.4 cm2, RVol 53 mL). Therefore, we decided to proceed with the TEER procedure using the Pascal Ace device (Edwards Lifesciences) and a classical 3-orifice technique (Video 4) with the insertion of 2 Pascal devices: one anteroseptally (8- to 2-o’clock orientation on transgastric view) and the other posteroseptally (9- to 3-o’clock orientation on transgastric view). TR was reduced to a mild degree (Figure 1C, Video 5) with a residual gradient across the tricuspid valve of 3 mm Hg after the combined procedure. At the 12-month follow-up, the patient was doing well with improved symptoms and a stable result on TTE.Case 2An 81-year-old man presented to our hospital 2 years ago with increasing peripheral edema and worsening dyspnea (NYHA functional class III). He had a history of coronary artery disease treated with coronary artery bypass graft 22 years ago, permanent atrial fibrillation with direct oral anticoagulation, and chronic kidney disease (grade 3b; glomerular filtration rate ∼34 mL/min).TTE showed a mildly reduced left ventricular ejection fraction (LVEF) (50%), moderate secondary mitral regurgitation (MR), and massive functional TR. Transesophageal echocardiography was performed after treatment with intravenous loop diuretic agents with decongestion and subsequent weight loss of 4 kg. This showed only moderate MR but still a massive TR (grade 4 of 5, VC biplane 17 mm, EROA 0.7 cm2, RVol 67 mL) with a septolaterally measured coaptation gap of 5 mm (Figure 2A, Video 6).Figure 2Periprocedural Images of Case 2(A) Initial transesophageal echocardiography: biplane view showing a coaptation gap and moderate leaflet tenting. (B) Transesophageal echocardiography showing good gap reduction after annuloplasty (septal [orange] and lateral [green] leaflets). (C) Follow-up TTE showing only mild TR.Coronary angiography showed stable disease with intact bypass vessels. Right heart catheterization showed isolated postcapillary pulmonary hypertension caused by slightly reduced LVEF, moderate MR, and atrial fibrillation. After discussion in the heart team, a decision for interventional treatment was recommended based on the prohibitive surgical risk (Society of Thoracic Surgeons 8%; TRI-SCORE 8 of 12, 48%). Because of the borderline coaptation gap with tethering of the septal leaflet, we decided against TEER. However, despite tethering of the septal leaflet, there was no excessive tenting of the entire valve, thus we decided to perform a percutaneous annuloplasty. The procedure was performed after 3 months, and the severity of TR decreased from grade 4 to grade 1 (Figure 2B, Video 7).Unfortunately, TR relapsed over time, probably because of the persistent tethering of the septal leaflet, and the patient was readmitted with cardiac decompensation 3 months later. TTE showed severe TR (grade 3 of 5). After decongestion and optimization of diuretic therapy, follow-up showed persistent severe TR (grade 3 of 5, VC biplane 9 mm, EROA 0.5 cm2, RVol 50 mL) (Video 8, Supplemental Figure 3). In addition, the patient had persistent dyspnea (NYHA functional class III). Therefore, we decided to treat this TR with TEER. Two devices were placed in a central position posteroseptally (Supplemental Figure 4) with a significant reduction of TR to grade 1 (Figure 2C, Video 9).At the 14-month follow-up, the patient was doing well with only mild dyspnea (NYHA functional class II). TTE showed only a mild residual TR with no relevant stenosis (mean trans-tricuspid pressure gradient 2.5 mm Hg).Case 3An 84-year-old woman with a history of isolated mitral valve replacement 1 year previously caused by severe primary MR presented with worsening dyspnea (NYHA functional class III) and leg edema. TTE showed preserved LVEF (55%) with a good function of the biological mitral valve prosthesis (mean pressure gradient of 3 mm Hg) with no MR. However, torrential (5 of 5) TR (Video 10) was evidenced (VC biplane 21 mm, EROA 1.4 cm2, RVol 86 mL) with a large coaptation gap of 12 mm and moderate tenting (Figure 3A). Preoperative TTE performed 1 year previously had shown only moderate TR with a tricuspid valve annulus diameter of 39 mm. Right heart catheterization showed isolated postcapillary pulmonary hypertension caused by valvular heart disease.Figure 3Periprocedural Images of Case 3(A) Initial transesophageal echocardiography: biplane view showing a big coaptation gap and a moderate tenting. (B) Transesophageal echocardiography biplane view showing improved coaptation of the tricuspid valve.(C) Follow-up transthoracic echocardiography showing only mild tricuspid regurgitation.The patient had a EuroSCORE (European System for Cardiac Operative Risk Evaluation) II of 11% and the TRI-SCORE was 4 of 12, 8%. In addition, because of the large gap, we decided against TEER. We performed a percutaneous tricuspid annuloplasty with an initial reduction from grade 5 to grade 3 (Video 11) and good gap reduction (Figure 3B). At the 1-month follow-up, there was only moderate TR (grade 2 of 5) (Supplemental Figure 5), and after 1 year TR had improved to mild (grade 1 of 5) (Figure 3C, Video 12) with marked reductions in right ventricular diameters. Clinically, she had improved to NYHA functional class I and was doing well.Expert TipsIn cases of advanced TR, a stepwise approach of transcatheter or surgical annuloplasty followed by TEER can extend the spectrum of patients able to access transcatheter tricuspid therapy. Annuloplasty could be performed as the first step, and close follow-up should determine whether subsequent TEER is required and feasible. Even when the anatomy is suboptimal for annuloplasty, the leaflet approximation provided by annuloplasty facilitates TEER, thereby providing more reliable results in advanced cases than TEER alone.DiscussionThis case series highlights several issues that are relevant for the treatment of patients with advanced TR. In patients with extensive annular dilation and large coaptation gaps that are not amenable to TEER, tricuspid annuloplasty represents a valuable treatment option and may provide durable TR reduction as a stand-alone therapy, only if there is mild to moderate leaflet tenting.1 As endorsed in the state-of-the-art paper on transcatheter tricuspid interventions,1 severe tenting was defined as a tenting height of >1.0 cm, measured from the leaflet tips to the annular plane performed at mid-systole. Surgical experience has shown that the degree of valve tenting correlated not only with TR severity and right ventricular dilatation, but also with persistent or recurrent TR after surgical repair.2 As such, an annuloplasty repair is less likely to treat this particular pathophysiology and additional techniques, such as leaflet augmentation or even valve replacement, should be considered.3 The same concept applies for transcatheter treatments: in patients with advanced tricuspid disease and more than moderate leaflet tenting, annuloplasty alone would not be sufficient. However, a staged approach of annuloplasty with consequent TEER could prove to be a valuable option. It is also important to note that additional factors, such as concomitant mitral and aortic disease, pulmonary pressure, atrial fibrillation, and, most importantly, right ventricular size and function, play an important role in determining the most appropriate treatment strategy and the success or failure of initial treatment with annuloplasty. Therefore, comprehensive assessment that takes all these factors into account is essential for good treatment decision making.Funding Support and Author DisclosuresSupport by the Deutsche Forschungsgemeinschaft Open Access Publication Funds of the Ruhr-Universität Bochum. Dr Friedrichs has received speaker honoraria from Abbott and Edwards. Dr Ivannikova has received speaker honoraria from Edwards. Dr Rudolph has received research grants from Abbott and Edwards. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

PMC

Plastic and Reconstructive Surgery Global Open

PMC10194783

5-19-2023

10.1097/01.GOX.0000937892.22859.d5

75. Evaluating the Long-term Outcomes Associated with the Use of Fresh Frozen Allograft Cartilage in Cosmetic and Reconstructive Rhinoplasty Procedures: A Prospective Controlled Clinical Trial

Wan Rou, Weissman Joshua P., Joshi Chitang J., Putnam Genevieve, Williams Tokoya, Galiano Robert D.

PURPOSE: Comparing the long-term outcomes of fresh frozen costal cartilage allograft (CCA) and traditional autologous costal cartilage (ACC) in cosmetic and reconstructive rhinoplasty proceduresMETHODS: This study was a prospective, non-randomized, open-label clinical trial. The protocol was approved by the IRB and registered on www.ClinicalTrials.gov: #NCT05566808. The fresh frozen cartilage sheets (Profile®) were provided by MTF biologics. Objective assessment to evaluate warping and resorption of the cartilage was achieved by measuring the differences of values (deviation angle, nasofrontal angle, total facial convexity, nasofacial angle, and nasolabial angle) obtained at 6 months and 12 months postoperative follow-up on standard 2D photos (Δ= ∣measurement6-measurement12∣). Subjective assessment was measured by the Face-Q assessment filled out by patients.RESULTS: A total of 50 patients were included. Objective assessment: In the control group, the changes in the deviation angle, nasofrontal angle, total facial convexity, and nasolabial angle were all greater than in the CCA group. The differences in the deviation angle (P=0.02) and nasolabial angle (P=0.02) at 6 and 12 months were statistically significant. Subjective assessment: In the CCA group, the mean score of Satisfaction with Nose improved from 33.82 preoperatively to 65.73 six months postoperatively (P=0.001) and 48.25 twelve months post-operatively (P=0.007). The mean score of Satisfaction with Nostrils increased from 44.32 preoperatively to 70.55 six months postoperative (P=0.037) and 62.0 twelve months postoperatively (P=0.3). The mean score of satisfaction with overall facial appearance changed from 39.41 preoperatively to 61.18 six months postoperatively (P=0.010) and 39.52 twelve months post-operatively (P=0.98). In the control (ACC) group, the mean score of Satisfaction with Nose improved from 49.25 preoperatively to 79.50 six months post-operatively (P=0.202) and 53.34 twelve months postoperatively (P=0.083). The mean score of Satisfaction with Nostrils increased from 49.25 preoperatively to 71.75 six months postoperative (P=0.346) and 61.63 twelve months postoperatively (P=0.639). The mean score of satisfaction with facial appearance overall was 64.27 preoperatively, 82.0 six months post-operatively (P=0.010), and 58.67 twelve months post-operatively (P=0.908). None of the score differences between the CCA and control groups were statistically significant (P>0.05).CONCLUSION: Non-terminally sterilized fresh frozen CCA is a useful, safe, and reliable source of cartilage graft in reconstructive and cosmetic rhinoplasty in comparison to ACC. It is aseptic, readily available, previously tailored, and free of donor site complications. Patients reported satisfactory results. Future prospective studies with larger sample sizes, longer follow-up time, or focusing on a [missing text.]

PMC

The Lancet Regional Health: Western Pacific

PMC10388838

7-27-2023

10.1016/j.lanwpc.2023.100869

Examining real world quality of care for Australia’s First Peoples presenting with chest pain

Williams Trent D., Ngo Doan T.M., Sverdlov Aaron L.

Australia’s First Peoples, Aboriginal and Torres Strait Islanders (Indigenous Australians), continue to face ongoing health, social and economic disadvantage when compared to non-Indigenous people, resulting in adverse health outcomes.1 This disadvantage is across the spectrum of health conditions and contributes to many chronic cardiovascular, respiratory, metabolic (including diabetes), mental health, and chronic kidney diseases. This disadvantage results in 67% of Indigenous people having at least on serious chronic health condition.2 The problem is particularly profound in cardiovascular disease (CVD)—it continues to be one of the leading causes of mortality in Australia, with ischaemic heart disease (IHD) remaining the leading cause of mortality in Australia’s First Peoples. Alarmingly young Indigenous people have higher mortality rates from IHD compared to non-Indigenous people.3 Furthermore, mortality rates from CVD for Indigenous Australians are 1.6 times higher than for non-Indigenous Australians. At the same time Indigenous Australians with CVD have lower access to specialised care than non-Indigenous Australians.4 This disparity is even higher outside metropolitan areas: the rates of presentation with CVD as the principal diagnosis is 2.3 times higher among as well as 1.6 times higher among people from remote or very remote areas as compared to major cities.5 Thus, any examination and insight into this inequality of CVD care is of the upmost importance.It is established that the further a person lives from metropolitan centre the greater their risk of an adverse outcome from cardiovascular disease.6 However, there are limited data regarding readmissions and mortality in rural patients. Data regarding cardiovascular outcomes of Indigenous people have largely been driven by large government agencies and peak bodies. In this context, Dawson et al.7 provides meaningful relevant real world data in a timely examination of this issue of critical importance.Clearly prescribed treatment pathways, guidelines and consensus statements exist within cardiology to guide clinical care, including acute coronary syndrome. Identifying any causes of deviation from accepted benchmarks and pathways remains critical to addressing treatment disparity.4 In this issue of the journal, Dawson et al.7 describe epidemiology and care quality for Indigenous Australians presenting to hospital via emergency medical services with chest pain in the state of Victoria in Australia. We thank and acknowledge these authors long term commitment to highlighting health inequality. This study is particularly noteworthy for several important findings, with the key one being the demonstration that age-standardized incidence rates for chest pain were 2.73-fold higher overall for Indigenous people (3128 vs. 1147 per 100,000 person-years)–this difference being particularly striking for younger patients, women, and those residing in outer regional areas. Importantly, adherence to care quality and process measures were lower for attendances involving Indigenous people, and alarmingly, these patients were not seen within accepted clinical time benchmarks and were less likely to be transferred to a PCI capable hospital. Unsurprisingly, these data resulted in long-term mortality being observed to be higher among Indigenous Australians following discharge.This paper is the first to examine prehospital care via ambulance in Victoria at a population-level. As such it provides important data to guide the urgent improvements required to address cardiovascular treatment disparity in Indigenous patients. Whilst the higher mortality data has been described, this paper provides important and much needed data in describing the higher rates of EMS reattendance for chest pain for Indigenous Australians following discharge from hospital. These data represent an ideal opportunity to address primary and secondary prevention strategies, with a focus on culturally appropriate education to address quality of care for Indigenous patients.Despite the rigorous methodology in this important work7 there are some notable limitations: the authors acknowledge that in this retrospective observational cohort study the study setting (Victoria) has a more metropolitan population with lower Indigenous population than other Australian state, limiting broad generalisation. Given the nature of the study and reliance only on administrative datasets, granular clinical data, such as presence of underlying risk factors, medication prescribing and diagnostic test results were not available. All these may have a meaningful impact on readmission and mortality data8 and provide valuable explanations9 for some of the differences observed in the study by Dawson et al.7 Further research taking these issues and parameters into account would be helpful in defining appropriate management of cardiovascular risk. This is particularly important due to high prevalence of modifiable cardiovascular risk factors observed in Indigenous populations. Any work that focuses on models of care that address this would make an important contribution to closing the gap in life expectancy: currently there is a paucity of data of successful culturally appropriate interventions, however some are showing promise.10The overall findings of the study should also give pause for thought about how we are addressing health outcomes for our Indigenous peoples. There is still an approximately 10-year gap in life expectancy in Australia between Indigenous and non-Indigenous members of the community, with cardiovascular disease being the leading cause of this disparity. Currently there is a lack of clinical pathways to guide healthcare workers to help close this gap. Despite the obvious benefits to developing such pathways, barriers to the development and implementation of these pathways are multifaceted and complex. These barriers include, heterogeneity of comorbidity presentation, distrust of current clinical models of care, early onset of chronic disease, as well as geographical limitations to receiving optimal healthcare. Involving indigenous health care workers at every stage of healthcare planning and delivery is essential to achieving improved healthcare outcomes.11We congratulate the authors for their important contribution to improving cardiovascular outcomes of Indigenous patients.7 The study is conducted in one state in Australia, yet it has wide national and international implications as it further supports an urgent need to address gaps and disparities in health care that Indigenous and other ethnic/cultural minority populations experience in many countries worldwide. Whilst we continue to document this, the onus must be on all of us to translate this data into meaningful clinical actions, solutions and change, guided by the input from and participation of the Indigenous people to improve health outcomes in our communities.ContributorsTrent D. Williams: Writing—original draft. Doan Ngo: Conceptualization; Writing—review & editing. Aaron Sverdlov: Conceptualization; Supervision; Writing—review & editing. All authors contributed equally.Declaration of interestsNo conflicts of interest declared.

PMC

World Journal of Gastroenterology

PMC10768411

12-21-2023

10.3748/wjg.v29.i47.6165

Liver decompensation after rapid weight loss from semaglutide in a patient with non-alcoholic steatohepatitis -associated cirrhosis

Peverelle Matthew, Ng Jonathan, Peverelle James, Hirsch Ryan D., Testro Adam

There is rapidly increasing uptake of GLP-1 (glucagon-like peptide-1) agonists such as semaglutide worldwide for weight loss and management of non-alcoholic steatohepatitis (NASH). remains a paucity of safety data in the vulnerable NASH cirrhotic population. We report herein the first documented case of liver decompensation and need for liver transplant waitlisting in a patient with NASH-cirrhosis treated with semaglutide. Rapid weight loss led to the development of ascites and hepatic encephalopathy and an increase in the patients Model for Endstage Liver Disease-Na (MELD-Na) score from 11 to 22. Aggressive nutritional supplementation was commenced and the semaglutide was stopped. Over the following months she regained her weight and her liver recompensated and her MELD-Na decreased to 13, allowing her to be delisted from the transplant waitlist. This case serves as a cautionary tale to clinicians using semaglutide in the cirrhotic population and highlights the need for more safety data in this patient group.

Core Tip: Patients with NASH cirrhosis who lose weight rapidly with semaglutide are at risk of liver decompensation. This complication requires the immediate cessation of semaglutide and aggressive nutritional rehabilitation with supplemental protein feeds and micronutrients. Restoration of lost weight can lead to liver recompensation; however, consideration of liver transplantation should be given to patients who fail to respond to treatment.TO THE EDITORWe read with interest the systematic review and meta-analysis by Zhu et al reporting on the efficacy and safety of semaglutide in patients with non-alcoholic fatty liver disease (NAFLD). Analyzing three randomized control trials involving 458 patients, they found that semaglutide was effective at improving histologic and radiologic markers of non-alcoholic steatohepatitis (NASH) activity but not histologic fibrosis. The risk of serious adverse events was similar compared to placebo, and importantly, no cases of hepatic decompensation occurred.We herein present a case of liver decompensation in a patient with NASH cirrhosis after the use of semaglutide. A 68-year-old female with compensated NASH cirrhosis [Model for Endstage Liver Disease-Na (MELD-Na) score 11] was prescribed semaglutide 2.4 mg once weekly to manage her diabetes and obesity. Her other medications at the time included salbutamol for asthma. The semaglutide led to 10 kg weight loss (11% body weight) within 8 wk of treatment before it was stopped. After approximately 8% body weight loss she developed new onset hepatic encephalopathy (HE) requiring the use of lactulose and rifaximin. She also developed ascites requiring diuretic therapy (spironolactone 100 mg and frusemide 40 mg; further increases limited by postural hypotension) and two large-volume paracenteses. Patient adherence to prescribed medications was confirmed by her family during this time. Due to her rapid weight loss, her semaglutide was stopped. Despite stabilization of her weight, she continued to decompensate and her MELD-Na continued to rise (Figure 1). On referral to our service her MELD-Na was 22 (bilirubin 40 µmol/L, creatinine 44, international normalized ratio 1.6, Na 128). Investigations for alternate causes of decompensation including infection, alcohol consumption, hepatocellular carcinoma and portal vein thrombosis were negative. We concluded her liver decompensation was most likely secondary to semaglutide-induced rapid weight loss and malnutrition. She was commenced on high energy and high protein supplementation consisting of 60 g Sustagen twice-daily and micronutrient replacement with thiamine. She also underwent assessment for liver transplantation. Over the following 3 mo, she was reviewed each month by a transplant hepatologist and dietitian to assess her clinical progress, nutritional intake and adherence to treatment. Over this period, she managed to regain 5 kg (6%) of her ideal body weight, and this was associated with an improvement in her ascites and HE and a reduction in MELD-Na to 19 (Figure 1). By 6 mo her weight had returned to baseline, she no longer required abdominal paracentesis and her MELD-Na was 13. She was de-listed from the transplant waitlist and remains compensated at last follow-up.Figure 1Patient’s weight change with semaglutide use and MELD-Na score over time.Our case highlights the potential risk of rapid weight loss with semaglutide in the vulnerable NASH cirrhosis population. In our case, the rapidity of weight loss was significantly greater compared to the studies included in Zhu et al’s meta-analysis (10 kg after 8 wk vs 6.5 kg after 48-72 wk). Loomba et al’s study, included within the meta-analysis, involved patients with NASH cirrhosis and did not report any cases of hepatic decompensation. Of note, our patients pre-semaglutide MELD-Na score was higher (11 for our patient vs. 7.6 in the study group), potentially conferring a greater predisposition to decompensation. It is interesting to note that despite stabilization of her weight after stopping semaglutide, our patient continued to decompensate until she was reviewed at our tertiary centre, as illustrated in Figure 1. Rapid weight loss is an established precipitant of hepatic decompensation in the post bariatric surgery population, with pathophysiological mechanisms thought to involve endogenous free-fatty acid oxidative damage, mitochondrial dysfunction and gut dysbiosis leading to hepatic inflammation and fibrosis[4-6]. However, it should be noted that decompensation has generally occurred later (up to 5 years post-surgery) and degree of excess weight loss (i.e. the amount of weight above the ideal body weight) was up to 110%. Furthermore, the use of glucagon-like peptide 1 agonists such a semaglutide causes delayed gastric emptying, which may impact the absorption of concomitantly administered oral medications and therefore their efficacy. This may have contributed to our patients’ diuretic-refractory ascites. In patients with liver cirrhosis, it is therefore important to consider inadequate absorption of medical therapies as a contributor to failure to respond to standard treatment.Clinicians should consider the use of semaglutide cautiously in patients with underlying NASH cirrhosis and should strictly adhere to the prescribing information and dose escalation protocols as recommended and consider using a lower dose of the drug. Failure to follow strict dose escalation protocol may lead to significant gastrointestinal side effects including nausea and vomiting, which may precipitate weight loss and decompensation. Furthermore, clinicians must exercise a low threshold for cessation should weight loss occur rapidly (≥ 1% of body weight/week) or signs of liver decompensation develop.

PMC

International Medical Case Reports Journal

PMC10198171

5-15-2023

10.2147/IMCRJ.S406901

19 Months Toddler with a Giant Oral Capillary Hemangioma, a Case Report

Kabagenyi Fiona, Anena Sandra Petti, Seguya Amina

AbstractHead and neck vascular tumors are common in children. Capillary hemangiomas are often easily confused with pyogenic granulomas due to histopathological resemblance. Furthermore, predisposing factors to pyogenic granulomas include an existing hemangioma, which may be co-existing entities. Surgical excision of large unsightly tumors causing functional deficits is a feasible management option. We report a case of a rapidly growing oral lesion in a toddler with feeding difficulties and anemia. It triggered a diagnostic dilemma as it was clinically consistent with a pyogenic granuloma but histologically diagnosed as a capillary hemangioma. It was successfully excised with no recurrence after 6 months.

IntroductionThe International Society for the study of Vascular Anomalies (ISSVA) classifies vascular lesions into vascular malformations and vascular tumors.1–3 Vascular tumors include congenital hemangiomas, infantile hemangiomas, and pyogenic granulomas.4 Hemangiomas are further pathologically classified as capillary, cavernous, or mixed hemangiomas.5,6 Unique clinical characteristics exist across the differential diagnoses of vascular tumors.7 However, histopathological resemblance between capillary hemangioma and pyogenic granuloma may cause diagnostic challenges. Fifty-six percent of lesions classified as capillary hemangiomas were found to be true pyogenic granulomas.8 In fact, the synonym for pyogenic granuloma is a lobular capillary hemangioma.9 This further complicates the tailored management of vascular tumors. Clinical judgment to differentiate between the two entities may contribute to initial management option. Although both capillary hemangiomas and pyogenic granulomas are common lesions in the head and neck region, which can affect the oral cavity,9,10 their natural history and predisposing factors differ.2,4,7,11 Capillary hemangioma appears gradually within the first year of life2 while the pyogenic granuloma typically appears rapidly after 1 year of life.4 Predisposing factors of capillary hemangioma include being female and Caucasian and born with low birth weight6 while those of pyogenic granulomas include having hemangiomas, dermatologic eczematous lesions, and trauma.12 These should be looked out for. Noteworthy, 77% of those with pyogenic granulomas may have no predisposing factor.11 Examination of congenital hemangiomas may show expansion in color alteration on crying, which is not the case in pyogenic granulomas.7Variable treatment options such as watchful monitoring, pharmacological and surgical options are given in both capillary hemangiomas and pyogenic granulomas.12–15 Surgical management is indicated for lesions causing functional deficits and causing complications such as ulcers and bleeding, periorbital sight threatening lesions, airway obstructive lesions, and threat to cosmesis.12–15 A 0–5% recurrence rate is reported following excision of pyogenic granulomas.12,13,16We present an interesting case of a young girl with a rapidly growing oral mass originating in the left floor of mouth, which was referred to our Paediatric Ear, Nose, and Throat (ENT) clinic from the Paediatric Oncology Department at Mulago National Referral Hospital. To the best of our knowledge, this is the first report in the literature of a giant capillary hemangioma arising from the floor of the mouth and postulated to cause anemia.Case ReportA 19-month-old female, otherwise healthy, presented with a month’s history of an oral mass. Her mother noticed a small oral mass that started growing from the left underside of the tongue. Initially, it did not affect her speech or feeding. She was given antibiotics from drug shops during the first fortnight with minimal improvement. In the next 2 weeks, the mass rapidly increased in size, covering more than two-thirds of her mouth, pushing the tongue to the side. She could no longer close her mouth, had trouble articulating words and was drooling all the time. However, no change in voice, stridor, or difficulty in breathing were reported. Due to her continued struggle to feed, her mother resorted to frequent pureed feeds. No choking or coughing episodes on feeds were reported. The mass was smelly, had a whitish coating with occasional contact bleeding. Save for the open mouth, she slept well. There was no history of fever, trauma, or other swellings or discolorations on the rest of the body or bleeding tendencies. Other medical, surgical, and family history were unremarkable.On clinical exam, she was well-nourished, afebrile but very irritable. She had facial symmetry with no stigmata for any syndrome. She had a normal cry, with moderate anterior drooling and a large oral mass (5*4*3 cm) arising from the left floor of mouth, which occluded three-quarters of her mouth (Figure 1A). It was a firm, non-fluctuant, oval mass coated with whitish debris on the surface and a pink to purple discoloration on the undersurface. Her tongue was mobile, free from the mass, and pushed to the right corner of the mouth. No petechiae or ulcers were seen. The ears, nose, neck, and other systemic examinations were normal. Figure 1Intra operative view; (A) Showing extensive lesion (yellow arrow) pushing tongue (black arrow) to the right. (B) Showing sessile base of oral lesion in the floor of mouth (yellow arrow showing oral lesion, black arrow showing tongue). (C) Showing the excised lesion (yellow arrow).After obtaining informed consent for an excisional biopsy, preoperative blood work up and imaging were done. Serum electrolytes, leukocytes and platelet counts were within normal ranges, but the hemoglobin level was 7 g/dl. Blood was therefore booked for intraoperative and post-operative transfusion after grouping and cross matching. The COVID-19 PCR was negative. A contrasted Computerized Tomography (CT) scan of the head and neck showed a regular mass in the left oral cavity that took up contrast. Fat planes in floor of mouth and tongue were intact. Preoperative Paediatric Anesthesiologist’s review was done for airway management plan.Intraoperatively, successful nasal intubation was performed. The easily friable lesion had a sessile base extending from 1 cm posterior to the frenulum, to the retro-molar trigone on the left with serpentine vascular vessels across the left floor of mouth (Figure 1B). The rest of the oral cavity was normal.The left lingual nerve and left submandibular duct were identified and spared. The lesion was completely enucleated (Figure 1C). Hemostasis was achieved with bipolar cautery and ligature ties. Blood loss was minimal. The defect in the floor of mouth was closed using interrupted absorbable sutures.Post-operatively, the child had blood transfusion with packed cells, oral antibiotics, analgesia, and oral care with saline rinses. She had mild hypoglossal paresis when she woke up but was able to feed with a graded diet 2-h post-operatively. She had an uneventful 24-h observation and was discharged the following day.Histology showed a diagnosis of capillary hemangioma secondarily infected (Figure 2). GLUT 1 testing is not locally available and thus was not done. Figure 2Under haematoxylin and eosin, x4 image shows proliferation of capillaries lined by normal epithelium and filled with blood, with a pyogenic membrane and a chronic inflammatory cellular filtrate.Follow-up at 2 weeks showed complete resolution of symptoms with normal tongue movement, and at 6 months she had normal oral cavity mucosa and tongue movement (Figure 3). Figure 3Pre-surgical view (A) Shows tongue pushed to the right (black arrow) by the oral lesion (yellow arrow). The post-surgical view (B) 6 months later shows healed floor of mouth (red arrow).The child’s parent provided informed consent for the case details and any accompanying images to be published, available upon request. Institutional approval was not required to publish the case details.DiscussionOral hemangiomas have been reported to include the gingiva,17 tongue,18–20 and hard palate,21–23 and to present more in females.6,24,25 This is similar to our case of a female child, with a lesion arising from the floor of mouth. There were no known predisposing factors to either capillary hemangioma or pyogenic granuloma. The clinical onset after 1 year of life, rapid growth, and tendency to bleed raised our suspicion of a pyogenic granuloma. To our surprise, histology came back as an infected capillary hemangioma.Paglial et al in their study of 128 children found that 5% of children with pyogenic granulomas had hemangiomas. Their average lesion size was 7mm, presenting for about 5 months on average with a 1–2-month delay before treatment.12 Our case did not seem to have a hemangioma before the current presentation, was more than 50mm in size, and progressed over a month with significant functional deficits, enough to render urgent surgical intervention. It is postulated to have caused significant anemia and required blood transfusion. Large lesions in hemangiomas that are greater than 10cm2 may lead to a 4% increase in the requirement for intervention and 5% increase in complication.26 Indications for surgery, though rare, include presence of ocular, airway, auditory, feeding complications, cosmetic problems, or even congestive cardiac failure.2 Our case had significant difficulty in feeding with occasional bleeding, and frequent drooling that was unsightly.Paglial et al found that shave excision and electrocautery were the most common treatment methods for pyogenic granulomas, followed by laser therapy. Other methods included punch excision, liquid nitrogen, and observation. Regression of the pyogenic granulomas was seen in 4.5% of patients that had no treatment, over 6–18 months.12 In a retrospective study, 80% of 408 patients with pyogenic granulomas had surgical complete excision, 19% were treated by curettage, shave excision, or cautery or a combination of these. The overall recurrence rate was 5% with shave excision, curettage, and cautery having a recurrence rate of 10% compared with 3.6% following excision and closure.13 Due to the large size of the lesion, complete surgical excision with electrocautery was used with no recurrence seen in our case.Histology remains the mainstay of diagnosis of vascular tumors. In one study where most cases were classified as capillary hemangiomas, 56% were in fact pyogenic granulomas.8 Another study utilizing GLUT-1 as a biomarker found that 34% of 77 biopsies for oral hemangiomas were true hemangiomas.9 Unfortunately, this biomarker is not locally available and thus was not done in our case, and our quest for answers remains. Luckily, this does not prognosticate clinical outcome. Biannual outpatient reviews will monitor any interval changes going forward.ConclusionAlthough vascular lesions of the oral cavity in children can be successfully observed, fast growing lesions that cause functional impairment and anemia should be considered for prompt surgical management to prevent further complications. Clinical correlation with histological findings is key in treatment planning.

PMC

Proceedings of the National Academy of Sciences of the United States of America

37579155

PMC10450430

10.1073/pnas.2305625120

A synergy between site-specific and transient interactions drives the phase separation of a disordered, low-complexity domain

Mohanty Priyesh, Shenoy Jayakrishna, Rizuan Azamat, Mercado-Ortiz José F., Fawzi Nicolas L., Mittal Jeetain

SignificanceThe RNA-binding protein TDP-43 is a component of neuronal inclusions associated with diseases such as amyotrophic lateral sclerosis, frontotemporal dementia, and Alzheimer’s disease. The C-terminal domain (CTD) of TDP-43 is a disordered, low-complexity domain which is aggregation-prone and undergoes liquid–liquid phase separation (LLPS). CTD contains a hydrophobic, conserved region (CR, aa:319-341) which is α-helical and critical for oligomerization and LLPS. Using multiscale simulations and experiments, we report a noncanonical mechanism of biomolecular recognition wherein transient interactions involving hydrophobic residues in CR flanking regions drive the phase separation of CTD through direct enhancement of site-specific, CR-mediated interactions. Our study uncovers rich, mechanistic insights into CTD phase separation which is essential for understanding TDP-43 function and pathology. TAR DNA-binding protein 43 (TDP-43) is involved in key processes in RNA metabolism and is frequently implicated in many neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. The prion-like, disordered C-terminal domain (CTD) of TDP-43 is aggregation-prone, can undergo liquid-liquid phase separation (LLPS) in isolation, and is critical for phase separation (PS) of the full-length protein under physiological conditions. While a short conserved helical region (CR, spanning residues 319-341) promotes oligomerization and is essential for LLPS, aromatic residues in the flanking disordered regions (QN-rich, IDR1/2) are also found to play a critical role in PS and aggregation. Compared with other phase-separating proteins, TDP-43 CTD has a notably distinct sequence composition including many aliphatic residues such as methionine and leucine. Aliphatic residues were previously suggested to modulate the apparent viscosity of the resulting phases, but their direct contribution toward CTD phase separation has been relatively ignored. Using multiscale simulations coupled with in vitro saturation concentration (csat) measurements, we identified the importance of aromatic residues while also suggesting an essential role for aliphatic methionine residues in promoting single-chain compaction and LLPS. Surprisingly, NMR experiments showed that transient interactions involving phenylalanine and methionine residues in the disordered flanking regions can directly enhance site-specific, CR-mediated intermolecular association. Overall, our work highlights an underappreciated mode of biomolecular recognition, wherein both transient and site-specific hydrophobic interactions act synergistically to drive the oligomerization and phase separation of a disordered, low-complexity domain.

Membraneless organelles, also referred to as biomolecular condensates, organize cellular biochemistry by sequestering functionally related macromolecular components from the bulk cellular environment (1–3). Experimental studies indicate that many of these organelles form via phase separation to create distinct subcompartments within either the nucleus or cytoplasm . These organelles include nucleoli, Cajal bodies, stress granules (SGs), and other ribonucleoprotein (RNP) granules . SGs, which primarily serve to sequester and halt the translation of mRNAs , recruit many distinct RNA-binding proteins (RBPs) and can form dynamically in response to physiological stress. In recent years, RBPs containing disordered, prion-like domains (e.g., FUS, hnRNPA2, hnRNPA1, TDP-43) have received considerable attention due to their involvement in SG assembly and maintenance . The prion-like, disordered regions (PrLDs) are typically enriched in polar residues (Ser, Thr, Asn, and Gln) and can undergo liquid–liquid phase separation (LLPS) in cells and as purified proteins in vitro via weak, multivalent interactions . Phase-separated droplets of prion-like domains can also mature to form fibril-like structures which may exert a toxic cellular effect.A constituent of SGs and other RNP granules that has received significant attention is TAR DNA-binding protein 43 (TDP-43), a primarily nuclear RBP that plays an essential role in the regulation of mRNA splicing . Mislocalization of TDP-43 to the cytoplasm due to chronic stress and/or in combination with mutations that disrupt its structure and interactions gives rise to the formation of ubiquitinated neuronal inclusions , a hallmark of amyotrophic lateral sclerosis (ALS). TDP-43 has a multidomain architecture consisting of an N-terminal folded domain that self-associates to form linear globular chains (12, 13), two RNA recognition motifs (RRMs) that together bind RNA , and a prion-like C-terminal domain (CTD) which is predominantly disordered (15, 16). Under in vitro conditions, the C-terminal domain readily phase separates in the presence of physiological concentrations of salt or RNA which is enhanced at low temperatures . Within the CTD, there is an evolutionarily conserved, hydrophobic sequence that takes on an α-helical structure (15, 16) and is flanked by disordered regions—IDR1, Q/N, and IDR2 (SI Appendix, Fig. S1). Homooligomerization through this conserved region (CR) makes a large contribution to phase separation of the CTD; changes in helical stability through either ALS-related or designed mutations (16–19) were shown to tune phase separation. In addition to CR, aromatic residues in the flanking regions (IDR1/2) were also shown to be critical for LLPS (20, 21) and fibrillation of CTD .A detailed bioinformatic analysis revealed the sequence conservation in IDR1/2 and the high conservation in the spacing of aromatic and hydrophobic residues . As shown in Fig. 1, TDP-43 CTD contains 8 phenylalanine (6 of them regularly spaced within the disordered flanking regions), 3 tryptophan, and 1 tyrosine. Both in vitro and in vivo experiments (17, 20, 21) point toward the essential nature of aromatic residues (F, W, Y) in driving the phase separation of TDP-43. While arginine-mediated interactions were found to be important for the condensate dynamics, neither electrostatics nor arginine-mediated interactions within the CTD were able to inhibit the LLPS of the TDP-43 reporter construct in vivo . While the importance of aromatic and charged residues in phase separation of TDP-43 CTD and other prion-like domains is well-established (23–26), the relative contributions of other residue types (e.g., aliphatic residues) to LLPS is less understood—previously viewed as playing a role in tuning the dynamics and solidification or aggregation but not in driving phase separation . Given the abundance of aliphatic hydrophobic residues within TDP-43 CTD—7 alanine, 5 methionine, and 2 leucine each in both the CR and the flanking disordered regions—their role in phase separation is important to define.Fig. 1.Sequence composition of the TDP-43 CTD. (A) Cartoon representation of the C-terminal domain of TDP-43 that contains aromatic and charged residues outside the CR (spanning residue 319–341) enriched in aliphatic residues. (B) TDP-43 CTD (aa: 267–414) sequence. Letters in green, red, and blue circles are aromatic, anionic, and cationic residues, respectively. The residues within CR are shown as orange circles.Significant advancements in the accuracy of enhanced sampling algorithms and atomistic force fields (28, 29) allow for an in-depth characterization of the conformational landscape of intrinsically disordered proteins (IDPs) and regions (IDRs) (30–33), which complements insights obtained from biophysical experiments such as NMR, SAXS, and FRET (34, 35). Further, coarse-grained simulations parameterized based on amino acid hydrophobicity scales (36, 37) have provided valuable insights into the relationship between sequence and phase behavior of IDPs/IDRs. Here, we employed a multiscale simulation strategy based on atomistic and coarse-grained simulations coupled with in vitro phase separation experiments and NMR spectroscopy to probe the relative contributions of prevalent hydrophobic (phenylalanine and methionine) residues in CR and flanking regions on TDP-43 oligomerization and phase separation. Our results provide a detailed picture of TDP-43 CTD phase separation that helps to understand its function and pathology in the context of neurodegenerative diseases.ResultsSingle-Chain Simulation Accurately Describes the Conformational Ensemble of TDP-43 C-Terminal Domain.The degree of compaction of disordered proteins at dilute concentrations positively correlates with their ability to undergo LLPS at higher concentrations (38, 39). Previously, we performed extensive characterization of small disordered fragments (six-fold increase in csat, indicating significant destabilization of the condensate. Next, the effects of methionine residues in the CR (5M→ACR) and disordered flanking regions (5M→AIDR) were tested by substituting them with alanine, keeping in mind that alanine has higher experimental helical propensity than methionine . Interestingly, mutational change in hydrophobicity by replacing methionine residues with alanine, both outside the CR (5M→AIDR) and inside the CR (5M→ACR), resulted in a lower phase separation propensity (~two-fold increase in csat) which indicates a favorable role for methionine residues in LLPS. Conversely, the substitution of methionines outside the CR with another aliphatic residue, leucine (5M→LIDR), shows a similar csat as WT, hinting that the contribution to phase separation may not be specific to methionine but that other large aliphatic (nonalanine) residues could substitute. Meanwhile, mutating all five methionine residues outside the CR region to tyrosine (5M→YIDR) displayed substantial enhancement of phase separation (>two-fold decrease in csat). Thus, even if methionine (and leucine) are important contributors to phase separation, their relative contribution is lower than that of tyrosine, an aromatic residue which is found to be crucial for LLPS of many IDPs, including the low-complexity domains of FUS and hnRNPA1 (23, 25). Importantly, these variants shed light on the residue-level determinants of CTD phase separation, which may be universal to other phase-separating prion-like domains. Overall, the variation in csat observed for CTD mutants suggests that phenylalanines in the flanking disordered regions and methionine residues present within and outside the CR region significantly contribute to the phase separation of CTD.Additional analysis of methionine residues via mutations using CG phase coexistence simulations suggests that methionines in both IDR1 (M307A and M311A) and IDR2 (M359A, M405A, and M414A) affect the phase behavior of CTD while substituting all methionine to alanine (10M→A) predicted to drastically weaken the phase separation compared to methionine variants examined above (SI Appendix, Fig. S9A). Positional mutational studies demonstrate that all individual methionine residues have contributions toward LLPS and collectively stabilize the condensed phase of TDP-43 CTD (SI Appendix, Fig. S9B). These computational results further support the importance of methionine residues, which have not been previously recognized as essential for LLPS.Phenylalanine and Methionine Residues Are Crucial for TDP-43 CTD Phase Separation In Vitro.To validate the observed trend in csat for WT CTD and its mutants observed in CG phase coexistence simulations, we tested the effect of these mutations on TDP-43 CTD phase separation in vitro by microscopy and droplet sedimentation assays (Fig. 4 and Methods). Droplet sedimentation assays provide a quantitative assessment of phase separation, wherein the concentration remaining in the supernatant after centrifugation of the suspended droplets provides a measure of the saturation concentration (csat) for the WT and variants that do not undergo rapid conversion into static aggregates. Under the given buffer conditions, phase separation occurs if the protein concentration is equal to or above its csat.Fig. 4.Phenylalanine and methionine residues are crucial in TDP-43 CTD phase separation in vitro. (A and B) Quantification of phase separation assay showing the protein remaining in supernatant after phase separation for WT and its variants measured from 0 to 300 mM NaCl. Standard deviations of three replicates are represented as error bars. (C) Saturation concentration (csat) for TDP-43 CTD WT and its variants are determined by measuring supernatant concentration at 300 mM NaCl. (D) DIC micrographs for WT TDP-43 CTD (40 μM) and designed variants at 300 mM salt. (Scale bar, 20 μm.)WT CTD did not undergo phase separation in the absence of salt and showed an enhanced propensity for droplet formation upon increasing salt concentration (Fig. 4A), as shown previously (16, 19). In contrast, 6F→A mutation impaired droplet formation up to 120 μM protein concentration across all salt concentrations (Fig. 4A). Similarly, 5M→ACR variant inhibits phase separation (csat > 120 μM). Hence, both mutants show much higher csat compared to WT (csat ~ 20 μM at 150 mM salt) (Fig. 4B). Notably, 5M→AIDR variant exhibited phase separation at or above 75 mM NaCl, with nearly two-fold higher csat than WT, but much lower csat than 5M→ACR (Fig. 4 A–C). Overall, the observed increase in csat for 6F→A and 5M→AIDR mutants is consistent with predictions from CG simulations, showing that both of these residues are important for CTD phase separation, despite being outside the CR. However, it is important to note that the effects of methionine residues within the conserved helical region are substantially greater than those outside, suggesting the dramatic enhancement of phase separation via helix–helix contacts stabilized through methionine residues in the CR. We note that this difference between 5M→ACR and 5M→AIDR is not captured in the CG model results (Fig. 3D), which highlights directions for future improvements. This may involve adding an extra bead for distinguishing protein backbone versus sidechain to capture site-specific interactions within helical structures and sequence-specific changes in secondary structure and its modulation by oligomerization.To test whether the importance of methionine residues generalizes to the alphabet of phase separation or only works for five evolutionarily conserved methionine positions within the flanking regions of CTD, 5 alanine residues within CTD flanks are mutated to methionine residues within the 5M→ACR mutant (5A→MIDR + 5M→ACR), boosting the total methionine content in the flanking regions from 5 total to 10 total. Interestingly, the resulting mutant has a much lower csat than 5M→ACR (~70 μM), showing that addition of methionine residues increases phase separation. DIC microscopy images confirm the formation of phase-separated droplets above 70 μM (SI Appendix, Fig. S10). However, in the context of wild-type CR, making the same substitution of alanine to methionine (5A→MIDR) results in the formation of non-moving round-shaped clusters at 150 mM NaCl and leads to irregularly shaped amorphous assemblies consistent with aggregation at 300 mM salt (Fig. 4D and SI Appendix, Fig. S11). Because this variant leads to rapid aggregation, we did not test the phase separation in the saturation concentration assay. The enhanced aggregation hints that the residues in the CTD sequence (especially methionine) are evolutionarily designed for an optimum number of occurrences. These two mutants further highlight the significance of methionine residues in modulating phase separation. A 5M→LIDR mutant showed almost no change compared to WT, whereas 5M→YIDR lowered csat by an order of magnitude (Fig. 4 A–C). These results are consistent with the predictions from the CG simulations (Fig. 3 D), highlighting the importance of aliphatic residues for phase separation along with the aromatic residues.Phenylalanine and Methionine Residues Are Important for CTD Self-Assembly.Finally, we evaluated the impact of these mutations inside and outside the CR that affect phase separation on the helical structure of the CR and its helix–helix contacts. NMR spectroscopy is a powerful technique that provides atomic resolution information on biomolecular structure and contacts. The 2D NMR spectrum is the fingerprint of a protein and provides direct information regarding the order/disorder of the protein as well as the formation of stable and transient contacts. Using NMR as a tool, we have previously established that TDP-43 CR α-helix self-assembly can be monitored as chemical shift perturbations (CSP) in CR residues upon increasing the protein concentration in conditions where TDP-43 CTD does not readily phase separate (i.e., low salt conditions) (Fig. 5A, WT CTD panel, ref. 16). To access the impact of mutations that alter TDP-43 CTD phase separation, we compared the 2D NMR spectrum of the variants with that of the WT CTD at 20 μM (a monomeric reference) and 90 μM (Fig. 5A). Interestingly, the resonances arising from residue positions in the CR for 5M→AIDR and 6F→A were nearly unperturbed compared to WT CTD at low protein concentration (20 μM), suggesting the α-helical structure of the monomeric CR is intact in these constructs. For WT CTD, we observed significant up-field 15N CSP at 90 μM for the CR residues corresponding to self-interaction and enhanced helicity (16, 54). However, the CSPs for CR residues in 5M→AIDR were reduced and were minimal in 6F→A compared to WT CTD (Fig. 5B), indicating disruption in the helix–helix assembly. Taken together, these data suggest that phenylalanine and methionine outside of the CR form contacts important in helix-mediated CTD oligomerization in the early stages of self-assembly preceding phase separation. Importantly, the substitution of these residues has no impact on CR helicity, consistent with the model that partial CR helical structure is stable and does not require contacts outside the CR.Fig. 5.Phenylalanine and methionine residues are crucial for TDP-43 self-assembly. (A) Overlay of 1H–15N HSQC spectra of the TDP-43 CTD at two concentrations 20 μM (red) and 90 μM (blue), showing chemical shift perturbations (arrows) associated with intermolecular interactions. The chemical shift deviations are minimal for 6F→A variant and reduced for 5M→AIDR variant, whereas 5M→ACR does not show chemical shift deviations up to 90 μM protein concentrations. The WT CTD and 6F→A spectra were recorded at 850 MHz, while the 5M→AIDR and 5M→ACR spectra were recorded at 600 MHz. (B) A comparison of 15N chemical shift differences (Δδ15N) for the CR of WT CTD versus its designed variants confirms the disruption of helix–helix interactions by these variants. (C) Secondary shifts (ΔδCα-ΔδCβ) of 5M→ACR mutant relative to WT CTD and helix-disrupting A326P show that 5M→ACR conserves the helix present in WT CTD.Previously, we showed that the helix-disfavoring M337V ALS-associated mutation disrupts helix–helix assembly, as does an engineered proline mutation M337P which is incompatible with helical structure . This observation prompted us to test whether the methionine residues play a critical role in helix–helix interactions beyond stabilizing the helix itself. To this end, we tested the 5M→ACR variant (that substitutes all five conserved region methionine positions to alanine—M322A, M323A, M336A, M337A, and M339A, see above). As expected, the two-dimensional spectral fingerprint at the CR was significantly perturbed by the substitution of the 5 methionine residues. In order to identify the CR residues, we carried out standard triple resonance NMR assignment experiments (including the HNN experiment, see Methods) and obtained backbone amide resonance assignments. Interestingly, substitution of all five conserved region methionine positions to alanine (5M→ACR) prevented assembly of the CR region, as little to no CSPs for the CR residues were observed upon increasing concentration (Fig. 5 A and B). Therefore, these data suggest the methionine residues in the CR are important for mediating helix–helix assembly probably due to the loss of methionine side-chain contacts. To test whether the mutations disrupt helicity, we calculated the secondary structure content of the CR in 5M→ACR. Secondary structure analysis was performed by comparing the experimental Cα and Cβ chemical shifts with their predicted random coil shifts, ΔδCα–ΔδCβ (55, 56), as we did previously for TDP-43 CTD WT and helix-enhancing mutants (16, 19). Surprisingly, our NMR data analysis revealed a significant population of helical structures based on residual 13C chemical shifts that are very similar to WT CTD. As a control, these values are also significantly higher than those in the helix-disrupting A326P variant (Fig. 5C). However, it should be pointed out that the secondary structure propensity of alanine residues at 322 and 323 positions are even more positive than those for methionine at these positions in WT CTD, possibly due to the higher helicity for polyalanine sequences . Still, the helicity was slightly smaller in other locations compared to WT CR, suggesting that these conserved methionines also form intramolecular contacts to stabilize the helix in TDP-43 CTD. Taken together, these data show that methionines are important for intermolecular contacts that mediate helical assembly and stabilize the helix.DiscussionWe adopted a hybrid simulation-based strategy coupled with in vitro experiments to identify the relative contribution of prevalent hydrophobic residues which modulate the phase separation of TDP-43 CTD. Our results indicate that the substitution of phenylalanine residues (6F→A) outside CR significantly reduces the phase separation of CTD. Along similar lines, F→S substitution within the CTD was previously shown to disrupt phase separation in vivo for a reporter TDP-43 construct and in vitro for full-length TDP-43 . TDP-43 CTD contains methionine residues which are equally distributed (five each) in the CR and the flanking regions. We observed that the substitution of methionines outside the CR (5M→AIDR) also weakens phase separation, while 5M→ACR mutant significantly disrupts phase separation.Further, reduced phase separation caused by removing the methionines inside the CR (5M→ACR) could be alleviated with the addition of methionine residues in the region flanking the CR (5A→MIDR + 5M→ACR). Along similar lines, M/V→A substitutions within the methionine-rich LC domain of Pab-1 were found to modulate its phase separation through an increase in the radius of gyration and demixing temperature . Additionally, methionines in the FUS RGG3 domain have been shown to make contacts with the FUS SYGQ low complexity domain within a cocondensed phase . In sum, methionine residues, surprisingly, substantially contribute toward interactions that drive the phase separation of CTD.It is important to consider the relative contribution of methionine towards phase separation compared to other aromatic and aliphatic residues. CG simulations and experimental data show that it has lower stabilizing contributions to phase separation than the aromatic residue—tyrosine (5M→YIDR). Substitutions of methionine with another aliphatic residue, leucine (5M→LIDR), yielded minor changes in phase separation compared to WT by experiment and simulation. Likewise, 5M→S mutation in IDR1/2 is shown to increase droplet dynamics without affecting its csat, while 5M→V behaved similarly to WT for a TDP-43 reporter construct under in vivo conditions . Our observations here provide direct evidence for the contribution of methionine to the thermodynamics of phase separation. Furthermore, our results suggest that other aliphatic residues except alanine may have similar contributions, expanding the molecular grammar of low-complexity domain phase separation.Our sequence alignment analysis over 93 homologs of TDP-43 CTD shows that the number of aliphatic residues (L = 2, I = 1, and M = 5) outside CR is mostly conserved and switched to another aliphatic residue (VLIM) in some homologs (e.g., L270, I383) (SI Appendix, Fig. S12). On the other hand, alanine, which is also aliphatic, is less conserved and tends to switch to or appear as a replacement for polar residues. Correspondingly, our in vitro experiments support the idea that alanine residues outside CR contribute less to LLPS than larger aliphatic residues. Although aliphatic residues may contribute to phase separation similarly based on their similar hydrophobicity and size, it is important to note that methionine has unique physicochemical properties that could broaden its role in phase separation .An important characteristic of methionine is its ability to undergo reversible oxidation (60, 61) which allows for the regulation of protein phase separation. This was probed in detail by the recent studies from the Tu and McKnight laboratories wherein the methionine-rich LC domain self-association of yeast ataxin-2 was demonstrated to be controlled by reversible methionine oxidation. Similarly, phase separation of TDP-43 CTD was shown to be disrupted in vitro due to the nonspecific oxidation of methionines in the presence of H2O2, an oxidizing agent, and restored via enzymatic reduction of methionine oxidation . However, these studies do not provide direct evidence of why methionine oxidation disrupts phase separation. Our findings based on explicit evaluation of csat for M→A variants directly attribute methionine’s role in CTD phase separation.Upon methionine composition analysis of 29 human RBP prion candidates , including ataxin-2, we found that one-third of those PrLDs contain at least 4% methionine (SI Appendix, Table S2, and Fig. S13). These observations suggest that the results reported in this study regarding the requirement of methionine residues in TDP-43 CTD phase separation could be general for other PrLDs. Interestingly, the cryo-EM structure of aggregated TDP-43 from the human brain of an individual affected by ALS with FTLD has a cluster of methionine and phenylalanine residues in the core (M307, M311, F313, M322, and M323) and numerous methionine-aromatic contacts can be seen at different locations. This implies that the presence of energetically stabilizing aliphatic-aromatic contacts may promote fibrillization from the condensed phase.Recently, it was shown that disordered regions flanking the binding interface could modulate the binding affinity between two transcriptional coactivators—CBP and NCOA, through transient hydrophobic interactions . Interestingly, our findings also lead us to propose a similar mode of biomolecular recognition wherein a synergy between site-specific (CR-mediated) and transient (IDR-mediated) interactions involving hydrophobic residues (phenylalanine/methionine) drive the oligomerization and subsequent phase separation of TDP-43 C-terminal domain (Fig. 6). Overall, these findings highlight the rich diversity of interaction modes which underlie protein–protein association and the formation of higher-order assemblies such as biomolecular condensates (68, 69).Fig. 6.LLPS of TDP-43 C-terminal domain is governed by synergistic interactions involving hydrophobic residues in both CR and flanking regions. Schematic highlighting the involvement of both phenylalanine (aromatic) and methionine (aliphatic) residues in driving the formation of the oligomeric intermediate enroute to LLPS of CTD. Both wild-type and 5M→LIDR (preserves hydrophobicity of IDR1/2) variant undergo oligomerization through the formation of transient IDR contacts and site-specific CR contacts. In contrast, the loss of hydrophobicity in 5M→AIDR, 6F→AIDR and 5M→ACR variants disrupts oligomerization and LLPS by weakening both IDR and CR contacts.MethodsRefer to SI Appendix for details. MD simulations were performed to predict the molecular interactions underlying the phase behavior of wild-type human TDP-43 CTD and relative changes in csat upon sequence mutations. Wild-type human CTD and its mutant variants were cloned from codon-optimized sequences into the pJ411 bacterial expression vector (N-terminal 6x-His tag), expressed in Escherichia coli and purified by affinity chromatography. DIC microscopy was used to qualitatively characterize the extent of phase separation of wild-type CTD and its mutant variants. To quantify phase separation in vitro, assays were conducted for protein samples with increasing salt concentration to determine csat. NMR experiments were performed to assess the impact of sequence mutations on CTD oligomerization and the helicity of the CR.Supplementary MaterialAppendix 01 (PDF)Click here for additional data file.

PMC

Advances in Radiation Oncology

PMC10316432

4-29-2023

10.1016/j.adro.2023.101228

Machine Learning for Predicting Clinician Evaluation of Treatment Plans for Left-Sided Whole Breast Radiation Therapy

Fiandra Christian, Cattani Federica, Leonardi Maria Cristina, Comi Stefania, Zara Stefania, Rossi Linda, Jereczek-Fossa Barbara Alicja, Fariselli Piero, Ricardi Umberto, Heijmen Ben

PurposeThe objective of this work was to investigate the ability of machine learning models to use treatment plan dosimetry for prediction of clinician approval of treatment plans (no further planning needed) for left-sided whole breast radiation therapy with boost.Methods and MaterialsInvestigated plans were generated to deliver a dose of 40.05 Gy to the whole breast in 15 fractions over 3 weeks, with the tumor bed simultaneously boosted to 48 Gy. In addition to the manually generated clinical plan of each of the 120 patients from a single institution, an automatically generated plan was included for each patient to enhance the number of study plans to 240. In random order, the treating clinician retrospectively scored all 240 plans as approved without further planning to seek improvement or further planning needed, while being blind for type of plan generation (manual or automated). In total, 2 × 5 classifiers were trained and evaluated for ability to correctly predict the clinician's plan evaluations: random forest (RF) and constrained logistic regression (LR) classifiers, each trained for 5 different sets of dosimetric plan parameters (feature sets [FS]). Importances of included features for predictions were investigated to better understand clinicians’ choices.ResultsAlthough all 240 plans were in principle clinically acceptable for the clinician, only for 71.5% was no further planning required. For the most extensive FS, accuracy, area under the receiver operating characteristic curve, and Cohen's κ for generated RF/LR models for prediction of approval without further planning were 87.2 ± 2.0/86.7 ± 2.2, 0.80 ± 0.03/0.86 ± 0.02, and 0.63 ± 0.05/0.69 ± 0.04, respectively. In contrast to LR, RF performance was independent of the applied FS. For both RF and LR, whole breast excluding boost PTV (PTV40.05Gy) was the most important structure for predictions, with importance factors of 44.6% and 43%, respectively, dose recieved by 95% volume of PTV40.05 (D95%) as the most important parameter in most cases.ConclusionsThe investigated use of machine learning to predict clinician approval of treatment plans is highly promising. Including nondosimetric parameters could further increase classifiers’ performances. The tool could become useful for aiding treatment planners in generating plans with a high probability of being directly approved by the treating clinician.

IntroductionRadiation therapy (RT) treatment planning for breast cancer focuses on reducing radiation exposure to healthy tissues (whole heart, left anterior descending coronary artery [LAD], lungs, and contralateral breast [CB]), while ensuring an adequate target coverage. Two phase 3 studies have shown significant toxicity reductions with intensity modulated RT (IMRT) compared with 3-dimensional (3D) conformal RT.1,2 Apart from regular C-arm linear accelerators, static beam IMRT for patients with breast cancer can also be delivered with TomoDirect, an IMRT modality delivered with TomoTherapy (Accuray, Madison, WI).3, 4, 5, 6In a standard clinical practice, treatment plans are generated by planners and presented to the treating clinician for approval. Often, the final approved plan is the product of an iterative procedure in which an initial plan is stepwise enhanced to best satisfy the clinician's requirements. If on the one hand this can be a process that can avoid human errors,7 it is also time-consuming and workload intensive.Automated planning has been proposed to enhance plan quality and reduce workload.8,9, 28 However, several studies with blinded plan comparisons have shown that clinicians do not always prefer the automatically generated plan.10, 11, 12 Recently, Cagni et al13 systematically investigated differences in plan scoring among planners and treating clinicians in a single department. Large differences in plan quality assessments were observed.In this study, we have investigated the ability of random forest (RF) or constraint logistic regression (LR) classifiers to use treatment plan dosimetry for correct prediction of clinicians’ plan evaluations for left-sided whole-breast RT (WBRT) with boost as approved without further planning to seek improvement or further planning needed. The basis of the study was treatment plans for previously treated patients. To enhance the statistical power of the study, for each patient the manually generated clinical plan and an automatically generated plan were included. For study purposes, the involved clinician retrospectively labeled in random order all clinical and automatically generated study plans as approved without further planning or further planning needed, while being blinded for type of applied plan generation (manual or automated).For both RF and LR, 5 different dosimetric feature sets (FS) were investigated (2 × 5 investigated classifiers in total) to assess dependence of prediction quality on selected plan parameters. Machine learning (ML) predictions for plans that were labeled “approved without further planning” were considered correct in case of a predicted probability P (approved without further planning) >.5.For each of the investigated 2 × 5 classifiers, nested cross-validation was used to establish both hyperparameters and assess model performance, using the same data set.14 Importance of included features for predictions was investigated to better understand in clinicians’ plan evaluations.To the best of our knowledge, this study is the first attempt of using ML with dosimetric plan parameters as input to predict clinicians’ plan evaluations. In a hypothesized future clinical application, a planner could then first assess the probability that the clinician would consider a generated plan approved. If this probability is low, the planner could then try to further improve the plan before presenting it to the clinician, thereby minimizing the time used by clinicians for plan evaluations.Methods and MaterialsPatient selection and treatment planningA total of 120 patients receiving adjuvant left-sided WBRT after breast-conserving surgery at the European Institute of Oncology (IEO) Institute between 2019 and 2020 were randomly selected from the institutional database. The study approved by the Ethical Committee of the IEO Institute (identification number UID2433). Institute (identification number UID2433). RT was delivered with TomoDirect in a TomoTherapy Hi-Art System (Accuray, Sunnyvale, CA).Clinical plans were manually generated with the VOLO treatment planning system (version 2.1.6; Accuray, Sunnyvale, CA), applying a jaw width of 2.5 cm, a pitch of 0.25, and modulation factors of 1.8 to 2.0 to keep the delivery time within the range of 10 to 15 minutes. Breast and tumor bed were contoured based on European Society for Therapeutic Radiation and Oncology guidelines for early breast cancer.15 Isotropic 5-mm expansions were added to create the corresponding planning target volumes (PTVs). Organs at risk (OARs) included left and right lung, CB, heart, and LAD.16 In line with the Radiation Therapy Oncology Group 1005 study protocol,17 40.05 Gy was delivered to the whole breast in 15 fractions over 3 weeks with a simultaneously integrated boost to the tumor bed that resulted in a total dose of 48 Gy. Dose objectives mainly followed those used in the previously mentioned protocol (Table 1).Table 1Dose-volume histogram constraints for clinical planning and recommended and maximum acceptable values for all considered targets and organs at riskTable 1Organ at riskIdealAcceptableHeartV16Gy 38 GyV90% >36 GyD50%D30%D0,03cc ≤46 GyD0,03cc ≤48 GyCIHID95% ≥45.6 GyD90% ≥43.2 GyPTV48.0Gy (boost volume)D5% ≤52.8 GyD10% ≤52.8 GyD0,03cc ≤55.2 GyD0,03cc ≤57.6 GyCIHIAbbreviations: CI = conformity index; HI = homogeneity index; PTV = planning target volume.Apart from obtained values for the constraints, obtained values for parameters in the table without recommended and maximum acceptable values were also used in this study.For each of the 120 study patients, automated plan generation was performed for the same planning computed tomography and structures as in the clinical plan. Autoplanning was performed with a for breast adapted version of the Guided Planning System10 in the RayStation TPS, version 11A (RaySearch, Stockholm, Sweden). This autoplanning module was not specifically tuned for generation of highest quality plans for the treatment approaches and traditions in the center where the included patients were treated, as comparison of autoplanning with manual planning was not a study aim (see Introduction section).Collected dataThe labeling of all 240 involved plans as approved without need for further planning to seek improvement or further planning needed was performed by a senior radiation oncologist with more than 20 years of experience in breast cancer treatment (IEO).The following 24 dosimetric plan parameters were gathered for all 240 plans: D0,03cc, D30%, D50%, D95%, conformity index (CI; defined as the ratio between the region of interest volume covered by the 95% isodose and the total patient volume covered by ≥95% of the prescribed dose), and homogeneity index (HI; defined as D95%/D5%) for the whole breast excluding boost PTV (PTV40.05Gy); D0.03cc, D5%, D95%, CI, and HI for the boost PTV (PTV48.0Gy); V20Gy, V8Gy, and Dmean for the heart and Dmean and D1% for the LAD; V16Gy, V8Gy, V4Gy, and Dmean for the left lung; V4Gy for the right lung; and D0,03cc, D5%, and Dmean for CB. See Table 1 for an overview.Apart from the previously mentioned dosimetric plan parameters, composite dosimetric scores (CPS) were collected for OARs and PTVs, as previously proposed by IEO investigators.18 In this scoring system, the involved 5 OARs and 2 PTVs each get a score of 0, 0.5, or 1, depending on the fulfilment of planning constraints reported in Table 1: 1 point was given if all dose constraints were within recommended values, 0.5 point if at least 1 dose constraint was respected, and no points otherwise. Parameters in Table 1 without acceptable values were not considered in this scoring system. Before classifier trainings, the 240 values for each dosimetric feature were first centered around zero by subtracting the mean value, and the values were scaled to unit variance.The full data set consisted of 240 rows (one for each plan) and 32 columns (24 dosimetric parameters, 7 composite scores, and the clinician's binary score [approved or not]). The Python scikit-learn library19 was used for all data analyses and model developments.ML models and trainingThe investigated 5 dosimetric FS used to train both the RF and LR classifiers (2 × 5 classifiers in total) consisted of the following:•FS1: 24 dosimetric parameters defined in the Collected Data section•FS2: 7 CPS defined in Collected Data section•FS3: 24 differences between dosimetric parameters and their objectives, as indicated in the “Ideal” column of Table 1. If this was missing (eg, left lung Dmean), the original value was maintained.•FS4: FS2 + FS3•FS5: FS2 + FS1For each of the 2 × 5 investigated classifiers (RF and LR, both combined with FSi with i = 1-5), model building was performed with nested cross-validation with an outer and an inner loop. The applied procedure is extensively described in Talbot14 and schematically presented in Fig. 1. Here a brief summary is presented: for the outer loop, the 240 available plans were equally and randomly distributed over 10 folds of 24 plans. Each of the 10 folds then served as a test set for model training based on the remaining (240-24) plans. However, before such a training, an inner-loop 5-fold cross-validation was performed to establish model hyperparameters such as the number and type of trees for RF and solver, penalty, and regularization strength for LR. Inner-loop cross-validations were performed using only the training set of the corresponding outer loop (Fig. 1). For each the 2 × 5 classifiers, the 10 outer-loop models were used to assess the prediction performance. The inner-loop models served only for establishment of model hyperparameters.Figure 1Schematic explanation of the applied nested cross-validation, consisting of 10-fold outer-loop cross-validation and 5-fold inner-loop cross-validation. Each of the 10 outer-loop model buildings is preceded by a paired 5-fold inner-loop cross-validation to establish hyperparameters using only the training patients of the corresponding outer-loop model. Nested cross-validation was performed for each of the 2 × 5 classifiers investigated in this study. For each classifier, the 10 outer-loop models were used to evaluate prediction performance.Figure 1The function “GridSearchCV” of the Python scikit-learn library19 was used in the inner loops to select optimal hyperparameters. For each of the 2 × 5 classifiers, prediction performance was assessed by calculating mean values and standard errors of the accuracy, area under the receiver operating characteristic curve (AUC), and Cohen’s kappa coefficient (κ)20 for the 10 outer-loop models.Landis and Koch21 proposed the following classification κ: κ .5 was considered as a correct prediction. Likewise, for plans with a label “further planning needed,” a probability <.5 was considered correct. The use of 5 different FS allowed us to investigate the sensitivity of RF and LR for the choice of applied dosimetric features. FS1 and FS3 both consisted of 24 dosimetric parameters that could be directly calculated from the dose distributions. The much smaller FS2 (7 parameters) contained for each of the 7 involved anatomic structures a composite score that was derived from related dosimetric parameters, as previously proposed.18 FS4 and FS5 were the largest FS, consisting of FS2 + FS3 and FS2 + FS1, respectively. For FS4, accuracy, AUC, and Cohen's κ for generated RF/LR models for prediction of approval without further planning were rather high: 87.2 ± 2.0/86.7 ± 2.2, 0.80 ± 0.03/0.86 ± 0.02, and 0.63 ± 0.05/0.69 ± 0.04, respectively. RF performance was basically independent of the applied FS (Table 2), meaning that FS2 with only 7 features performed as well as FS1 and FS3 with 24 features and FS4 and FS5 with 31 features. For LR, a dependency on FS was observed, with the large FS4 and FS5 overall performing the best. The possibility of using nonlinear combinations of available dosimetric features in RF modeling could make up for reduced availability of dosimetric features in FS2.Clinicians’ plan evaluations are not only based on plan parameters but consider also the full 3D dose distribution. This study shows that not explicitly considering the full 3D dose in the 2 × 5 investigated classifiers could still result in high-quality predictions of clinicians’ plan evaluations.As mentioned previously, all 240 study plans were retrospectively labeled by the clinician as “approved without further planning” or “further planning needed.” Apart from this labeling, the clinician also assessed plan acceptability. Although only 71.5% of plans were labeled as “approved without further planning,” the clinician found 100% of plans in principle acceptable for treatment. Apparently, for a large number of plans the clinician had a wish to further explore plan improvement even though the plan was in principle acceptable. This reflects the complex decision making that was modeled in this article; the label “further planning needed” was not related to unacceptable constraint violations but to more subtle desires for plan improvement.For all investigated 2 × 5 classifiers, PTV40.05Gy was by far the most important anatomic structure for predictions, reflecting the importance given to it by the clinician (Fig. 2), with D95% as the most important parameter for most classifiers having PTV40.05Gy D95% as feature (Fig. 3).In this study, all 240 available labeled treatment plans could be used for training, validation, and testing (classifier performance assessment) due to the applied nested cross-validation (Talbot,14 Fig. 1). With this procedure, inner-loop cross-validation was used for establishment of hyper parameters, to be used for model trainings in the outer-loop cross-validation.A limitation of this study is that the analyses were performed for a single clinician. Generalizability of these prediction models for use by more clinicians is a topic of future research. The endeavor of developing a single model for all clinicians in the center could result in higher consistency of the treatments delivered in the study center. Another limitation is the lack of nondosimetric patient data in the performed analyses, including age, performance status, previous or concomitant treatments, surgery results, and comorbidities. Future investigations will include such factors that could further enhance the reliability of the predictive models.ConclusionWe have investigated several ML approaches for prediction of clinician approval of treatment plans for left-sided WBRT plus boost based on plan dosimetry. Results are encouraging for future workflows in which treatment planners will only present treatment plans to treating clinicians if they have a high probability of being directly approved, that is, without an additional round of planning and plan evaluation.

PMC

bioRxiv

37293062

PMC10245734

5-18-2023

10.1101/2023.05.18.541300

Heavy Metal Pollution From a Major Earthquake and Tsunami in Chile Is Associated With Geographic Divergence of Clinical Isolates of Methicillin-Resistant

Martínez Jose RW, Alcalde-Rico Manuel, Jara-Videla Estefanía, Rios Rafael, Moustafa Ahmed M., Hanson Blake, Rivas Lina, Carvajal Lina P., Rincon Sandra, Diaz Lorena, Reyes Jinnethe, Quesille-Villalobos Ana, Riquelme-Neira Roberto, Undurraga Eduardo A., Olivares-Pacheco Jorge, García Patricia, Araos Rafael, Planet Paul J., Arias César A., Munita Jose M.

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority pathogen listed by the World Health Organization. The global spread of MRSA is characterized by successive waves of epidemic clones that predominate in specific geographical regions. The acquisition of genes encoding resistance to heavy-metals is thought to be a key feature in the divergence and geographical spread of MRSA. Increasing evidence suggests that extreme natural events, such as earthquakes and tsunamis, could release heavy-metals into the environment. However, the impact of environmental exposition to heavy-metals on the divergence and spread of MRSA clones has been insufficiently explored. We assess the association between a major earthquake and tsunami in an industrialized port in southern Chile and MRSA clone divergence in Latin America. We performed a phylogenomic reconstruction of 113 MRSA clinical isolates from seven Latin American healthcare centers, including 25 isolates collected in a geographic area affected by an earthquake and tsunami that led to high levels of heavy-metal environmental contamination. We found a divergence event strongly associated with the presence of a plasmid harboring heavy-metal resistance genes in the isolates obtained in the area where the earthquake and tsunami occurred. Moreover, clinical isolates carrying this plasmid showed increased tolerance to mercury, arsenic, and cadmium. We also observed a physiological burden in the plasmid-carrying isolates in absence of heavy-metals. Our results are the first evidence that suggests that heavy-metal contamination, in the aftermath of an environmental disaster, appears to be a key evolutionary event for the spread and dissemination of MRSA in Latin America.

IntroductionMethicillin-resistant Staphylococcus aureus (MRSA) infections are a major global public health problem, with the World Health Organization considering the development of new therapeutic alternatives against MRSA a top priority (WHO, 2017; Murray et al. 2022). MRSA has distinctive epidemiologic patterns with specific genetic lineages restricted to particular geographical areas.(Arias et al. 2017; Challagundla et al. 2018). The widespread dissemination of MRSA over time is often driven by “waves” of clonal replacements, where novel lineages replace predominant regional clones (Planet et al. 2017). While the underlying factors driving clonal replacement of MRSA remain unclear, this phenomenon has been widely reported in different parts of the world, including Latin America. One of the most successful MRSA lineages in Latin America has been a healthcare-associated, ST5-SCCmecI lineage, which was first described in 1998 in Chile and Argentina (designated Chilean-Cordobes clone, [ChC])(Medina et al. 2013; Martínez et al. 2019). By the end of the 2000s, however, this lineage was almost completely replaced by a community-acquired MRSA (CA-MRSA) clone identified as USA300-Latin American variant (USA300-LV) in Colombia and Ecuador. In contrast, the ChC clone has remained largely dominant in countries like Chile and Peru, located on the South Pacific coast of Latin America (Aires de Sousa et al. 2001; Reyes et al. 2009; Arias et al. 2017).USA300-LV belonged to an ST8-SCCmecIVc lineage that was closely related to the CA-MRSA USA300 clone (ST8-SCCmecIVa) responsible for a major epidemic of CA-MRSA infections in the United States in the late 1980s and 1990s (Planet et al. 2015). Interestingly, the evolutionary divergence observed between these lineages was associated with the independent acquisition of two horizontally-acquired genetic elements: the Arginine Catabolic Mobile Element (ACME) in the North American USA300 clone, and the Copper (Cu) and Mercury (Hg) resistance mobile element (COMER) in South American lineage (Planet et al. 2015). Of note, apart from the arginine metabolism machinery, ACME also harbored copX(B), a Cu resistance gene also observed in other successful MRSA clones (Saenkham-Huntsinger et al. 2021). Furthermore, previous studies have suggested a possible evolutionary advantage of acquiring heavy metal resistance traits in the emergence of new MRSA lineages (Kernberger-Fischer et al. 2018; Zapotoczna et al. 2018). These observations suggest that acquiring mobile genetic elements harboring heavy-metal resistance genes (HMRGs) might play a key role in the emergence and dissemination of successful MRSA clones. However, the possible underlying environmental causes driving the appearance and dissemination of MRSA lineages remain unclear.Studies involving non-pathogenic bacteria suggest environmental contamination with heavy metals promotes horizontal gene transfer of antimicrobial resistance genes and selects for organisms harboring plasmids that carry heavy metal resistance traits, which are frequently co-transferred with other antimicrobial resistance determinants (Xu et al. 2017; Rodgers et al. 2018; Zhang et al. 2018). In addition, extreme natural events such as volcano eruptions, heavy rainfalls, earthquakes, and tsunamis release high amounts of heavy metals into the environment (Shruti et al. 2018; Brizuela et al. 2019; Ji et al. 2021; Ota et al. 2021). However, the potential role of such events as drivers of the evolution of clinically-relevant antimicrobial-resistant pathogens remains unclear.On February 27, 2010, the sixth-largest earthquake ever recorded (Mw 8.8) occurred off the coast of central Chile. The earthquake, which was also felt in some parts of Argentina and Peru, triggered a subsequent tsunami and landslides that affected several coastal towns and cities. The disaster resulted in about 0.5 million homes damaged, thousands of people injured, 525 deaths, and 23 people missing. It also had a major environmental impact. The tsunami severely damaged the industrialized port of Talcahuano, Concepción Bay, which includes an oil refinery, steel, and cement production, petrochemical industries, coal power stations, and military and civilian shipyards. Data suggest that the disaster resulted in a higher-than-average concentration of heavy metals in marine sediments, urban soils, and marine fauna (Luz María Fariña; Cristián Opaso; Paulina Vera 2012; Tume et al. 2018; Tapia et al. 2019).In this study, we aimed to explore the possible role of the 2010 earthquake and tsunami, and the release and resuspension of heavy metals into the environment in the industrialized coastline of Concepción, as a driving force for the selection and evolution of MRSA genomic lineages circulating in Chile. We performed a detailed phylogenomic reconstruction of 113 ChC clone MRSA clinical isolates recovered from bloodstream infections,(Arias et al. 2017) obtained from seven healthcare centers in six countries in Latin America, including one in Concepción, Chile. We used hybrid assemblies combining short- and long-read sequencing to evaluate the potential impact of mobile genetic elements carrying horizontally transferable Heavy Metal Resistance Genes (HMRGs) in the evolution of the MRSA ST5-SCCmecI ChC clone.ResultsCharacteristics of the ChC MRSA strains collectionOur study included 113 ChC MRSA bacteremia isolates recovered from seven hospitals in Lima, Perú (n=37, 32.7%); Santiago, Chile (n=28, 24.8%); Concepción, Chile (n=25, 22.1%); Caracas, Venezuela (n=13, 11.5%); Sao Paulo, Brazil (n=5, 4.4%); Bogotá, Colombia (n=3, 2.7%); and Buenos Aires, Argentina (n=2, 1.8%). As typically described for the ChC MRSA clone, isolates exhibited high rates of resistance to ciprofloxacin, gentamicin, erythromycin, and clindamycin, along with susceptibility to tetracyclines, cotrimoxazole, rifampicin, vancomycin, and linezolid (Table S1). A total of 27% of the isolates were susceptible to ceftaroline, while the remaining 73% exhibited a minimal inhibitory concentration in the susceptible dose-dependent range, as per CLSI breakpoints (Table S1), consistent with a previous report (Khan et al. 2019). We observed no statistically significant differences in antimicrobial susceptibility across geographical locations (Table S1).WGS analyses and phylogeographical relatedness of ChC MRSA in LAThe in-silico sequence type (ST) determination revealed that all the isolates belonged to clonal complex 5 and identified 112 out of the 113 isolates (99%) as ST5; the remaining isolate was classified as ST105. Out of the 112 ST5 strains, 109 (97%) were considered ChC clones (carried mecA on SCCmecI); the remaining three isolates harbored a non-classical ChC clone SCCmecIV cassette. The ST105 isolate carried mecA in SCCmecII.Our core genome-based phylogenomic reconstruction revealed that the 109 genomes belonging to the ST5-SCCmecI (ChC) clone grouped into three well-defined clades that followed a marked geographic pattern (Fig. 1). Clade I (ChC-I, n=9) predominantly consisted of MRSA isolates from Chile (n=8), with one from Peru. Clade II (ChC-II, n=28) only contained isolates recovered from patients in Chile. Finally, clade III (ChC-III, n=72), the largest and most diverse, was further split into three sub-clades: i) ChC-IIIa (n=21) including 16 isolates from Chile and all five strains from Brazil; ii) ChC-IIIb (n=16) grouped all isolates from Venezuela (n=10), Colombia (n=3) and Argentina (n=2), along with one from Peru; and iii) ChC-IIIc (n=35) only included isolates recovered from Peru (Fig. 1).High prevalence of pSCL4752 plasmid-encoded HMRGs in Latin American ST5-SCCmecI MRSATo evaluate the potential role of HMRGs in the divergence of ST5-SCCmecI MRSA, we performed an in silico search of horizontally-acquired HMRGs involved in the processing of heavy metals. A total of 80/113 (71%) isolates harbored at least one set of acquired HMRGs associated with resistance to As (arsBC), Cd (cadACD), or Hg (merABTR). Among them, 72 out of 80 (90%) co-carried all the resistance determinants for As, Cd, and Hg (Fig. 1). Of the remaining eight isolates, three carried genes encoding resistance to As and Cd, four only carried As resistance genes, and one harbored cadmium resistance genes alone. Interestingly, all 113 isolates lacked copX(B), an acquired determinant involved in Cu resistance previously found in other MRSA clones.Since co-detection of horizontally acquired HMRGs was frequently observed among our isolates (Fig. 1), we sought to determine the genomic context of these HMRGs by performing a hybrid assembly (short-read and LRS) of a representative strain (SCL 4752) harboring As, Cd, and Hg resistance traits. Our results generated a complete genome of 3.052.503 bp with a GC content of 32.9%, composed of two contigs, including the chromosome and a plasmid that we designated pSCL4752. This plasmid consisted of a total of 36,660 bp with a GC content of 32.5% (Fig. 2), and shared extensive identity (99.9%) with a rep20_3_rep(pTW20)/rep21_20_p020(pLGA251) plasmid designated pCM05. Of note, pCM05 had been previously identified in a linezolid-resistant ST5-SCCmecI MRSA strain isolated in Colombia (NC_013323.1)(Arias et al. 2008). pSCL4752 was predicted to encode a total of 41 CDSs, including all horizontally-acquired HMRGs described above (arsBC, cadACD, merABTR), and a copy of the blaIRZ operon, which encodes the expression of the staphylococcal penicillinase, BlaZ (Fig. 2). pSCL4752 also contained two duplicated invertases (bin3 and hin), five transposases (IS431L, IS431R, ISSau6, ISBli29, IS481), and a plasmid replication initiator protein (repA), along with three replication proteins and several hypothetical proteins (Fig. 2). merABTR, resA, garB, and one hypothetical protein were flanked by two IS26 family transposases (IS431L and IS431R), potentially suggesting the presence of a mobile mercury resistance transposon.Geographical divergence of the ST5-SCCmecI MRSA clone in Chile is associated with the presence of pSCL4752A total of 71 out of the 113 (63%) isolates harbored the pSCL4752 plasmid. Noteworthy, the frequency of this plasmid in clades ChC-I (77%) and ChC-III (81%) strongly varied compared to clade ChC-II (35%) (Fig 1). Interestingly, clade ChC-II was mainly composed of isolates recovered from Santiago (central Chile). In contrast, the isolates grouped in clades ChC-I and ChC-III were obtained from Concepción (southern Chile). Thus, we aimed to study the possible role of pSCL4752 as a driver of MRSA evolutionary divergence. Since the major divergence was observed in MRSA isolates from Chile, we focused the analysis on the genomes of the 53 Chilean MRSA isolates recovered from Santiago and Concepción.An in-depth Bayesian molecular clock analysis using the 53 Chilean genomes estimated the most recent common ancestor in 2008 (95% high posterior density interval [HPD] 2007.03–2008.77) (Fig. 3). The molecular clock revealed a major divergence event in March 2010 (following the February 27 earthquake), which was quickly followed by two secondary divergence events occurring in parallel between September and November of 2010. As shown in Fig. 3, these events grouped isolates into four clades highly associated with the city of origin (Santiago and Concepción). This geographical divergence was also linked to the presence of pSCL4752 (Fisher’s exact test p<0.0001) (Fig. 3). Indeed, the prevalence of carriage of pSCL4752 was 88% for isolates recovered from Concepción and only 29% for Santiago. These results suggest that the environmental heavy metal pollution associated with the 2010 earthquake and subsequent tsunami was a major driver of the geographic divergence observed in Chilean ST5-SCCmecI MRSA.Isolates harboring the pSCL4752 plasmid exhibited increased resistance to heavy metalsTo assess the functionality of the HMRGs contained in pSCL4752, we performed susceptibility testing for As, Cd, Hg, and Cu by broth microdilution in the 53 Chilean isolates (Figs. 3 and 4). Overall, plasmid-harboring strains exhibited significantly higher MICs to Hg, Cd, and As (p<0.0001). Indeed, the MIC50/90 for Hg, Cd, and As in isolates harboring pSCL4752 were 25/25 μM, 800/1600 μM, and 100/200 μM, respectively, and 1.5/6.25 μM, 25/25 μM, and 50/100 μM, in those lacking the plasmid (Fig. 4). In concordance with the absence of Cu resistance genes on the plasmid, the MIC50/90 values to Cu did not vary between strains with or without pSCL4752 (Fig. 4). Hence, our results support the notion that MRSA isolates harboring pSCL4752 could be positively selected in environments with high concentrations of heavy metals, such as observed after the 2010 tsunami in the coast of Concepción in southern Chile.The presence of pSCL4752 carries a fitness cost in the absence of heavy metalsTo determine the role of pSCL4752 in resistance to heavy metals and to evaluate a possible fitness cost associated with its carriage, the plasmid was “cured” in one representative strain from each of the four clades established by the molecular clock analysis (Fig. 3). The loss of the plasmid was observed in all the isolates after two days of growth in trypticase soy broth medium, which was confirmed by polymerase chain reaction. All isogenic strains in which pSCL4752 was cured presented a statistically significant reduction in the minimal inhibitory concentrations of Hg (p=0.001), Cd (p=0.0005), and As (p=0.0313), as compared to their plasmid-harboring counterparts (Figs. 5A–C). In addition, plasmid-cured strains grew faster (average doubling time 42 min±5.7 vs. 59 min±3.8, respectively; p=0.0005) and reached a higher OD600 (1.638 ±0.1; p<0.05) than their isogenic parental strains harboring the plasmid (Fig. 5, lower panel). These results suggest that the presence of pSCL4752 confers an evolutionary advantage to ChC isolates in the presence of sub-inhibitory concentrations of heavy metals but introduces a fitness cost when this selection pressure is removed.DiscussionIncreasing evidence suggests an association between the acquisition of heavy metal resistance determinants and the rise of antimicrobial-resistant pathogens (Xu et al. 2017; Zhang et al. 2018; Biswas et al. 2021). Indeed, the chromosomal acquisition of horizontally-transferred HMRGs has been linked to the evolutionary divergence of North and South American epidemics of USA300-LV and USA300, two major CA-MRSA clones (Planet et al. 2015). However, data on the potential role of plasmids harboring HMRGs as drivers of the evolution and spread of clinically relevant MRSA lineages are scant. Herein, we describe a major evolutionary divergence event in clinical isolates of the ST5-SCCmecI ChC clone MRSA that was associated with the presence of a plasmid (pSCL4752), harboring heavy metal resistance determinants. This divergence, estimated to have occurred in 2010, followed a distinct geographical distribution, clustering isolates recovered from Santiago and Concepción, two Chilean cities. Additionally, a unique phylogeographic analysis of the ST5-SCCmecI MRSA clone in Latin America revealed a distinct geographic clustering highly associated with the country of bacterial isolation.Our results align with previous studies describing the emergence of new MRSA lineages associated with possible evolutionary advantages of heavy metal resistance traits (Kernberger-Fischer et al. 2018; Zapotoczna et al. 2018). Environmental contamination with heavy metals has been associated with horizontal gene transfer and the selection of non-pathogenic organisms harboring plasmids that carry heavy metal resistance traits (Xu et al. 2017; Zhang et al. 2018). The divergence observed could be partly driven by environmental selective pressure. Indeed, historical records report high levels of heavy metal pollution in urban soils and marine sediments in Concepción (Barrios-Guerra 2004; Tume et al. 2008; Luz María Fariña; Cristián Opaso; Paulina Vera 2012; Tume et al. 2018). Research has shown that tsunamis and other major catastrophic events release and resuspend heavy metals from marine sediments and land-based pollutants (Brizuela et al. 2019; Ota et al. 2021). A previous study found a significant increase in heavy metals observed in mollusks collected off the coast of Concepción following the 2010 tsunami (Tapia et al. 2019). Our molecular clock analyses estimated that the initial divergence event leading to the selection of HMRGs-harboring plasmid pSCL4752 occurred between March and September 2010. Altogether, these data suggest that the increase in environmental heavy metals released by the 2010 earthquake and subsequent tsunami contributed to the selective pressure driving the divergence events observed in the ST5-SCCmecI MRSA clone.Phenotypically, isolates containing pSCL4752 exhibited higher tolerance to As, Cd, and Hg, suggesting an active role of the HMRGs harbored on the plasmid. However, in the absence of heavy metals, clinical isolates containing pSCL4752 exhibited slower growth than those not carrying the plasmid, suggesting that pSCL4752 could provide an evolutionary advantage in environments containing heavy metals, increasing the survivability of MRSA. On the other hand, maintenance of pSCL4752 in the absence of heavy metals resulted in a fitness burden. Interestingly, previous data have shown some MRSA clones have maintained mobile genetic elements containing HMRGs despite a fitness cost, due to an adaptative advantage beyond heavy metal resistance. Indeed, a horizontally transferred copper-resistant locus provided increased survival in macrophages and was associated with co-carriage of crucial antimicrobial resistance determinants in USA300 (Zapotoczna et al. 2018; Rosario-Cruz et al. 2019). We detected a blaIRZ operon present in pSCL4725, suggesting it may be related to the selection of the plasmid. However, we found the blaIRZ operon in 57% (n=24) of the isolates not carrying the pSCL4725 plasmid, suggesting that the plasmid was most likely selected by heavy metals and not by a potential advantage provided by this antimicrobial resistance operon. Furthermore, our genomic analyses revealed that Hg resistance is likely transposable since Hg resistance genes were contained within a transposon-like structure flanked by two IS26 family transposases (IS431L and IS431R). This element has been found in the chromosome linked to the SCCmec element in other MRSA lineages, including the COMER element (Planet et al. 2015). This, further suggest that mobile Hg resistance determinants might play a major role in the selection of successful MRSA lineages.Our core genome-based phylogeographic analyses of isolates belonging to the ST5-SCCmecI ChC clone MRSA revealed a substantial genomic heterogeneity strongly associated with the city of origin. These results align with previous data suggesting an inherently higher geographical diversity in MRSA isolates belonging to clonal complex 5 (which includes ST5-SCCmecI) as compared to other MRSA lineages (Challagundla et al. 2018). Geographic genomic heterogeneity has also been observed in other MRSA lineages such as ST105 and ST239, both of which underwent marked divergence within different regions of Brazil (Botelho et al. 2019; Viana et al. 2021). The divergence events that generated the North and South American USA300 clones subsequently led to further rapid clonal expansion across different geographic regions (Reyes et al. 2009). Furthermore, the appearance of two predominant variants of the USA300 clone in an outbreak in New York suggested that MRSA clones may undergo genomic divergences even within the same geographical area and genetic lineage (Copin et al. 2019).This study has some limitations. First, Chile was the only country where isolates were collected from two different cities. Therefore, we cannot discard the possibility that similar divergence events associated with the loss of pSCL4752 occurred in other regions of Latin America. A larger sample size with a more geographical and temporal representation of isolates would improve our understanding of the evolutionary history of the ST5-SCCmecI MRSA lineage and the impact of horizontally-acquired HMRGs, and the pSCL4752 plasmid in particular, on the evolution of this clone in Latin America. Second, there is no record of heavy metal concentrations in the clinical settings in Santiago or Concepción in the aftermath of the 2010 earthquake. Therefore, we cannot compare the minimum inhibitory concentrations obtained in this study with real-world conditions.In conclusion, we used genomic data from clinical isolates of the ST5-SCCmecI ChC MRSA clone to describe a major evolutionary divergence event associated with plasmid-harbored heavy metal resistance genes. We observed that the divergence follows a spatiotemporal pattern probably associated with heavy metal pollution associated with an extreme natural event, the 2010 earthquake, and tsunami. Indeed, we found suggestive evidence of a possible link between the release of higher quantities of heavy metals in the aftermath of an environmental disaster and the divergent evolution of the ChC MRSA in the region. Improving our understanding of how chronic exposure and adaptation to environmental pollution associated with extreme events could affect the emergence of antimicrobial resistance determinants is critical for avoiding a potential future health crisis. Our results highlight the urgent need for additional research on environmental risk factors associated with the emergence of antimicrobial resistance.Materials and MethodsStrain collection, antibiotic and heavy metal susceptibility testingWe studied a collection of 113 MRSA isolates recovered from adult patients diagnosed with S. aureus bacteremia between January 2011 and July 2014 in Argentina, Brazil, Chile, Colombia, Peru, and Venezuela. Susceptibility testing of antimicrobials and heavy metals was performed using either agar dilution or broth microdilution method according to the 2019 Clinical and Laboratory Standards Institute (CLSI) (details in Appendix 1).Whole-genome sequencing (WGS) and Phylogenomic analysisMethods for DNA extraction, WGS, and in silico characterization for the 113 isolates are described in Appendix 1. The genome of a representative isolate (SCL 4752) was sequenced by long-read sequencing (LRS) (MinION, Oxford Nanopore Technologies, Oxford, UK) using the SQK-LSK208 kit following the manufacturer’s instructions and a consensus hybrid assembly using both Illumina and LRS reads was obtained. The phylogenomic relationships were assessed with a maximum likelihood phylogenomic tree and a Bayesian molecular clock using a GTR substitution model (Appendix 1).Plasmid curing and Growth curvesThe plasmid curing protocol consisted of consecutive 24 hours passages of cultures growing with shaking at 44°C in tubes containing fresh MH broth (May et al. 1964). Growth curves were measured in three replicates for 24 hours at 37°C (Details in Appendix 1).

PMC

Proceedings of the National Academy of Sciences of the United States of America

PMC10466086

10.1073/pnas.2312256120

Emil Gotschlich: Physician–scientist and vaccine pioneer (1935–2023)

Rappuoli Rino

Emil Claus Gotschlich, a physician–scientist well known for the development of the first meningitis vaccine, was born in a family with a long tradition in medicine. His grandfather, Carl Gotschlich, a German doctor who specialized in hygiene and tropical diseases, had been a physician in Alexandria, Egypt, and director of the Hygiene Institute of the University of Heidelberg, while his great uncle had isolated the Vibrio cholerae El Tor strain, the causative agent of the deadly diarrheal disease, during the Muslim Hajj in 1906. Both of Emil’s parents were German physicians, Emil Clemens Gotschlich and Magdalene, née Holst. His father worked in Bangkok, Thailand, in 1933 where Emil was born in January 1935. His mother had very strong, anti-Nazi convictions, and her citizenship was revoked when she was blacklisted by the Gestapo because she refused the invitation to Aryian people to travel to Germany to see the Munich Olympic games in 1936. In 1939, they traveled to Zurich, Switzerland, and when the Second World War began, his father moved back to Thailand, while his mother, without a passport, was trapped in Switzerland and eventually found a job in the Canton Ticino, in a home for children rescued from the Nazi death camps. When Emil was 12, he went to school at the Colegio Papio in Ascona, on the Lago Maggiore. The school, run by the Benedictine Order priests, was taught in Italian, and he received an Italian education until he was 15 when his family moved to the United States in 1950. Emil spoke French, German, and English, but loved Italian. His love for the Italian language lasted for his entire life, and being afraid of losing his mastery of the language, he regularly attended Italian classes and continued to read books in Italian.Emil Gotschlich. Image credit: Kathleen Gotschlich.In the United States, Emil studied medicine and graduated from New York University in 1959. Shortly after, in 1960, he joined the laboratory of Maclyn McCarty and Rebecca Lancefield at the Rockefeller University. McCarty, together with Oswald Avery and Colin MacLeod, had discovered in 1944 that DNA, rather than proteins, constituted the chemical nature of the genetic material. The key to the discovery had been the biochemical skills of McCarty who obtained pure DNA from virulent type III Streptococcus pneumoniae. The purified DNA allowed him to show that DNA alone was able to transform the nonvirulent type II-R pneumococci into fully virulent bacteria expressing type III capsular polysaccharide. Because of this revolutionary finding, McCarty was offered a professorship and his own laboratory at Rockefeller. In the 1960 s, his lab was still fully engaged in the study of the virulence of streptococci and had developed all the biochemical know-how to purify bacterial polysaccharides and DNA. Emil’s first project was C-reactive protein, a poorly understood plasma protein found in response to tissue injury, that had been crystalized in the McCarty lab. Emil used McCarty’s method to purify CRP and published a series of seminal papers which were critical for the measurement of CRP in current blood tests, as an indicator of tissue inflammation.In 1966, Emil was drafted into the Army and served as a captain in the Medical Corps at the Walter Reed Army Institute of Research in Bethesda, Maryland, which at the time was battling the problem of meningococcal meningitis in American troops engaged in the Vietnam war. His knowledge of polysaccharide biochemistry and chemistry and the ability to measure the immune response to them became a key asset. In a famous paper by Irving Goldscheneider and Emil C. Gotschlich published in the Journal of Experimental Medicine in 1969, they reported that people with antibodies against the meningococcal capsular polysaccharide induced by the asymptomatic nasopharingeal carriage of meningococci were protected from disease (1, 2). They concluded that a vaccine able to induce antibodies against a polysaccharide would be the solution. Unfortunately, the way polysaccharides were purified at that time generated quite low-molecular-weight molecules which were not immunogenic. Using his profound knowledge of biochemistry, Emil developed a strategy to purify high-molecular-weight polysaccharides from culture supernatants by precipitating them before they were degraded, using the cationic detergent Cetavlon . However, when he tested these high-molecular-weight polysaccharides in animal models, he was quite disappointed to find that they were not immunogenic. Confident that he had the solution, he then decided to inject himself with the high-molecular-weight polysaccharide and made the seminal discovery that the vaccination induced a strong immune response. Today, we know that polysaccharides are T cell-independent antigens, which, in naive animals and in children, are not immunogenic. However, they become immunogenic in adolescents, adults, and the elderly, which with age develop memory B cell against polysaccharides. When the polysaccharide immunization was repeated in six human volunteers, similar results were obtained . This fundamental discovery allowed Emil to develop vaccines against meningococcus A and C and to show that in military recruits the vaccine-induced bactericidal antibodies and protection from disease . The technology was then transferred to the Institute Merieux, a French commercial manufacturer which produced more than 80 million doses of meningococcus AC polysaccharide vaccine that were used in Brazil to halt the ongoing meningococcal epidemic in the country and in several African regions. This discovery pioneered the development of many other bacterial capsular polysaccharide vaccines such as the polysaccharide vaccine against S. pneumoniae in the 1970 s followed by a polysaccharide vaccine against a Haemophilus influenzae and Salmonella typhi. Emil’s discovery was immediately adopted by another giant in vaccinology, John Robbins, who pioneered the conjugation of the capsular polysaccharides to proteins to make them immunogenic, particularly in infants. They both became very close friends for the rest of their lives and from that friendship arose all subsequent vaccines against encapsulated bacteria. These include vaccines against the ACW and Y serogroups of meningococcus, 20 serotypes of pneumococcus, H. influenzae, and S. typhi.The remarkable development of an effective meningococcal vaccine was published in a series of five consecutive papers in the same issue of the Journal of Experimental Medicine (1–5). The first in the series described human immunity against the meningococcus and reported a study on an outbreak of meningococcal meningitis in military recruits at Fort Dix, NJ. The data clearly showed that those who developed the disease did not have antibodies able to kill the bacteria in a bactericidal assay, while the presence of bactericidal antibodies correlated with protection from disease. The discovery of a simple laboratory method to predict protection or susceptibility to meningococcal disease marked a milestone in vaccine history. Following that paper, the FDA licensed all meningococcal vaccines using only data from the bactericidal assay, without requiring long and expensive efficacy trials. This revolutionized the vaccinology field for meningococcus. Still, today, finding a “correlate of protection,” the ability to predict immunity from disease, is the dream of every vaccinologist.When I joined Emil’s lab at Rockefeller in the winter of 1979, I was not fully aware of how famous he was at that time. He had just received the Lasker award for devising innovative chemical techniques which permitted the development of an effective meningococcal vaccine, and his lab was busy running the serology of the vaccine trials. My project was to develop a vaccine for gonococcus, a bacterium related to meningococcus, which causes a sexually transmitted disease. Emil was very friendly and very proudly introduced me to the legendary Maclyn McCarty and Rebecca Lancefield and to Vincent Fischetti who had his lab next door. Emil often invited my wife and me to dinner in his apartment on the Rockefeller University campus, where he personally prepared every dinner. His wife Kathy, my wife Angela, and Emil and I became close friends. Outside of the lab, we discussed Italian literature, opera, and music. In the laboratory, I was somewhat new to microbiology and biochemistry, and every day I had many new questions. I was surprised by the time he would spend answering my often-silly questions. I am not exaggerating when I say that he would spend more than one hour per day mentoring me. Since I left his laboratory, we continued to remain friends and I would visit him almost every year, and often, Emil, Kathy, John Robbins, and his wife Joan would go out for dinner. During the many years I spent developing conjugate vaccines for meningococcus and a protein-based vaccine for meningococcus B, he always supported me with his scientific advice and friendly encouragement. He was also very engaged in scientific discussions with young investigators from my laboratory. He provided advice, suggestions, and different perspectives on projects and somehow positively influenced the course of our research. This was Emil, generous, smart, and visionary. He had the ability to think out of the box and bring to the discussion ideas that nobody else would be able to do. During meetings, he would be mostly silent, listening to others incorporating their obvious comments into the discussion, and then, after assimilating it all, he would come out with original and provocative ideas.Although Emil truly loved to work in the lab, at some point he decided to dedicate his full time to chair the Institutional Review Board (IRB) of The Rockefeller University Hospital. This was a major administrative job that he did for 15 y, from 2002 to 2017. His activity as chair of the IRB is perhaps one of Emil’s greatest contributions. He was able to strategically address and influence the experimental medicine of The Rockefeller University and make it one of the most innovative worldwide. It is impressive how such a small hospital could do so much and so well. The hospital came out with very original research protocols, using creative interpretations of regulations to ease the needs of investigators, but designed with the highest bioethical standards, scientific quality, and rigor. During this period, early-phase studies in humans performed at The Rockefeller University often led to many basic discoveries. Looking at the publications that came from investigators at Rockefeller University during this period, we can recognize a common theme behind the innovative nature of the studies from giant investigators, such as Michel Nussenzweig, John Laurent Casanova, Jeff Ravetch, and Leslie Vosshall. Somehow, we can imagine Emil acting as the conductor of The Rockefeller University experimental medicine orchestra, although he never took credit for these studies. It would have been interesting to hear Emil’s comments on this piece in his memory. For sure, he would have appreciated how much he has influenced science, global health, and people like me who will always be grateful for the immense legacy he unconditionally gifted us.

PMC

HemaSphere

PMC10428925

8-08-2023

10.1097/01.HS9.0000969616.68483.49

P678: EARLY WARNING RESPONSES IN CML PATIENTS: A REAL-LIFE TURIN EXPERIENCE

Giai Valentina, Bisio Matteo, Rosso Tiziana, Urbino Irene, Frairia Chiara, Olivi Matteo, Lanzarone Giuseppe, D’agostino Mattia, Beggiato Eloise, D’ ardia Stefano, Audisio Ernesta, Cerrano Marco, Ferrero Dario, Pregno Patrizia, Freilone Roberto

Background: In the last decades, tyrosine kinase inhibitors (TKI) have not only improved biological responses, but also survival and quality of life of chronic myeloid leukemia (CML) patients. In the large registration studies, results are usually exceptional and sometimes not so reproducible in a real-life setting.Aims: Our project aims to analyze the role of “warning” category in a real-life experience.Methods: In our retrospective study, we collected molecular responses and clinical data of CML patients followed at Città della Salute e della Scienza in Turin, Italy. Responses were evaluated according to CML ELN 2020 recommendations (Hochhaus A., Leukemia 2020).Results: One-hundred-sixty-two patients were analyzed: 101 were males (62%), median age was 62 years. Ninety-seven percent of patients was in chronic phase. ELTS score was high in 10.5%, intermediate in 34%, low in 54.3% and missing in 1.2% of patients. Median follow up was 5.5 years.At 3 months, 17% (23/135) of patients showed a warning. Thirteen were receiving Imatinib (IMA), 7 Dasatinib (DAS) and 3 Nilotinib (NIL). All patients except one continued the same treatment. At 6 months, 9 of these patients stayed in the warning category (8 on IMA, 1 on DAS), while 9 reached the failure group (2 on IMA, 4 on DAS, 3 on NIL) and 4 gained the optimal response (2 on IMA, 2 on DAS). Six of the 9 failure patients switched to Ponatinib (PON), 1 to DAS and 2 to NIL. At the 12th month, 5 of the 9 warning patients jumped into the failure category and 4 stayed in the warning group. Six of the 3 months warning patients (6/23, 26,1%) underwent to an allogeneic stem cell transplantation and 5 patients (5/23, 21,7%) died for CML related causes.At 6 months, 24/145 (18%) patients were in the “warning” group. Half of the 6th month warning patients stayed in the warning group even at the 12th month (50%, 15/24). Just 4/24 6th months warning patients changed treatment.The higher warning percentage was detected at 12 months: 39,1% (52/137). 27 were on first line IMA of which 5 changed therapy before the 12 month because of an insufficient response at 6 months; 15 were on first line DAS of which 4 switched therapy before 12 months; 10 were on first line nilotinib (NIL) of which just 1 changed treatment before the 12 months. Half of patients (26/52, 50%) reached optimal response at 24 months, only 2 switched therapy between 12th and 24th month, one for extra hematologic toxicity and the other for warning response. Nine of them were still on first line treatment, even if they were in the warning category already at 3-6 or 12 months (1 on DAS and 8 on IMA). Just 3 patients switched therapy before the 12th month (Fig.1).Global overall survival (OS) in our population was 85,7% at 5 years and 75% at 10 years. Three months warning patients showed a worst OS, compared to the optimal response patients (Fig. 2). No differences in OS were detected among patients treated with different TKI.Summary/Conclusion: In our real-life study, we confirm that warning patients at 6 and 12 months can reach deeper molecular responses at following timepoints even without switching to a second line TKI. OS of warning patients at 6 and 12 months show no significant differences compared to optimal response patients OS. In our registry, all 5 patients that died for CML related causes showed a warning response at 3 months, confirming that early responses can anticipate later responses. Further studies are needed to elucidate role of early “warning” category in a larger setting of CML patients with longer follow up.Keywords: Molecular response, BCR-ABL, Tyrosine kinase inhibitor, Chronic myeloid leukemia

PMC

Investigative Ophthalmology & Visual Science

38306106

PMC10851174

2-02-2024

10.1167/iovs.65.2.6

Central and Peripheral Changes in Retinal Vein Occlusion and Fellow Eyes in Ultra-Widefield Optical Coherence Tomography Angiography

Zhao Xin-yu, Zhao Qing, Wang Chu-ting, Meng Li-hui, Cheng Shi-yu, Gu Xing-wang, Sadda Srinivas R., Chen You-xin

PurposeTo explore the central and peripheral retinal and choroidal changes in retinal vein occlusion (RVO) and fellow eyes using ultra-widefield swept-source optical coherence tomography angiography (UWF-SS-OCTA).MethodsFifteen ischemic central RVO (CRVO), 15 branch RVO (BRVO), and 15 age-matched healthy controls were prospectively recruited. Retinal and choroidal parameters, including retinal vessel flow density (VFD) and vessel linear density (VLD), choroidal vascularity volume (CVV), choroidal vascularity index (CVI), and VFD in the large and medium choroidal vessels (LMCV-VFD), were measured in the central and peripheral regions of the 24 × 20-mm UWF-SS-OCTA images.ResultsIschemic CRVO and BRVO eyes showed increased foveal avascular zone area, perimeter, and acircularity index (AI) compared to their fellow eyes and healthy control eyes, and RVO fellow eyes also had larger AI values than controls (P < 0.05). For ischemic CRVO and BRVO eyes versus control eyes, VFD, VLD, CVV, CVI, and LMCV-VFD decreased, but retinal thickness and volume in the superficial capillary plexus, deep capillary plexus, and whole retina increased (P < 0.05). Moreover, RVO fellow eyes also showed significantly decreased retinal VFD, LMCV-VFD, and CVI, as well as increased retinal thickness and volume, compared with control eyes (P < 0.05). Alterations were not consistent throughout the retina, as they involved only the peripheral or central regions in some cases.ConclusionsThe affected and unaffected fellow eyes of RVO patients both demonstrated central and/or peripheral structural and vascular alterations in the retina and choroid. Because UWF-SS-OCTA enables visualization and evaluation of the vasculature outside the posterior pole, it presents a promising approach to more fully characterize vascular alterations in RVO.

Retinal vein occlusion (RVO) is one of the most common retinal vasculopathies, with a global prevalence of 0.77% among people 30 to 89 years of age, which is equivalent to 28.06 million affected people.1 The pathogenesis of RVO is characterized by thrombosis and obstruction within the retinal venous system, resulting from either retinal vein compression or vessel wall damage. Based on the site of the occlusion, RVO can be categorized as central RVO (CRVO) or branch RVO (BRVO). RVO may markedly impair retinal circulation, causing capillary damage that leads to complications such as macular edema (ME), neovascularization, retinal or vitreous hemorrhage, and eventually severe vision loss. RVO has been identified as a possible indicator of systemic vascular abnormalities, as conditions such as hypertension, hyperlipidemia, diabetes mellitus (DM), and atherosclerosis have been established as systemic risk factors for RVO.1,2 Additionally, RVO patients also face an increased risk of cerebrovascular diseases.2Optical coherence tomography angiography (OCTA) has been used to evaluate pathologic vascular changes in RVO3–6; however, several issues remain unresolved. Notably, alterations in the choroid associated with RVO have been poorly characterized, with conflicting findings regarding subfoveal choroidal thickness (SFCT) and flow density in the choriocapillaris.7,8 Additionally, there is also a lack of information about alterations in the large and medium choroidal vessels. Moreover, pathologic alterations in the fellow eyes of patients with RVO have been incompletely studied despite the systemic vascular associations of RVO, which might be expected to have impacts on the fellow eye. In prior studies, RVO fellow eyes were mainly used as controls or references for comparison against alterations evident in RVO eyes,9–11 but these fellow eyes were not compared to healthy control eyes.12 Furthermore, the choroidal vascularity index (CVI), a parameter calculated as the ratio of the choroidal luminal area to the total choroidal area in a single OCT B-scan, has recently been introduced to quantitatively analyze choroidal structure in the setting of various retinal diseases.13 More recently, this two-dimensional (2D) CVI has been replaced by a three-dimensional (3D) CVI assessment using the entire OCT volume, but alterations in the 3D CVI in the setting of RVO have not been evaluated. Finally, most previous studies evaluating the retina and choroid using conventional OCTA were limited to a field of view (FOV) ranging from 3 × 3 mm to 12 × 12 mm,14–16 without evaluating regions beyond the posterior pole, despite the fact that RVO commonly involves these more peripheral retinal regions. Given these gaps in knowledge, it would appear that a more comprehensive assessment of the retina and choroid using ultra-widefield imaging techniques would provide better insights into the angiographic and structural alterations and pathophysiology of RVO. In this study, we applied the novel ultra-widefield swept-source OCTA (UWF-SS-OCTA) with a FOV of 24 × 20 mm, which enabled visualization of the fundus beyond the posterior pole, allowing us to assess the peripheral retina and choroid in both RVO and RVO fellow eyes.MethodsStudy DesignThis observational cross-sectional study was conducted in accordance with the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Peking Union Medical College Hospital (K3885). All included participants provided informed consent at enrollment.Sample Size CalculationBased on our preliminary evaluation, the mean foveal avascular zone (FAZ) area in the ischemic CRVO eyes and the healthy control eyes was found to be 0.50 and 0.30, respectively. With a type I error set at 0.05 and a power of 0.90, the minimum required sample size in each group was determined to be 12.17Participant EnrollmentThe CRVO and BRVO groups were each comprised of 15 unilateral RVO patients with the diagnosis confirmed by two experienced ophthalmologists (XZ, YC) at the Ophthalmology Department of Peking Union Medical College Hospital in Beijing, China, from August 2021 to May 2022. CRVO eyes exhibiting a deep capillary plexus (DCP) vessel density of ≤38.4% or retinal non-perfusion involving ≥10 disc areas in the acquired UWF-SS-OCTA images were categorized as ischemic CRVO.18,19 The unaffected CRVO fellow and BRVO fellow eyes were also included. In addition, the healthy control group was comprised of 15 age-matched individuals without any ocular diseases (except for cataracts or non-pathologic myopia), with one eye randomly enrolled.Exclusion criteria included the following: 1. Previous ocular treatment history, including intravitreal injection, laser therapy, or ocular surgery (except for cataract extraction)2. Any other ocular comorbidities, such as diabetic retinopathy (DR), hypertensive retinopathy, retinal arterial occlusion, glaucoma, uveitis, etc. Participants with keratoconus, high myopia (≥−6.0 D), or high astigmatism (≥3 D) were also excluded.3. Coexisting systemic diseases such as systemic autoimmune diseases that could be associated with RVO. In addition, DM cases were also excluded because patients with DM without clinically visible DR may also have abnormal fundus microcirculation.204. RVO-induced vitreous hemorrhage or pre- or intraretinal hemorrhage that could impair visualization of the retinal and choroidal vasculature5. Participants with poor-quality OCTA images or incomplete medical records regarding their systemic historyAll enrolled participants underwent comprehensive ophthalmic examinations, including assessments of best-corrected visual acuity (BCVA), intraocular pressure (IOP), slit-lamp examination, mydriatic ophthalmoscopy, UWF-SS-OCT, and UWF-SS-OCTA.UWF-SS-OCTA Image Acquisition and AnalysisThe UWF-SS-OCT and UWF-SS-OCTA images were captured using a BMizar (BM-400K) instrument (TowardPi Medical Technology, Beijing, China). It utilizes a 1060-nm-wavelength, swept-source vertical-cavity surface-emitting laser with a speed of 400,000 A-scans per second, providing a transverse resolution of 10 µm and an axial resolution of 3.8 µm. The A-scan depth within the tissue is 6.0 mm (2560 pixels). For UWF-SS-OCTA, a fovea-centered 24 × 20-mm scan pattern was chosen. This pattern consisted of 1536 A-scans per B-scan at 1280 B-scan positions, resulting in a 15.625-µm A-scan and B-scan separation. Central macular thickness (CMT) and SFCT were measured in UWF-SS-OCT images using instrument caliper tools. Two independent ophthalmologists (XZ, QZ) performed these measurements, and the mean value of each measurement was recorded. The FAZ was automatically identified in UWF-SS-OCTA images using the built-in instrument software, with errors manually corrected when required. FAZ-related parameters, including area, perimeter, AI, and flow density in a 300-µm annulus surrounding the FAZ (FD-300), were measured using the full retinal slab (from the inner limiting membrane to 6 µm below the outer plexiform layer) (Fig. 1A). The AI was defined as the ratio of the FAZ perimeter to the standard circular perimeter of the equal FAZ area.Figure 1. Schematic diagram of an ischemic CRVO participant. (A) Full retinal layer. All FAZ parameters, including the FAZ area, perimeter, AI, and FD-300, were measured on the full retinal layer (from the inner limiting membrane to 6 µm below the outer plexiform layer). FD-300 refers to the vessel flow density (VFD) in a 300-µm annulus surrounding the FAZ (yellow arrowhead). (B–G) The 24 × 20-mm UWF-SS-OCTA images of each retinal and choroidal layer were divided into 24 × 20 squares, each 1 × 1 mm, and each parameter was measured in the 1 × 1-mm square. The centered 12 × 12 squares were defined as the central region (red box), and other squares were defined as the peripheral region. (B) Superficial capillary plexus (SCP) layer, showing VFD. (C) DCP layer, showing VFD. (D) Choriocapillaris (CC) layer, showing VFD. (E) Large and medium choroidal vessel (LMCV) layer, showing VFD. (F) LMCV layer, showing choroidal vascularity volume (CVV). (G) LMCV layer, showing the CVI.The retina and choroid were automatically segmented into sublayers by the built-in instrument software (Fig. 2), with artifacts minimized by employing volumetric projection artifact removal approaches. The retinal sublayers included the superficial capillary plexus (SCP) and DCP, and the choroidal sublayers consisted of the choriocapillaris (CC) and the large and medium choroidal vessel (LMCV) layers. All of these segmentations were manually inspected and corrected as needed by two ophthalmologists (XZ, QZ) before any calculation. The acquired 24 × 20-mm UWF-SS-OCTA images of each layer were divided into 24 × 20 squares, each measuring 1 × 1 mm. The squares within the centered 12 × 12 region were defined as the central region, equivalent to the FOV of conventional 12 × 12 mm OCTA images. Squares outside this region were defined as the peripheral region (Figs. 1B–1G).Figure 2.Schematic diagram of different retinal and choroidal sublayers in the UWF-SS-OCT and UWF-SS-OCTA images. In the UWF-SS-OCT images, the area marked in red represents the retinal vascularity and CC; the LMCV is marked in yellow; and the boundaries of sublayers are indicated by green lines. (A, B) SCP layer, from the inner limiting membrane to 9 µm below the inner plexiform layer. (C, D) DCP layer, from 6 µm below the inner plexiform layer to 9 µm below the outer plexiform layer. In the UWF-SS-OCTA images, the peripheral retinal arteries and veins remain visible in the DCP layer due to the thinner nature of the peripheral retina and the fact that the retinal vessels are partially situated in the DCP layer, rather than being caused by projection artifacts from SCP. This assertion finds support in the evident presence of retinal arteries and veins in the DCP layer as seen in the UWF-SS-OCT B-scan images. (E, F) CC layer, from Bruch's membrane to 29 µm below Bruch's membrane. (G, H) LMCV layer, from 29 µm below Bruch's membrane to the choroidoscleral interface.Retinal parameters, including vessel flow density (VFD), thickness, and volume, were measured within the SCP, DCP, and the full retina. VFD was calculated as the ratio of the area occupied by the blood vessels divided by the total image area.21 Following skeletonization, vessel linear density (VLD), defined as the ratio of the length occupied by the blood vessels to the total area in the linearized vessel map,22 was measured within the SCP. Choroidal parameters measured from the UWF-SS-OCTA images included CC-VFD, LMCV-VFD, and choroidal vascularity volume (CVV) and CVI measured for the LMCV. CVV represents the volume of LMCV, and the CVI is the ratio of CVV to the total choroidal volume.23 Each parameter was measured in the whole scan, central region, and peripheral region. Representative UWF-SS-OCTA images of CRVO and BRVO eyes are shown in Figures 3 and 4.Figure 3.Representative UWF-SS-OCTA images of an ischemic CRVO participant. (A) Full retinal layer. (B) SCP layer, showing VFD. (C) DCP layer, showing VFD. (D) CC layer, showing VFD. (E) LMCV layer, showing VFD. (F) LMCV layer, showing CVV. (G) LMCV layer, showing the CVI.Figure 4.Representative UWF-SS-OCTA images of a BRVO participant. (A) Full retinal layer. (B) SCP layer, showing VFD. (C) DCP layer, showing VFD. (D) CC layer, showing VFD. (E) LMCV layer, showing VFD. (F) LMCV layer, showing CVV. (G) LMCV layer, showing the CVI.Data AnalysisNumerical and categorical data are displayed as mean ± SD and frequency (percentages), respectively. The Snellen BCVA was converted to the respective logMAR equivalent for statistical analysis.24 The χ2 test or Fisher's exact test was performed for comparisons of categorical variables. The dependent-variables t-test or Wilcoxon signed-rank test was utilized to compare numerical data between the affected and unaffected eyes in RVO patients, depending on the data distribution. For the comparison of RVO and RVO fellow eyes with control eyes, the independent-variables t-test or Mann–Whitney U test was chosen. All statistical analyses were performed using Stata/SE 12.0 software (StataCorp, College Station, TX, USA), with P 0.05). The average RVO durations in the ischemic CRVO and BRVO groups were 7.96 ± 10.43 and 6.56 ± 5.27 months, respectively. Ischemic CRVO and BRVO eyes had significantly worse BCVA compared to both fellow eyes and healthy control eyes (P < 0.001). Systemic hypertension was present in 13 ischemic CRVO patients (86.67%) and 12 BRVO patients (80.00%), but none was observed in controls (P < 0.001).Table 1.Demographics of the ParticipantsIschemic CRVO (n = 15)BRVO (n = 15)VariableControl (n = 15)Ischemic CRVO Eyes P vs. Control P vs. FellowFellow Eyes P vs. ControlBRVO Eyes P vs. Control P vs. FellowFellow Eyes P vs. ControlAge (y), mean ± SD49.83 ± 9.1451.92 ± 17.440.68451.92 ± 17.440.68455.50 ± 14.180.20455.50 ± 14.180.204Male gender, n (%)7 (46.67)8 (53.33)0.7158 (53.33)0.7157 (46.67)1.0007 (46.67)1.000RVO duration (mo), mean ± SD7.96 ± 10.436.56 ± 5.27BCVA, mean ± SD0.86 ± 0.230.25 ± 0.28<0.001*<0.001*0.67 ± 0.290.0580.46 ± 0.30<0.001*<0.001*0.88 ± 0.180.793IOP (mmHg), mean ± SD16.11 ± 3.4117.15 ± 8.110.6600.34114.67 ± 5.510.39914.87 ± 2.030.2360.41915.38 ± 1.250.439Spherical equivalent (D), mean ± SD–2.20 ± 1.58–1.31 ± 1.250.0980.944–1.28 ± 1.080.073–1.23 ± 1.130.0630.815–1.32 ± 0.950.075Pseudophakia (eyes), n (%)0 (0.00)2 (13.33)0.4831 (6.67)1.0000 (0.00)NA1 (6.67)1.000Systemic comorbidity, n (%) Hypertension0 (0.00)13 (86.67)<0.001*13 (86.67)<0.001*12 (80.00)<0.001*12 (80.00)<0.001* Diabetes mellitus0 (0.00)0 (0.00)NA0 (0.00)NA0 (0.00)NA0 (0.00)NA Hyperlipidemia0 (0.00)6 (40.00)0.022*6 (40.00)0.022*4 (26.67)0.0504 (26.67)0.050* P 0.05).Both affected and unaffected fellow eyes of ischemic CRVO and BRVO patients had significantly lower SCP-VFD than controls in the whole scan, peripheral region, and central region (P 0.05). BRVO fellow eyes had a lower central DCP VFD than controls (31.09 ± 3.19 vs. 34.32 ± 5.11; P = 0.047). The SCP-VLD in ischemic CRVO and BRVO eyes was significantly lower than in their fellow eyes and controls across the whole scan areas of UWF-SS-OCTA images (P 0.05). When comparing BRVO eyes with BRVO fellow eyes, greater thickness in the DCP and full retina and a higher volume in all retinal layers were observed in the BRVO eyes (P < 0.05). The comparisons of retinal parameters among groups are summarized in Table 2.Table 2.Retinal Parameters of RVO, Fellow, and Healthy Control EyesIschemic CRVO (n = 15)BRVO (n = 15)VariableControl (n = 15)Ischemic CRVO Eyes P vs. Control P vs. FellowFellow Eyes P vs. ControlBRVO Eyes P vs. Control P vs. FellowFellow Eyes P vs. ControlCMT (µm), mean ± SD232.37 ± 24.52301.46 ± 155.570.1020.224241.33 ± 105.550.752241.19 ± 128.340.7960.901237.00 ± 10.810.509FAZ, mean ± SD Area (mm2)0.32 ± 0.130.53 ± 0.330.031*0.006*0.26 ± 0.120.1680.45 ± 0.160.023*0.010*0.32 ± 0.100.837 Perimeter (mm)2.34 ± 0.583.45 ± 1.220.003*0.002*2.02 ± 0.410.0922.89 ± 0.680.024*0.032*2.41 ± 0.470.718 AI1.20 ± 0.081.39 ± 0.200.002*<0.001*1.28 ± 0.080.013*1.33 ± 0.09<0.001*0.032*1.26 ± 0.080.049* FD-300 (%)0.31 ± 0.060.29 ± 0.050.2870.5500.30 ± 0.040.5960.30 ± 0.060.5980.6240.31 ± 0.050.999VFD (%), mean ± SD SCP Whole scan33.96 ± 3.1229.13 ± 5.190.005*0.13431.24 ± 1.070.004*30.33 ± 5.950.046*0.47331.58 ± 2.970.041* Central region39.83 ± 3.2135.70 ± 5.210.014*0.22137.52 ± 2.150.028*36.43 ± 4.690.028*0.21638.01 ± 1.150.048* Peripheral region30.88 ± 3.2426.14 ± 4.020.001*0.029*28.65 ± 1.250.019*27.88 ± 4.160.036*0.32129.010 ± 1.220.046* DCP Whole scan31.44 ± 4.8925.703 ± 7.010.015*0.11528.19 ± 3.500.046*26.65 ± 6.130.025*0.22629.03 ± 4.230.160 Central region34.32 ± 5.1127.28 ± 7.780.007*0.08831.10 ± 3.110.046*28.71 ± 5.990.010*0.18531.09 ± 3.190.047* Peripheral region28.78 ± 3.8424.78 ± 5.750.033*0.47526.01 ± 3.190.040*25.29 ± 5.350.0496*0.36327.01 ± 4.820.275 Retina Whole scan33.91 ± 3.3930.11 ± 5.440.029*0.51531.16 ± 2.900.024*30.55 ± 5.120.043*0.35532.04 ± 3.370.141 Central region39.16 ± 3.2934.79 ± 5.350.012*0.06237.83 ± 2.820.24535.51 ± 5.680.040*0.14137.94 ± 2.510.263 Peripheral region31.16 ± 3.5725.28 ± 5.800.002*0.11528.04 ± 3.070.016*26.81 ± 6.420.030*0.23229.13 ± 3.580.131VLD (%), mean ± SD SCP Whole scan8.52 ± 0.827.28 ± 1.310.004*0.007*8.30 ± 0.290.3317.29 ± 1.640.015*0.016*8.52 ± 0.890.999 Central region9.89 ± 0.788.97 ± 1.250.022*0.1299.53 ± 0.600.1668.84 ± 1.600.031*0.009*10.10 ± 0.670.436 Peripheral region7.81 ± 0.876.51 ± 1.430.006*0.004*7.70 ± 0.190.6586.59 ± 1.660.018*0.026*7.75 ± 0.940.862Thickness (µm), mean ± SD SCP Whole scan65.53 ± 4.3185.18 ± 10.94<0.001*0.09679.15 ± 8.02<0.001*77.52 ± 9.84<0.001*0.007*68.97 ± 5.730.074 Central region65.37 ± 8.45119.62 ± 19.99<0.001*0.395113.19 ± 20.75<0.001*109.38 ± 21.13<0.001*0.043*97.35 ± 5.99<0.001* Peripheral region65.60 ± 5.0970.83 ± 8.080.043*0.008*64.56 ± 2.680.49063.87 ± 7.560.4670.008*56.80 ± 5.89<0.001* DCP Whole scan157.40 ± 8.90176.42 ± 14.00<0.001*0.576179.15 ± 12.36<0.001*177.23 ± 14.53<0.001*0.028*168.02 ± 5.06<0.001* Central region155.93 ± 5.21192.99 ± 19.27<0.001*0.220201.16 ± 16.27<0.001*194.76 ± 17.56<0.001*0.001*177.991 ± 3.32<0.001* Peripheral region158.03 ± 10.98169.31 ± 13.780.019*0.934169.71 ± 12.450.011*169.72 ± 18.430.044*0.254163.76 ± 7.390.105 Retina Whole scan222.93 ± 9.37261.60 ± 22.60<0.001*0.665258.31 ± 18.29<0.001*254.75 ± 21.00<0.001*0.004*236.99 ± 5.69<0.001* Central region221.30 ± 10.61312.61 ± 37.49<0.001*0.897314.37 ± 36.19<0.001*304.13 ± 35.32<0.001*0.004*275.34 ± 7.43<0.001* Peripheral region223.62 ± 12.34239.74 ± 19.320.011*0.385234.29 ± 14.090.036*233.58 ± 23.470.1570.049*220.56 ± 7.060.411Volume (mm3), mean ± SD SCP Whole scan0.065 ± 0.0040.084 ± 0.011<0.001*0.0890.078 ± 0.008<0.001*0.077 ± 0.010<0.001*0.007*0.068 ± 0.0050.041* Central region0.065 ± 0.0080.120 ± 0.020<0.001*0.4030.113 ± 0.021<0.001*0.109 ± 0.021<0.001*0.042*0.097 ± 0.006<0.001* Peripheral region0.065 ± 0.0050.069 ± 0.0080.0980.011*0.063 ± 0.0020.2090.063 ± 0.0070.3890.008*0.056 ± 0.005<0.001* DCP Whole scan0.155 ± 0.0100.173 ± 0.0150.001*0.6760.175 ± 0.012<0.001*0.175 ± 0.016<0.001*0.0780.166 ± 0.0070.001* Central region0.155 ± 0.0050.193 ± 0.019<0.001*0.2250.201 ± 0.016<0.001*0.195 ± 0.017<0.001*0.001*0.178 ± 0.003<0.001* Peripheral region0.155 ± 0.0130.164 ± 0.0160.0780.9360.164 ± 0.0120.0550.166 ± 0.0210.0910.4700.161 ± 0.0100.129 Retina Whole scan0.220 ± 0.0100.257 ± 0.024<0.001*0.6130.253 ± 0.018<0.001*0.251 ± 0.022<0.001*0.009*0.235 ± 0.006<0.001* Central region0.221 ± 0.0110.313 ± 0.038<0.001*0.9060.314 ± 0.036<0.001*0.304 ± 0.035<0.001*0.004*0.275 ± 0.007<0.001* Peripheral region0.219 ± 0.0140.233 ± 0.0210.046*0.3430.227 ± 0.0140.1480.228 ± 0.0260.2380.1270.218 ± 0.0090.655* P 0.05). Both ischemic CRVO and ischemic CRVO fellow eyes had significantly lower whole and central CVI values compared to controls (P 0.05) (Table 3).Table 3.Choroidal Parameters of the RVO, Fellow, and Healthy Control EyesIschemic CRVO (n = 15)BRVO (n = 15)VariableControl (n = 15)Ischemic CRVO Eyes P vs. Control P vs. FellowFellow Eyes P vs. ControlBRVO Eyes P vs. Control P vs. FellowFellow Eyes P vs. ControlSFCT (µm), mean ± SD236.90 ± 77.81223.92 ± 119.570.7280.357261.33 ± 94.500.447264.81 ± 138.240.5010.887270.83 ± 83.600.260CC-VFD (%), mean ± SD Whole scan43.54 ± 1.1243.33 ± 0.990.5940.69743.13 ± 1.540.41442.84 ± 1.280.1190.39943.14 ± 0.520.219 Central region45.48 ± 0.8145.97 ± 1.510.2640.28345.45 ± 1.050.92245.16 ± 1.250.4120.76545.27 ± 0.660.443 Peripheral region42.57 ± 1.5242.09 ± 1.230.3600.94942.05 ± 2.040.52841.76 ± 1.690.1790.63042.09 ± 0.880.298LMCV-VFD (%), mean ± SD Whole scan63.84 ± 2.5560.14 ± 4.040.006*0.10862.04 ± 1.810.035*60.11 ± 5.700.028*0.040*63.47 ± 2.040.669 Central region68.29 ± 2.1564.52 ± 6.060.031*0.13367.33 ± 3.490.37265.06 ± 5.150.033*0.14067.25 ± 2.510.233 Peripheral region61.52 ± 3.1458.75 ± 4.060.020*0.51359.49 ± 1.490.003*58.57 ± 4.110.036*0.039*60.96 ± 2.040.570CVV (mm3), mean ± SD Whole scan72.87 ± 15.2265.13 ± 24.270.3050.97664.85 ± 26.670.32182.89 ± 41.030.3830.46474.32 ± 17.820.813 Central region81.68 ± 17.5277.05 ± 32.450.6020.57470.16 ± 33.840.25295.00 ± 48.810.3280.33881.88 ± 18.120.976 Peripheral region68.21 ± 15.0062.62 ± 21.940.4560.98162.43 ± 23.670.43177.09 ± 36.810.3990.54270.55 ± 18.0550.702CVI (%), mean ± SD Whole scan30.94 ± 3.6827.78 ± 4.660.049*0.29026.31 ± 2.47<0.001*29.63 ± 5.160.4320.52430.74 ± 4.160.890 Central region34.41 ± 4.3430.29 ± 4.550.018*0.15828.08 ± 3.69<0.001*31.19 ± 4.120.047*0.19733.03 ± 3.480.343 Peripheral region29.02 ± 3.8726.60 ± 4.310.1160.33525.47 ± 1.990.004*28.25 ± 5.020.6450.45029.59 ± 4.550.715* P < 0.05.DiscussionThe present study is the first, to the best of our knowledge, to utilize UWF-SS-OCTA to evaluate both retinal and choroidal alterations in the central and peripheral regions of RVO and RVO fellow eyes. Our findings indicate that both ischemic CRVO and BRVO eyes showed FAZ enlargement and irregularity; we also detected FAZ irregularity in ischemic CRVO fellow and BRVO fellow eyes. Both the affected and unaffected eyes of ischemic CRVO and BRVO patients had decreased VFD, increased thickness, and increased volume in the central and/or peripheral retinal regions. Lower LMCV-VFD and CVI were also observed in ischemic CRVO, ischemic CRVO fellow, and BRVO eyes.The FAZ is the macular capillary-free zone surrounded by interconnected capillaries, with an average area of 0.20 to 0.40 mm2 in healthy individuals.25 Wons et al.26 reported that the size of FAZ may be an indicator of the status of foveal circulation in retinal vasculopathy. In the present study, ischemic CRVO and BRVO eyes had increased FAZ area and perimeter compared to both fellow eyes and controls, which is consistent with previous findings.26–28 The AI is a recently introduced parameter to evaluate deviation of the FAZ shape from a perfect circle. Deng et al.29 found that CRVO eyes had significantly higher AI than controls. Similarly, our study reported increased AI in both ischemic CRVO and BRVO eyes compared to their fellow eyes and healthy control eyes. In addition, we found significantly decreased VFD in ischemic CRVO and BRVO eyes in the SCP, DCP, and full retina compared with the healthy control eyes, consistent with previous findings.11,30–33 It should be noted that the DCP is the principal venous outflow system for the retinal capillary plexuses, and collateral vessels typically develop in the DCP during the evolution of RVO.34 Previous investigations have pointed out that the development of these collateral vessels is negatively associated with retinal VFD.35 RVO could impair retinal venous outflow, cause dilation and tortuosity of vascular segments, and increase the retinal capillary pressure. These various pathologic alterations may promote the exudation of blood and fluid into the intercellular space,36 which might explain the increased retinal thickness and volume observed in RVO eyes compared to healthy control eyes in our study.The choroid, containing the most extensive vasculature in the eye, plays a critical role in supplying oxygen and nutrition to the outer retinal layers. Given the interactions between the retina and the choroid, it is perhaps not surprising that RVO may have an impact on the choroid. Previous studies on SFCT in RVO eyes have yielded inconsistent results, with several reporting increased SFCT in RVO eyes8,37–39 and others finding no difference between RVO and their fellow eyes.14,40,41 Our study found a similar SFCT in ischemic CRVO and BRVO eyes compared to their fellow eyes and controls. Whether RVO could impact the CC-VFD is also a topic of controversy. Some OCTA studies have observed lower CC-VFD in RVO eyes than their fellow eyes and control eyes and the utilization of anti-vascular endothelial growth factor (VEGF) agents could normalize the CC-VFD.9,42,43 Nevertheless, other studies found similar CC-VFD in CRVO and BRVO eyes compared to their respective fellow eyes.15,44 In our analysis using UWF-SS-OCTA, no differences were observed in CC-VFD in ischemic CRVO and BRVO eyes compared to their fellow eyes and control eyes. Although the reasons for the discordant findings regarding the choroid in RVO among these various studies are not well understood, we speculate that differences in gender, axial length, type and stage of RVO, and RVO-associated complications such as ME may play a role.Aribas et al.9 proposed that the size of larger choroidal vessels was either not influenced or only slightly increased in RVO eyes, based on an evaluation of the ratio of the thickness of the Haller layer to the whole choroid,. In this study, we introduced a novel three-dimensional parameter, CVV, to gain a more comprehensive understanding of structural alterations in the LMCV in RVO eyes. Our findings showed that the CVVs in ischemic CRVO and BRVO eyes were similar to their fellow eyes and control eyes. The CVI is a two-dimensional parameter introduced in previous studies to quantitatively analyze choroidal composition and was defined as the ratio of choroidal luminal area to the total choroidal area in OCT B-scans.45 A lower 2D CVI was observed in RVO eyes compared to their fellow eyes and controls.9,13,45 Hwang et al.13 further noted that the CVI in BRVO eyes increased and became similar to that of their fellow eyes after intravitreal injection of anti-VEGF agents. In our study, we computed a more comprehensive 3D CVI as the ratio of CVV to total choroidal volume. Using this 3D approach, we noted that ischemic CRVO eyes had significantly lower CVI values than healthy control eyes in the whole OCTA image and the central region. In addition, BRVO eyes exhibited lower 3D CVI values than healthy control eyes in the central choroidal region, although not in the whole scan or the peripheral region.However, the underlying mechanisms and pathogenesis behind the decreased CVI in RVO eyes remain unclear. The decreased CVI with unchanged CVV in RVO eyes may imply an increased choroidal stromal volume, which may suggest the possibility of choroidal swelling or congestion affecting the stroma. The lower LMCV-VFD in ischemic CRVO and BRVO eyes compared with healthy control eyes might also be explained by the increased choroidal stromal volume. Previous studies have reported that the impaired venous flow in RVO may cause blood and fluid exudation into the intercellular space.36 We speculate that impaired retinal venous outflow may promote extracellular fluid movement toward the choroid, contributing to choroidal stroma swelling and an increase in volume. An alternative hypothesis is that the ischemia-induced production of VEGF may also impact the adjacent choroid, leading to increased choroidal vascular fluid leakage and attendant choroidal stromal edema.46 Julien et al.47 found that anti-VEGF agents could reduce the number of choriocapillaris endothelial cell fenestrations in monkeys. This finding might suggest that anti-VEGF agents have the potential to reduce fluid leakage through fenestrated vascular walls, possibly thereby relieving choroidal stromal swelling. Mitamura et al.48 did find that intravitreal aflibercept injection could significantly reduce the choroidal stromal area in RVO eyes. The observation of a decreased CVI with unchanged CVV in the unaffected fellow eyes of RVO patients compared to healthy control eyes raises the possibility that the reduced CVI might also be attributed to systemic conditions, such as hypertension or other cardiovascular risk factors. Whether isolated choroidal stromal expansion is specific to RVO or may be observed in other conditions requires further study. It is notable that eyes with central serous chorioretinopathy typically feature choroidal luminal expansion,49 but diabetic ME is associated with increased luminal and stromal volumes.50Our findings revealed notable alterations in the retina and choroid of the unaffected fellow eyes of RVO patients. For example, ischemic CRVO fellow eyes were associated with significantly decreased retinal VFD, increased retinal thickness and volume, increased LMCV-VFD, and decreased CVI compared to healthy controls. These findings align with previous research by Park et al.,10 who also reported microvascular impairment in the retina and choroid of RVO fellow eyes. Collectively, these results lend support to the hypothesis that RVO is not solely a localized ocular event but may instead be indicative of broader systemic vascular changes affecting both eyes. The observed retinal microvascular impairment in RVO eyes could arise from a combination of the influence of systemic risk factors prior to the onset of occlusion and the non-perfusion area that develops after vein occlusion. A meta-analysis conducted by Song et al.1 reported various systemic factors associated with a higher risk of RVO, such as advanced age, hypertension, myocardial infarction, stroke, higher total cholesterol, and higher creatinine. Therefore, it may be speculated that the management of systemic factors may be relevant to preventing the progression of RVO.The utilization of UWF-SS-OCTA imaging in this study allowed for the comprehensive visualization of both central and peripheral fundus regions in a single examination. For example, we noticed that, compared to controls, ischemic CRVO fellow eyes had significantly lower peripheral LMCV-VFD, but no significant difference existed in the central region. In BRVO eyes, a lower LMCV-VFD than in their fellow eyes was found in the peripheral region but not in the central region, which would typically be the only region evaluated by conventional OCTA imaging. The importance of employing UWF imaging assessments has also emerged in other retinal vascular diseases, such as in DR, where peripheral lesions are thought to be of prognostic importance and may impact future staging systems for DR.51,52 UWF-SS-OCTA, enabling the visualization of the peripheral fundus region, is a promising imaging technique to characterize the retinal and choroid vasculature.In OCTA images, minimizing volumetric projection artifacts from the SCP is crucial for achieving clear visualization and accurate quantification of vessel flow in the outer retinal layers, particularly in the DCP layer. In our study, we observed that the peripheral retinal arteries and veins remained visible in the DCP layer of UWF-SS-OCTA images. This finding is consistent with the report by Hormel et al. (IOVS 2023;64:ARVO E-Abstract 4422), who also observed the visualization of peripheral retinal arteries and veins in UWF-SS-OCTA images. The visibility of these vessels in the DCP layer can be attributed to the thinner nature of the peripheral retina and the partial location of retinal vessels within the DCP layer, rather than as a result of projection artifacts from SCP. Further supporting this explanation is the clear presence of retinal arteries and veins in the DCP layer, as demonstrated in the UWF-SS-OCT B-scan images of our study.Fluorescein angiography has traditionally served as the standard imaging modality for assessing retinal perfusion in cases of RVOs within clinical practice, but it presents important limitations, including invasiveness, time-consuming procedures, and the potential for severe systemic risks. The introduction of OCTA has revolutionized the non-invasive evaluation of fundus vasculature, eliminating the need for invasive dye administration.53 Our study found that OCTA could offer clear visualization of non-perfusion areas in RVO-affected eyes, demonstrating its potential to distinguish between ischemic and non-ischemic RVO cases. This distinction is invaluable for informing the prognosis and guiding the management of RVO. Furthermore, OCTA provides depth-resolved angiographic images, enabling quantitative analysis of choroidal sublayers, which are largely overlooked in fluorescein angiography. Our study, utilizing the innovative UWF-SS-OCTA, has revealed specific choroidal alterations in RVO eyes that have the potential to ignite researchers’ interest in further exploring the relationship between these alterations and the progression of RVO in prospective studies. These choroidal alterations could potentially serve as prognostic parameters for the effective management of RVO cases. Moreover, the notable retinal and choroidal alterations observed in the unaffected fellow eyes of RVO patients suggest a link between RVO and systemic vascular changes and underscore the importance of systemic management for RVO patients in clinical practice.Our study is not without limitations that should be considered when assessing our findings. First, our sample size was relatively small; thus, we may have been underpowered to detect small differences in parameters between groups. However, despite this, a number of significant differences, including those between fellow eyes and healthy control eyes, were observed. Second, as this was a hospital-based study, the population, particularly the healthy controls, may not reflect the broader community. Third, RVO patients included in this study only underwent single time-point OCTA imaging, and this may have occurred at different time points following the RVO diagnosis. Variability in disease duration may impact our results, as longitudinal alterations in the circulation may affect these patients. Further prospective longitudinal studies with larger study populations will be necessary to address these limitations. Nonetheless, we believe our findings underscore the importance and value of widefield SS-OCTA imaging.In conclusion, the affected and unaffected fellow eyes of RVO patients showed both central and/or peripheral structural and vascular changes affecting the retina and choroid. UWF-OCTA, enabling the visualization of the more peripheral fundus regions, offers a promising approach to more fully characterize vascular alterations in RVO.

PMC

Case Reports in Oncology

37384202

PMC10294126

5-31-2023

10.1159/000530066

Malignant Struma Ovarii (Papillary Carcinoma) with Hyperthyroidism: A Case Report and Literature Review

Yang Bo, Zhong Lijuan, Peng Lilin, Huang Ting, Zhu Di, Lu Yuanzhi

AbstractMalignant struma ovarii (MSO) is an extremely rare monodermal ovarian teratoma. Preoperative diagnosis and intraoperative freezing diagnosis are exceedingly difficult due to the rarity of the disease and its clinically noncharacteristic manifestations with less than 200 reports in the current literature. In this paper, a case of MSO (papillary carcinoma) with hyperthyroidism was discussed in terms of its epidemiology, clinicopathology, molecular features, treatment, and prognosis.

IntroductionMalignant struma ovarii (MSO) is an extremely rare monodermal teratoma of the ovary; the early description of this disease was in 1889 . Most tumors occur in single ovary, and only <5% of cases occur in both ovaries . The most common histological subtypes of MSO were papillary carcinoma (70%) and follicular carcinoma (30%) . There are few reports in the literature. Here we found a case of MSO (papillary carcinoma) with hyperthyroidism. In addition, the immunohistochemical results of the patient indicated positive BRAF, which suggested that the patient was highly likely to have BRAF mutations. Currently, BRAF mutations occur in common diseases such as melanoma, thyroid papillary carcinoma, and colorectal cancer and have also been reported to be found in biliary tract carcinoma and lung adenocarcinoma in recent years [4–6].Case PresentationA 41-year-old woman was referred to our hospital on August 16, 2021, for further treatment after B-ultrasound examination at another hospital revealed a left ovarian cyst. The patient had a history of chronic nephritis for 12 years, and long-term urine routine examination showed positive for occult blood. The patient was found to have hyperthyroidism at another hospital in February 2021 and was treated with the “I131” protocol and subsequently hypothyroidism in July 2021. Since the patient was treated in another hospital, no information such as 131I thyroid imaging was obtained. The patient is currently receiving regular oral thyroxine treatment. After hospital admission, B-mode ultrasonography indicated that a cystic solid occupation with undetermined nature was observed in the left ovary, while the size of the uterus was normal with no obvious lesions, and serological examination showed no obvious abnormality.After admission to hospital, the patient received laparoscopic left adnexectomy. Freezing section H&E staining showed that adenoid structure was observed in the cystic wall tissue under microscope accompanied by mild atypia of epithelia, and ovarian serous cystadenoma was considered as the preliminary diagnosis during operation. Borderline lesions and low-grade cystadenocarcinoma were further excluded by H&E staining and immunohistochemistry. Postoperative specimens were generally found to be cystic solid masses, with a volume of 3.7 cm × 3.3 cm × 1.0 cm and a thickness of 0.1–0.4 cm. The surface was covered with coffee-like substances, and the section was grayish-brown (Fig. 1). Microscopically, tumor cells with papillary structure were observed in the ovarian tissue with infiltrating growth pattern, abnormal cell polarity, and overlapping and enlarged nuclei, which were ground-glass like, with nuclear sulcus, nuclear pseudoinclusion body, and gravel body, and slightly normal thyroid follicular epithelium could be seen in some areas (Fig. 2a–d). Immunohistochemical staining showed that tumor cells in the region of papillary carcinoma were positive for TTF-1, CK19, galectin-3, PAX-8, and BRAF (Fig. 3a–e) but negative for TPO (Fig. 3f), CK20, P16, and WT1 and approximately 5% positive for Ki-67. The final pathological diagnosis was consistent with MSO (papillary carcinoma).Fig. 1.Gross mass was cystic solid. The surface was covered with coffee-like substance, and the section was gray-brown.Fig. 2.H&E staining. a At low magnification, the tumor tissue was dominated by papillary structures (HE×40). b Tumor nuclei are crowded or overlapped (HE×100). c Ground-glass nuclei, nuclear sulcus, and nuclear pseudoinclusion bodies (HE×200) were observed at high magnification. d Sand and gravel bodies are seen in the interstitium (HE×200).Fig. 3.Immunohistochemical staining for CK19 (a), galectin-3 (b), BRAF (c), TTF-1 (d), and PAX-8 (e) was positive and for TPO (f) was negative in tumor tissue as indicated in the figures.After surgery, combined with the pathological results, the clinician informed the patient that the tumor was of reproductive origin and had a low risk of malignant potential. After comprehensive discussion by multidisciplinary team, the patient was suggested to be enter into regular follow-up (once every 3 months within the first year) program. Patient’s condition was stable until June 1, 2022, and no tumor recurrence was observed.DiscussionMSO tends to occur in middle-aged patients. According to statistics of a previous study , the age of patients diagnosed with MSO ranged from 12 to 78 years, with an average age of 42.55 years. In the study, involving 144 female eligible patients from 1970 to 2020, all patients came from 27 countries and five continents, including China (42 cases, 29.37%), the USA (32 cases, 22.38%), and Spain (9 cases, 6.29%), making up the top three. Asia has the largest proportion of cases (45.45%), with no cases reported in Africa. The highest incidence occurs between the ages of 31 and 40 years. Patients over 60 years of age accounted for only 13.98% of cases. In addition, they found that the age at which patients were diagnosed was normally distributed. Both benign and MSO may have no specific clinical symptoms. An observed and described study reported that 41.2% of 34 patients were asymptomatic and the diagnosis was incidental . In the same study, the most common chief complaint, if symptomatic, was lower abdominal pain (20.6%); other related symptoms included abnormal vaginal bleeding, low back pain, and frequent urination. It has been reported that only 8% of patients may also have hyperthyroidism.MSO has been reported to be molecular-genetically similar to primary thyroid carcinoma. In 2018, Gomes Lima et al. reported the rare occurrence of two synchronous independent papillary thyroid carcinomas in the ovary and thyroid with different RAS mutations (NRAS Q61R and HRAS Q61R, respectively). In addition, second-generation sequencing (NGS) detection demonstrated that germline mutation of KIT V530I was detected in ovarian papillary thyroid carcinoma and concurrent papillary thyroid microcarcinoma . These findings suggest that similar molecular mechanisms may contribute to the development of thyroid carcinoma in both orthotopic and ectopic thyroid tissues. Roberta Poli et al. reported that activation of driver mutations was detected in 5 of 6 cases of ovarian thyroid papillary carcinoma by second-generation sequencing (NGS), including 2 cases with NRAS mutation, 2 cases harboring BRAF mutations, and one case with JAK3 oncogene mutation. Chung et al. found that a patient with MSO had high microsatellite instability after late relapse. Although there are several reports of molecular changes in MSO, the conclusion of genetic determinants in MSO is still controversial. Therefore, more investigations are needed to be conducted to elucidate the genetic drivers of MSO.There is no universal treatment guideline for MSO. The current clinical treatment regimens for this disease include unilateral adnexectomy, abdominal hysterectomy plus bilateral adnexectomy, and adjuvant chemotherapy. In addition, there is a lack of evidence for thyroid management with thyroidectomy and radioiodine ablation following surgical removal of primary ovarian tumors. Some authors recommend routine thyroidectomy followed by I131 treatment, regardless of the presence of metastasis at diagnosis, to avoid local recurrence and distant metastasis. Asc et al. reported a case of MSO treated with cell depletion and thermal intraperitoneal chemotherapy. Favorable survival rates (95.3%, 88.7%, and 88.7% at 5, 10, and 20 years of OS, respectively) were found regardless of treatment. Therefore, the overall prognosis of MSO is relatively good, but pulmonary and bone metastases of MSO have been reported in recent years [14, 15]. Therefore, all cases of MSO should be followed regularly for at least 10 years after operation and adjuvant therapies.In conclusion, this case of MSO (papillary carcinoma) with hyperthyroidism is very meaningful and worthy of our deep understanding and consideration. First, MSO poses a diagnostic challenge. Both benign and malignant ovarian masses may be asymptomatic or nonspecific. Tumor markers, including CA125, are of no value. Ultrasound, CT, and MRI lack distinct features, suggesting that multidisciplinary team input is critical. Second, this case report is positive for BRAF in immunohistochemistry, suggesting that the possibility of BRAF mutation is extremely high. I personally think that BRAF mutation has a certain relationship with MSO, but whether BRAF mutation is the direct driving factor of MSO remains to be determined; further studies are needed to prove it. Finally, with the vigorous development of molecular testing, more and more diseases have been detected with gene changes. At present, BRAF mutations are common in melanoma, papillary thyroid cancer, colorectal cancer, and other diseases. In recent years, it has also been reported in biliary tract cancer, BRAF mutations have been found in lung adenocarcinoma, and they have also done some targeted therapy trials, and some trials have shown good results [4–6]. Although the prognosis of MSO is very good, there have been more and more reports of lung metastasis and bone metastasis of MSO in recent years. Should we also use targeted therapy for patients with MSO with BRAF mutation? More cases and further studies are needed to be sure. Hope to bring good news to patients with MSO in the near future.ConclusionMSO is a very rare monodermal teratoma of the ovary. We report a case of MSO (papillary carcinoma) with hyperthyroidism. This patient was found incidentally by physical examination and had no clinical symptoms other than hyperthyroidism. Therefore, the great challenge for clinicians is that it is difficult to detect the disease, which delays the treatment of patients and shortens the survival time of patients. In addition, immunohistochemical BRAF positivity indicated a high probability of BRAF mutation, which is similar to papillary thyroid carcinoma occurring in the thyroid. Finally, we hope that this disease will get the attention of gynecologic oncologists, endocrinologists, surgical oncologists, and pathologists. The CARE Checklist has been completed by the authors for this case report, attached as online supplementary material (for all online suppl. material, see authors thank Professor Lu Yuanzhi for his guidance in this paper.Statement of EthicsThis study protocol was reviewed, and the need for approval was waived by the Ethics Review Board of Jinan University First Affiliated Hospital. Written informed consent was obtained from the patient for publication of this case report and any accompanying images.Conflict of Interest StatementThe authors declare that they have no conflict of interest regarding the publication of this case report.Funding SourcesThis case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Author ContributionsBo Yang conceived, designed, and took entire responsibility for publishing this study under supervision of Yuanzhi Lu. Bo Yang, Lijuan Zhong, Lilin Peng, Ting Huang, and Di Zhu helped in drafting and editing the manuscript. All authors provided intellectual input to the study and approved the final version of the Manuscript.

PMC

HemaSphere

37234821

PMC10208707

5-23-2023

10.1097/HS9.0000000000000886

Bleeding and Thrombosis in Patients With Cirrhosis: What’s New?

Lisman Ton

The liver is a central organ in the hemostatic system as it is the site of synthesis of many proteins involved in the activation and regulation of coagulation and fibrinolysis. In addition, the liver synthesizes thrombopoietin, which is a key hormone involved in the production of platelets. Patients with advanced chronic liver disease or acute liver failure develop alterations in their hemostatic system that are at least partly related to decreased synthetic capacity of the diseased liver. In addition, hemostatic changes in patients with liver disease may be related to systemic or intrahepatic activation of coagulation with consumption of hemostatic components. Finally, chronic endothelial cell activation resulting in elevated levels of endothelial-derived proteins such as von Willebrand factor contributes to the hemostatic changes associated with liver disease.Although historically liver diseases were considered as bleeding disorders, nowadays it is recognized that liver diseases are not only associated with bleeding but also with thrombotic complications.1,2 Because of the absence of high-quality clinical evidence, it is still unclear how to best prevent or treat bleeding and thrombosis in patients with liver diseases. The combination of laboratory studies with clinical observations, however, has led to a more rational approach to hemostatic management. In recent years, a number of international societies have issued clinical guidance documents in this area that share a number of concepts.3–7 First, the concept of rebalanced hemostasis has become widely embraced.8 The recognition that patients with liver disease have concomitant changes in both prohemostatic and antihemostatic systems leading to a relatively preserved hemostatic system has led to a much more restrictive approach to prophylactic correction of hemostasis with the aim to prevent bleeding, for example, before invasive procedures. It is now widely accepted that routine diagnostic tests of hemostasis, such as the platelet count and the prothrombin time, are unsuitable as indicators of hemostatic capacity in patients with cirrhosis.9,10 As a consequence, routine prophylactic correction of a low platelet count and a prolonged prothrombin time by infusion of platelet concentrates or fresh frozen plasma is increasingly discouraged.3–7 Second, the recognition of a hypercoagulable state in patients with cirrhosis, for example evidenced by enhanced in vitro thrombin generating capacity and an elevated risk for development of venous thromboembolism,11,12 has led to increased awareness for the role of thromboprophylaxis, even in those patients with thrombocytopenia and/or prolonged prothrombin time.3,7Here, the author outlines the recent developments in the prevention and management of bleeding and thrombosis in patients with liver disease.HEMOSTATIC VERSUS NONHEMOSTATIC BLEEDINGSpontaneous and procedure-related bleeds have been feared complications of patients with liver disease. The most dramatic bleeding events have likely been witnessed during the early days of liver transplantation. A report from the 1980s on blood product use in >600 patients receiving a liver transplant in Pittsburg showed massive blood product use with red blood cell (RBC), fresh-frozen plasma (FFP), and platelet concentrate requirements of 29, 37, and 32 units per transplant on an average.13 Interestingly, blood product use during liver transplantation has decreased substantially over time, and a 2015 report on >700 patients receiving a liver transplant in Quebec, Canada showed transfusion-free liver transplantation in the majority of patients with RBC, FFP, and platelet concentrate requirements of 0.5, 0.3, and 0.2 units per transplant on an average.14 Spontaneous bleeds, notably upper gastrointestinal bleeds, complicate chronic liver disease until today. Such bleeds may still be dramatic and are associated with morbidity and mortality.15Historically, bleeding complications in patients with cirrhosis were directly attributed to the hemostatic changes in these patients. The prolonged prothrombin time and a low platelet count were at that time interpreted as evidence for a profound hypercoagulable state. Nowadays, it is recognized that patients with cirrhosis are in a rebalanced hemostatic status, even in the presence of profound changes in the prothrombin time and platelet count. More importantly, it is now recognized that many bleeding complications in patients with cirrhosis are unrelated to hemostatic failure.4 For example, variceal bleeding is related to portal hypertension with a negligible role for the hemostatic system. The observations that anticoagulant treatment is not associated with a high risk of variceal bleeding16 and that the severity and outcome of variceal bleeding is similar in patients who were or were not using anticoagulant drugs at the time of the bleed17 support the concept that variceal bleeding is independent of hemostatic dysfunction. Also bleeding during liver transplantation may in part be related to portal hypertension, and some centers perform preoperative phlebotomy with the aim to reduce pressure-related bleeding.14 Bleeding may also be caused by mechanical injury. Such bleeds include inadvertent puncture or laceration of vessels during invasive procedures including liver transplantation. Finally, bleeds that are likely related to hemostatic failure may occur, including bruising, epistaxis, oozing from puncture wounds, and dental bleeds. Such bleeds are largely mild and rarely necessitate hemostatic therapy.Recent clinical guidance documents advise against prophylactic administration of blood products to prevent spontaneous or procedure-related bleeding.3,4,6,18 When bleeding occurs, clinicians are inclined to use prohemostatic treatment with blood products, factor concentrates, or antifibrinolytics. However, as many bleeds in patients with cirrhosis are unrelated to hemostatic failure, it has been argued that hemostatic treatment should not be used in the initial phases of treatment.19 Rather, portal hypertension-related bleeds should be managed by pharmacological interventions that reduce portal pressure combined with local measures to stop the bleeds (eg, endoscopic interventions to treat a ruptured varix). Mechanical bleeds may be treated by local measures, for example, local pressure in case of a bleed following dental extraction or ligation of an injured vessel during (transplant) surgery. Recent studies in patients with variceal bleeding have shown that hemostatic treatment with, for example, fresh frozen plasma or tranexamic acid is ineffective and may do harm,20,21 which supports a restrictive approach to hemostatic therapy in bleeding cirrhosis patients. Only in those patients with intractable bleeds or in patients with significant bleeding during transplant surgery, prohemostatic treatment is indicated. Low volume products such as fibrinogen concentrate and prothrombin complex concentrate may be preferred over fresh frozen plasma and platelet concentrate, as the latter may increase portal pressure and exacerbate portal hypertension-related bleeds.22 Furthermore, large quantities of fresh frozen plasma are required to normalize coagulation parameters, and the yield of platelet concentrates may be low.23In aggregate, the recognition that bleeding in patients with cirrhosis frequently is unrelated to hemostatic failure has led to a restrictive approach to prohemostatic therapy, both in prophylactic and in a treatment setting. Figure 1 summarizes the types of bleedings and potential therapeutic strategies.Figure 1.Categories of bleeding in liver disease. Bleeding in patients with liver disease may be due to mechanical injury (by inadvertent laceration of a vessel during surgery or a minor invasive procedure), portal hypertension-related causes (eg, variceal bleeding), or hemostatic failure (bleeding following dental extraction, bruising, and bleeding from puncture wounds). Shown are strategies to prevent and treat these different types of bleeding complications. FFP = fresh-frozen plasma.BLEEDING RISK IN RELATION TO HEMOSTATIC CHANGES IN THE CRITICALLY ILLThe concept of rebalanced hemostasis has been developed using studies in patients with compensated cirrhosis or in stably decompensated patients.24–26 Studies in patients with acute liver failure (without underlying chronic liver disease) suggested that rebalanced hemostasis also remains in these critically ill patients, which have profound hemostatic abnormalities.27,28 Importantly, bleeding complications in patients with acute liver failure are rare. In a study of >1700 patients with acute liver failure, clinically significant bleeding occurred in only 11% of patients, despite a median international normalized ratio of 2.7 and platelet count of 96 × 109/L on admission.29More recent studies have assessed hemostatic changes in critically ill patients with cirrhosis.30–33 Also in these patients, there is laboratory evidence for rebalanced hemostasis, although there are notable hypercoagulable and hypocoagulable features in the hemostatic system of these patients.32 The combination of both hypercoagulable and hypocoagulable features appears exaggerated in patients with additional complications including infection, renal failure, and progression from acute decompensation to acute-on-chronic liver failure.34Bleeding risk in critically ill patients with cirrhosis is modest. In a study of >600 patients, bleeding occurred in 14%, and was mostly related to portal hypertension.35 Indeed, hemostatic changes are not associated with bleeding risk in critically ill patients with cirrhosis. Rather, systemic inflammation was associated with bleeding risk. Similarly, bleeding in patients with acute liver failure was independent of hemostatic changes, but also seems related to inflammation.27Although bleeding risk in critically ill cirrhosis patients appear independent of hemostatic changes, it has been proposed that decompensating events such as acute kidney injury, infection, and progression of disease are associated with hypocoagulable changes that contribute to bleeding.34 There is some clinical evidence that acute kidney injury in patients with cirrhosis indeed is associated with an increase bleeding risk,36 though the clinical evidence is based on a limited number of patients that bled. Although advanced hemostatic testing with viscoelastic whole blood tests or whole blood platelet function tests have been proposed to examine the hemostatic status of critically ill cirrhosis patients with decompensating events,34 there is little evidence that prohemostatic interventions guided by such laboratory tests are useful in reducing bleeding risk. For now, it is important to recognize the limitations of hemostatic testing in predicting bleeding risk, and to understand that we do not have clear evidence that hemostatic therapy may be helpful. Additional study clearly is required to understand the best practices to prevent or treat bleeding in critically ill cirrhosis patients.PORTAL VEIN THROMBOSISAlthough historically bleeding was the main concern in patients with cirrhosis, in recent years the focus has shifted toward thrombotic disease. Patients with cirrhosis not only experience bleeding complications, but are also at risk for deep vein thrombosis and pulmonary embolism, stroke, and myocardial infarction.1 Prevention and treatment of these complications is complicated by the cirrhosis-associated hemostatic changes.37 Next to these well-known thrombotic diseases, cirrhosis may be complicated by portal vein thrombosis (PVT), which is a rare disease in the general population, but occurs frequently in the sicker cirrhosis patients. Figure 2 outlines the recent insights in the pathogenesis and treatment of PVT that will be discussed below.Figure 2.Pathogenesis of portal vein thrombosis. The portal vein wall develops intimal thickening in patients with cirrhosis, whereas no intima is present in a healthy portal vein wall. In patients with portal vein thrombosis, the intima thickening has progressed and may be accompanied by a fibrin-rich thrombus within the portal vein lumen. PVT = portal vein thrombosis.PVT is an unusual thrombotic disease, as it is frequently asymptomatic and recognized incidentally during routine imaging procedures. There are other notable differences between PVT and other venous thrombotic diseases. For example, the portal vein lacks venous valves, which are points of origin of deep vein thrombosis of the leg. In addition, whereas acquired or hereditary hypercoagulable states are recognized risk factors for deep vein thrombosis and pulmonary embolism, and there is accumulating evidence that hypercoagulability does not form a risk factor for PVT.38–40 Rather, a reduction of flow in the portal vein, related to portal hypertension, appears to be the major risk factor for PVT.38 In line with these observations, it has recently been shown that portal vein thrombi often do not contain classical thrombus components such as fibrin and platelets.41 Rather, portal vein thrombi appear to consist principally from a pronounced thickening of the portal vein wall, notably the intima. Only in 30% of portal vein thrombi, a fibrin-rich structure was observed on top of the thickened intima. As intimal thickening of the portal vein also occurs in patients with cirrhosis without PVT, but not in healthy individuals, it is tempting to speculate that intimal thickening rather than thrombus formation is the initiating trigger of PVT development.My group has, therefore, proposed that PVT may be a misnomer, and that portal vein stenosis or portal vein obstruction may be a better term for this complication in patients with cirrhosis.41,42 Importantly, anticoagulant treatment frequently is not effective in dissolving a portal vein thrombus, reinforcing the notion that this may not be a classical thrombotic disease. In meta analyses, ~40% of portal vein thrombi recanalize without anticoagulation, whereas around 60%–70% recanalizes with anticoagulation.43,44 Thus, although there is a definite effect of anticoagulation, the proportion of patients that benefit from anticoagulation is modest compared with the benefit of anticoagulation in the treatment of deep vein thrombosis and pulmonary embolism.A recent individual patient data meta-analysis has demonstrated that anticoagulation in patients with cirrhotic nontumoral PVT provides a clear survival benefit in patients with cirrhosis.43 Interestingly, this effect of anticoagulation was independent of PVT severity or recanalization, but was proportional to the duration of anticoagulant treatment. As previous studies have demonstrated that PVT per se does not increase mortality risk,40 these data strongly suggest that anticoagulation has a PVT-independent effect on outcome. Thus, this study suggests that portal vein recanalization should not be considered as the goal of anticoagulant treatment in cirrhotic PVT. As anticoagulant therapy also is associated with a survival benefit in patients with cirrhosis without PVT,45 anticoagulant therapy should perhaps be broadly considered with the aim of delaying decompensation and reducing mortality. The ongoing multicenter cirroxaban randomized trial (clinicaltrials.gov NCT02643212) is specifically testing the effects of anticoagulant treatment on the outcome of cirrhosis.CONCLUSIONThe combination of clinical and fundamental studies has tremendously increased our understanding of the complex hemostatic changes in patients with cirrhosis, and have led to a more rational approach to the prevention or treatment of both bleeding and thrombotic complications. Ongoing work will further refine the current clinical guidance documents, which hopefully will improve the quality of life and outcome of patients with liver diseases.AUTHOR CONTRIBUTIONSTL conceived of and wrote the manuscript.DISCLOSURESThe author has no conflicts of interest to disclose.SOURCES OF FUNDINGThe author declares no sources of funding.

PMC

Heliyon

PMC10907525

2-13-2024

10.1016/j.heliyon.2024.e26077

Exogenous application of nano-silicon, potassium sulfate, or proline enhances physiological parameters, antioxidant enzyme activities, and agronomic traits of diverse rice genotypes under water deficit conditions

Abd-El-Aty Mohamed S., Kamara Mohamed M., Elgamal Walid H., Mesbah Mohamed I., Abomarzoka ElSayed A., Alwutayd Khairiah M., Mansour Elsayed, Ben Abdelmalek Imen, Behiry Said I., Almoshadak Ameina S., Abdelaal Khaled

Water deficit is a critical obstacle that devastatingly impacts rice production, particularly in arid regions under current climatic fluctuations. Accordingly, it is decisive to reinforce the drought tolerance of rice by employing sustainable approaches to enhance global food security. The present study aimed at exploring the effect of exogenous application using different biostimulants on physiological, morphological, and yield attributes of diverse rice genotypes under water deficit and well-watered conditions in 2-year field trial. Three diverse rice genotypes (IRAT-112, Giza-178, and IR-64) were evaluated under well-watered (14400 m3/ha in total for the entire season) and water deficit (9170 m3/ha) conditions and were exogenously sprayed by nano-silicon, potassium sulfate, or proline. The results showed that drought stress substantially decreased all studied photosynthetic pigments, growth traits, and yield attributes compared to well-watered conditions. In contrast, antioxidant enzyme activities and osmoprotectants were considerably increased compared with those under well-watered conditions. However, the foliar application of nano-silicon, potassium sulfate, and proline substantially mitigated the deleterious effects of drought stress and markedly enhanced photosynthetic pigments, antioxidant enzyme activities, growth parameters, and yield contributing traits compared to untreated stressed control. Among the assessed treatments, foliar spray with nano-silicon or proline was more effective in promoting drought tolerance. The exogenous application of proline improved chlorophyll a, chlorophyll b, and carotenoids by 21.4, 19.6 and 21.0% followed by nano-silicon treatment, which enhanced chlorophyll a, chlorophyll b, and carotenoids by 21.1, 17.6 and 9.5% compared to untreated control. Besides, the application of proline demonstrated a superior improvement in the content of proline by 52.5% compared with the untreated control. Moreover, nano-silicon exhibited the maximum enhancement of catalase and peroxidase activity compared to the other treatments. The positive impacts of applied exogenously nano-silicon or proline significantly increased panicle length, number of panicles/plant, number of grains/panicle, fertility percentage, 1000-grain weight, panicle weight, and grain yield, compared to untreated plants under water deficit conditions. In addition, the physiological and agronomic performance of evaluated rice genotypes significantly contrasted under drought conditions. The genotype Giza-178 displayed the best performance under water deficit conditions compared with the other genotypes. Consequently, the integration of applied exogenously nano-silicon or proline with tolerant rice genotype as Giza-178 is an efficient approach to ameliorating drought tolerance and achieving agricultural sustainability under water-scarce conditions in arid environments.

1IntroductionRice (Oryza sativa) is the staple food for almost half of the world's people . It is grown on about 165 × 106 ha, yielding 787 × 106 tonnes of rice annually . Rice production should be doubled by 2050 due to global population growth . With an estimated production of 6.1 million tonnes per year and an area of over 0.5 million hectares, Egypt is the biggest rice producer in the Middle East . However, this production is endangered by limited water supply and rising population. Drought is widely recognized as the greatest threat to global rice production and food security. Water scarcity causes yield losses of up to 50% worldwide [5,6]. Climate change will increase drought and damage water resources. Rice requires around 2500 L of water to produce 1 kg of grain during its life cycle . Water scarcity disrupts plant biochemical and physiological processes, reducing growth and production. Water deficits during germination, seedling growth, tillering, flowering, and grain filling cause high yield losses. At the vegetative stage, drought causes senescence of leaves, lessened photosynthesis, limited leaf extension and tillering, stunted plant growth, and ultimately decreased grain yield [8,9]. Water shortages generate reactive oxygen species (ROS) which can destroy plant metabolism by denaturing enzyme activity [10,11]. ROS peroxides biological membranes disrupting transport . Plants have adaptation mechanisms to cope with water scarcity, such as increased leaf water potential, improved root architecture, improved osmotic adjustment, increased proline accumulation, and increased leaf rolling and stomatal closure [13,14]. They also have effective enzymatic and non-enzymatic antioxidant defenses against ROS-induced oxidative damage [15,16]. Consequently, it is necessary to enhance the drought tolerance of field crops and alleviate the negative effects of water scarcity by utilizing innovative appropriate approaches. Various agricultural practices including applied exogenously bio-stimulative chemicals, growth regulators, or osmoprotectants, can protect plants from drought stress and enhance tolerance [, , , ].The exogenous application of safe and effective substances is a crucial strategy for improving plant growth, development, production, and quality under drought stress . Silicon application boosts rice root growth, physiological processes, shoot weight, and crop yield by 10–20% . It also helps in maintaining mineral nutrition, membrane integrity, stress tolerance defense, and photosynthesis efficiency. Nano-materials have recently developed and become a popular approach to alleviate various environmental challenges in multiple fields . Research has shifted toward green nanoparticle manufacturing and its use to alleviate abiotic and biotic stresses. Silicon nanoparticles (Si-NPs) improve silicon uptake and confer stress tolerance in crops against various abiotic and biotic stresses. Due to its small microscopic size, it possesses better physicochemical properties than bulk silicon [24,25]. It displays a higher surface area, increased surface solubility and reactivity, and many well-categorized surface characteristics . Particle size affects absorption and transit into plant cells . Additionally, nanoparticles interact with plant cells to transport chemicals that regulate plant metabolism and various physiological functions. Hence, the applied foliar Si-NP improves growth and yield for rice plants grown in water-deficit conditions . It also boosts leaf-relative water content and antioxidant enzyme activity and decreases oxidative stress under drought stress . However, there is still a lack of understanding of how Si-NP minimizes and mitigates drought stress damage in plants.Proline as a compatible solute plays a dual role in maintaining osmotic balance and preserving the integrity of cellular components and organelles [30,31]. Proline scavenges free radicals, protecting plants from drought-induced ROS damage [32,33]. Subsequently, the exogenous application of proline effectively mitigates the detrimental effects of drought [34,35]. This is achieved by enhancing turgor potential, reducing oxidative stress caused by ROS, maintaining osmotic balance, and boosting the activity of antioxidant enzymes . As a result, photosynthesis is improved, and the amount of oxidative damage is reduced .Potassium (K) is a vital nutrient for plants, as it regulates numerous physiological processes that govern plant growth, yield, and quality attributes . It regulates the function of stomata in transpiration and photosynthesis, including the maintenance of photophosphorylation, plant turgor, enzyme activation, and the transfer of photoassimilates. applied exogenously of K can attenuate the deleterious effects of drought on rice by improving various physiological processes including protein synthesis, enzyme activation, photosynthesis, water relations, and stomatal movement . Moreover, it enhances water stress tolerance by increasing nutrient and water uptake and reducing transpiration water loss . Consequently, K foliar spray enhances plant growth, crop productivity, and drought tolerance of rice plants grown under water-stress conditions [40,41]. Research focusing on the exogenous application of nano-silicon, potassium sulfate, and proline and their potential contributions to alleviating the detrimental effects of drought stress while enhancing rice productivity under water-deficit conditions, particularly in field settings, is currently limited. Drawing on insights from earlier studies, we posited that the exogenous application of nano-silicon, potassium sulfate, or proline could significantly enhance rice plant growth and productivity by improving the efficiency of physiological parameters and antioxidant enzyme activities. In light of this, our study aimed to explore the impact of exogenously applied nano-silicon, potassium sulfate, or proline on photosynthetic characteristics, antioxidants, growth, and yield parameters of diverse rice genotypes grown under water deficit conditions. This knowledge can help identify effective drought tolerance inducers to enhance crop performance and tolerance to drought stress.2Materials and methods2.1Experimental site and plant materialsThe trial was performed during the two summer growing seasons of 2021 and 2022 at Sakha Agricultural Research Station, Egypt (31°6′N 30°56′E). The site is described by an arid and hot climate with no precipitation in the summer season. The maximum and minimum temperatures and relative humidity of both growing seasons at the experimental site were collected and displayed in table S1. Soil samples were collected before sowing (0–30 cm depth) and were analyzed (table S2). The soil analysis displayed that the soil was clay throughout the profile (56.0% clay, 12.0% silt, and 32.0% sand). Electrical conductivity, organic matter, and pH were 3.04 dS m−1, 1.35 g kg−1, and 8.15, in the same order. Three diverse rice genotypes; IRAT-112 (drought tolerant); Giza −178 (moderately tolerant) and IR-64 (drought sensitive) were utilized in the current study. The selected genotypes were chosen based on their drought tolerance from previous preliminary screening trials (unpublished data). The origin and pedigree of the evaluated genotypes are shown in table S3.2.2Experimental design and treatmentThe field trial was applied in a strip-split-plot design with three replications. The vertical plots were designated for irrigation treatments, the horizontal plots were dedicated to foliar applications, and the rice genotypes as subplot factor. The vertical plots were represented by 72 units (720 m2) and the horizontal plots were represented by 36 experimental units (360 m2). The seeds of each genotype were sown in the nursery on May 20th. At 30 days from sowing, seedlings were pulled from the nursery and transplanted into plots (10 m2) in 20 × 20 cm spacing using one seedling per hill. The irrigation was applied according to the standard practice in the studied region using surface irrigation. The amount of irrigation water for rice cultivation is determined annually in various regions of Egypt based on soil type, climatic variables, and water requirements by the Department of Water Requirement and Field Irrigation belongs to the Egyptian Ministry of Agriculture and Land Reclamation. The recommended irrigation amount, set at 14400 m3/ha, was intentionally decreased by 35% to induce water stress, resulting in an application of 9170 m3/ha. The applied water amount for each irrigation treatment was measured employing a flow meter. The applied foliar treatments were potassium sulfate K2SO4 (at the rate of 2 ml/L); proline (at the rate of 2 g/L) and nano-silicon (at the rate of 0.4 g/L) as nano-silicon dioxide 20–30 nm particle size with a purity of 99.5 %, versus untreated control. The untreated control plants were applied with distilled water)and a spreading agent only. Treatments were sprayed twice, at mid-tillering and panicle initiation. was applied in the present study. Calcium superphosphate (15.5% P2O5) was applied in nursery land at the rate of 50 kg P2O5 ha−1 before plowing. After the last ploughing and directly before sowing Nitrogen was added at 165 kg N/ha using urea form (46.0% N). The field trial was fertilized before plowing with 50 kg P2O5 ha−1. Potassium fertilizer was applied in two equal doses after transplanting at 30 and 45 days by adding 60 kg K2O/ha using potassium sulfate form (48% K2O).2.3Studied traits2.3.1Physiological MeasurementsChlorophyll a and b and carotenoid (mg/g FW) contents were recorded at the heading stage following the method of Peng . To begin, five fresh leaves were carefully washed to eliminate impurities. Subsequently, 2 g of leaf tissue was homogenized in 80% acetone using a mortar and pestle. After centrifugation, the resulting supernatants were utilized to measure absorbance at 663 nm, 645 nm, and 470 nm using a spectrophotometer. From these absorbance readings, the concentrations of chlorophyll a, chlorophyll b, and carotenoids were calculated (mg/g fresh weight).Proline content was recorded at the panicle initiation stage as described in the method of Bates et al. . Samples of 0.5 g of rice leaf were collected and homogenized in 4 ml of 3% sulfosalicylic acid using a mortar and pestle. After storing the homogenates at 5 °C for 24 h, they were centrifuged at 3000 rpm and room temperature for 5 min. The resulting supernatants were combined with 4 ml of acidic ninhydrin reagent, mixed, and heated in a water bath at 100 °C for 60 min. Following cooling, 4 ml of toluene was added. The upper toluene layer was separated, and its absorbance was measured at 520 nm using a spectrophotometer. Proline concentration was determined using a standard curve and expressed as μmol of proline per gram of fresh weight.At the panicle initiation stage, the enzymatic antioxidant activity was assessed by freezing leaf samples in liquid nitrogen to prepare extracts. Catalase activity (unit/mg protein) was determined following the technique outlined by Aebi . For the enzyme extract, 0.5 mL of 0.2 M H2O2 in 10 mM K-phosphate buffer (pH 7.0) was employed before the analysis. The catalase enzyme activity was measured using a spectrophotometer at 240 nm, tracking the consumed H2O2. Peroxidase activity (unit/mg protein) was recorded as described by Vetter et al. . This involved combining 100 μL of enzyme extract with 2.9 mL of assay solution composed of 50 mM phosphate-citrate buffer (pH 6.5), 0.03% hydrogen peroxide, and 0.1% o-phenylenediamine. The reaction was initiated with the extract, and the increase in absorbance at 430 nm was monitored for 5 min. The change in absorbance at 430 nm was determined over a 5-min period.2.3.2Agronomic traitsTen plants were randomly collected from each plot to record flag leaf area (cm2) according to Yoshida et al. at the heading stage. Besides, plant height (PH) was recorded from the soil surface to the tip of the main panicle of each plant. Moreover, at harvesting, ten panicles were harvested at random from each plot to record panicle length, number of panicles/plant, number of grains/panicle, 1000-grain weight, panicle weight, and spikelet fertility. Spikelet fertility was calculated by dividing the filled spikelets from a panicle by the total spikelets. The grain yield was determined from a 6-m2 area in each experimental unit adjusted to 14% moisture content and converted to ton/ha.2.4Statistical analysisThe analysis of variance (ANOVA) was applied to explore the significant differences among applied treatments. R statistical software version 4.2.1 was utilized to analyze the obtained data. A strip-split-plot design with three replications, irrigation treatments as vertical strip, foliar applications as horizontal strip, and rice genotypes as the subplot factor. Tukey's HSD test was utilized for post-hoc analysis (p < 0.05). Moreover, the heatmap was applied with the package of RColorBrewer and biplot of the principal component with ggplot2 in R software to determine the relationship among studied traits and treatments.3Results3.1Physiological traitsPhotosynthetic pigments were significantly (P ≤ 0.05) affected by irrigation treatments, foliar application, genotypic performance, and their interactions (Table 1). Chlorophyll a, chlorophyll b, and carotenoids levels significantly (P ≤ 0.05) reduced by 18.0, 29.5, and 14.3% under drought conditions, compared to regularly irrigated plants (Table 1). However, the foliar treatments using nano-silicon, potassium sulfate, or proline led to significant (P ≤ 0.05) increase in chlorophyll a, chlorophyll b, and carotenoids in comparison with untreated stressed plants. The best treatment was the foliage application of proline which improved chlorophyll a, chlorophyll b, and carotenoids by 21.4, 19.6 and 21.0% followed by nano-silicon treatment, which enhanced chlorophyll a, chlorophyll b, and carotenoids by 21.1, 17.6 and 9.5% compared with untreated control. The evaluated rice genotypes exhibited highly significant (P ≤ 0.01) variations in their responses to irrigation water treatments. The genotypes IRAT-112 and IR-64 displayed the highest chlorophyll a, chlorophyll b, and carotenoids under well-watered conditions, whereas Giza-178 possessed superior values under water deficit conditions (Fig. 1A–C). Likewise, the assessed genotypes exhibited contrasting responses to foliar-supplied treatments. The uppermost contents of chlorophyll a, chlorophyll b, and carotenoids were assigned for the genotype Giza-178 and IRAT-112 treated with foliar application of nano-silicon and proline, compared to their corresponding controls under water deficit conditions.Table 1Impact of irrigation regimes and different exogenously applied substances on photosynthetic pigments of diverse rice genotypes over two summer seasons of 2021 and 2022.Table 1Studied FactorChlorophyll a(mg g−1 FW)Chlorophyll b(mg g−1 FW)Carotenoids(mg g−1 FW)IrrigationWell-watered3.72 a2.61 a1.26 aDrought stress3.05 b1.84 b1.08 bFoliar applicationUntreated control2.94 c1.99 c1.05 cNano-silicon3.56 a2.34 a1.15 bPotassium sulfate3.47 b2.20 b1.21 abProline3.57 a2.38 a1.27 aGenotypesIRRAT3.33 b2.36 a1.19 aG1783.51 a2.28 ab1.21 aIR643.32 b2.04 b1.12 bANOVAdfP valueIrrigation (IR)10.012<0.001<0.001Foliar application (FA)30.015<0.001<0.001Genotype (G)2<0.001<0.001<0.001Year (Y)10.3420.0920.072IR × FA30.007<0.001<0.001IR × G2<0.001<0.001<0.001IR × Y10.0660.0350.164FA × G6<0.001<0.001<0.001FA × Y30.1530.0830.048G × Y20.0290.1940.179IR × FA × G6<0.001<0.001<0.001IR × FA × G × Y30.3370.6940.952Fig. 1Impact of different exogenously sprayed substances on chlorophyll a (A), chlorophyll b (B), and carotenoids (C) in diverse rice genotypes under well-watered and water deficit conditions. The bars on the top positioned above the columns of treatments correspond to Tukey's HSD (p ≤ 0.05). When the difference between two treatments extends beyond the HSD bar, it signifies their significant difference.Fig. 1The irrigation treatments, exogenous application, genotypic performance, and their interactions exhibited highly significant (P ≤ 0.01) effects on the antioxidant enzymatic activity (peroxidase and catalase) and proline content (Table 2). Water deficit caused a substantial elevation in the activities of catalase, peroxidase, and proline content, by 42.08%, 33.23 %, and 140.20%, respectively, compared to well-watered treatment. The exogenous application of nano-silicon, potassium sulfate, and proline significantly (P ≤ 0.05) enhanced these parameters compared with untreated plants. The maximum enhancement of catalase and peroxidase activity was recorded by the foliar spray of nano-silicon (35.1 and 32.2 respectively). Otherwise, the application of proline demonstrated a superior improvement in the content of proline by 52.5% compared with the untreated control. The evaluated rice genotypes exhibited highly significant (P ≤ 0.01) variations in their responses to irrigation water treatments and foliar applications. It was noticed that the genotype Giza-178 showed the maximum values of catalase, peroxidase, and proline content. Moreover, both genotypes Giza-178 and IRAT-112 whether treated with nano-silicon or proline treatments achieved the uppermost values of catalase, peroxidase, and proline content under drought stress conditions (Fig. 2A–C)Table 2Impact of irrigation regimes and different exogenously applied substances on the antioxidant enzymatic activity of diverse rice genotypes over two summer seasons of 2021 and 2022.Table 2Studied FactorCatalase(Unit mg/protein)Peroxidase(Unit mg/protein)Proline content(μmol g/DW)IrrigationWell-watered0.638 b1.58 b0.505 bDrought stress0.817 a1.93 a0.977 aFoliar applicationUntreated control0.627 d1.49 c0.552 cNano-silicon0.847 a1.97 a0.799 abPotassium sulfate0.685 c1.76 b0.772 bProline0.752 b1.80 b0.842 aGenotypesIRRAT0.674 b1.70 b0.623 cG1780.792 a1.85 a0.835 aIR640.717 ab1.71 b0.765 bANOVAdfP valueIrrigation (IR)1<0.001<0.001<0.001Foliar application (FA)3<0.001<0.001<0.001Genotype (G)2<0.001<0.001<0.001Year (Y)10.0520.4020.059IR × FA3<0.001<0.001<0.001IR × G2<0.001<0.001<0.001IR × Y10.0930.2670.402FA × G6<0.001<0.001<0.001FA × Y30.0590.0460.267G × Y20.0760.0570.065IR × FA × G6<0.001<0.001<0.001IR × FA × G × Y30.7710.6080.608Fig. 2Impact of different exogenously sprayed substances on catalase activity (A), peroxidase activity (B), and proline content (C) in diverse rice genotypes under well-watered and water deficit conditions. The bars on the top positioned above the columns of treatments correspond to Tukey's HSD (p ≤ 0.05). When the difference between two treatments extends beyond the HSD bar, it signifies their significant difference.Fig. 23.2Agronomic traitsThe evaluated agronomic traits were significantly (P ≤ 0.05) affected by irrigation treatment, exogenous application, genotypic performance, and their interactions (Table 3). Water deficit displayed an adverse impact on plant height and flag leaf area in comparison with well-watered conditions. There was a considerable reduction in plant height and flag leaf area by 19.4% and 36.8%, respectively, under water deficit conditions in comparison with those under complete irrigation conditions. Notwithstanding, the application of nano-silicon, potassium sulfate, and proline significantly increased flag leaf area by 10.1%, 7.5%, and 12.1%, respectively, and plant height by 5.1%, 3.4%, and 4.8%, in the same order, compared to untreated plants. The rice genotypes responded differently to irrigation treatments and foliar applications. The highest flag leaf area and plant height values belonged to IRAT-112 genotype, while IR-64 had the lowest values. Moreover, the genotype IRAT-112 treated with nano-silicon or proline treatments achieved superior flag leaf area and plant height under drought stress conditions (Fig. 3A–D).Table 3Influence of irrigation regimes and different exogenously applied substances on some yield attributes of diverse rice genotypes over two summer seasons of 2021 and 2022.Table 3Studied FactorLeafarea (cm2)Plantheight (cm)No of paniclesper plantPaniclelength (cm)IrrigationWell-watered45.11 a106.82 a21.18 a21.92 aDrought stress28.51 b86.05 b15.88 b19.17 bFoliar applicationUntreated control34.27 c93.33 c17.50 c19.75 cNano-silicon37.74 a98.12 a18.68 b20.73 bPotassium sulfate36.84 b96.47 b18.84 ab20.63 bProline38.40 a97.82 a19.09 a21.07 aGenotypesIRRAT43.86 a109.48 a12.29 b19.45 bG17834.08 b90.07 b22.03 a20.12 bIR6432.49 b89.75 b21.26 a22.07 aANOVAdfP valueIrrigation (IR)1<0.001<0.0010.011<0.001Foliar application (FA)3<0.0010.0320.006<0.001Genotype (G)2<0.001<0.001<0.001<0.001Year (Y)10.0610.0440.3310.325IR × F3<0.001<0.0010.0220.008IR × G2<0.001<0.001<0.001<0.001IR × Y10.0820.0210.6110.984FA × G6<0.001<0.0010.379<0.001FA × Y30.0510.8550.4980.021G × Y20.0670.0540.7610.768IR × FA × G6<0.001<0.0010.002<0.001IR × FA × G × Y30.2250.0650.5270.093Fig. 3Impact of different exogenously sprayed substances on leaf area (A), plant height (B), number of branches per plant (C), and panicle length (D) in diverse rice genotypes under well-watered and water deficit conditions. The bars on the top positioned above the columns of treatments correspond to Tukey's HSD (p ≤ 0.05). When the difference between two treatments extends beyond the HSD bar, it signifies their significant difference.Fig. 3Deficient irrigation significantly decreased panicle length, number of panicles/plant, number of grains/panicle, fertility percentage, 1000-grain weight, panicle weight, and rice grain yield by 12.6%, 25.1%, 16.3%, 6.5%, 11.4%, 34.3%, and 28.2%, in the same order, in comparison with well-watered conditions (Table 4). However, the application of nano-silicon, potassium sulfate, and proline mitigated the devastating impacts of water deficit and considerably promoted all the aforementioned characteristics. Foliar treatment of proline displayed a superior boost of yield attributes followed by nano-silicon treatment. It significantly increased panicle length, number of panicles/plant, number of grains/panicle, fertility percentage, 1000-grain weight, panicle weight, and grain yield by 9.1, 6.7, 8.8, 13.1, 4.58, 18.9, and 14.9% compared to untreated plants. The evaluated genotypes displayed varied responses to irrigation treatments. The genotypes Giza-178 and IR-64 recorded superior yield traits under well-watered conditions, while Giza-178 and IRAT-112 possessed superior performance under drought stress conditions. Besides, the assessed genotypes displayed contrasting responses to the exogenous application. The highest yield traits were assigned for the genotypes Giza-178 and IRAT-112 treated with the foliar application of nano-silicon and proline, compared to their corresponding controls under water deficit conditions (Fig. 4A–E). Otherwise, the genotype IR64 exhibited the lowest agronomic performance under water deficit conditions under different foliar applications.Table 4Influence of irrigation regimes and different exogenously applied substances on grain yield contributing traits of diverse rice genotypes over two summer seasons of 2021 and 2022.Table 4Studied FactorNo of grainsper panicleFertility(%)1000 grainWeight (g)Panicleweight (g)Grain yield(ton/ha)IrrigationWell-watered147.46 a84.77 a29.13 a4.31 a10.10 aDrought stress123.49 b80.15 b25.81 b2.83 b7.25 bFoliar applicationUntreated control128.40 c76.09 d26.87 c3.18 c8.01 bNano-silicon141.00 a84.19 b27.42 b3.75 a9.06 aPotassium sulfate132.78 b82.03 c27.50 b3.58 b8.44 bProline139.72 a86.08 a28.10 a3.78 a9.20 aGenotypesIRRAT132.58 b82.56 b36.15 a4.66 a7.92 bG178160.94 a84.14 a22.38 b3.39 b9.86 aIR64112.91 c80.09 c23.89 b2.67 b8.25 bANOVAdfP valueIrrigation (IR)10.018<0.001<0.001<0.001<0.001Foliar application (FA)3<0.001<0.001<0.001<0.001<0.001Genotype (G)2<0.001<0.001<0.001<0.001<0.001Year (Y)10.0710.0720.1120.0390.041IR × F3<0.001<0.001<0.001<0.001<0.001IR × G2<0.001<0.001<0.001<0.001<0.001IR × Y10.2250.0180.0890.0480.022FA × G6<0.001<0.001<0.0010.051<0.001FA × Y30.0830.0610.0870.0640.073G × Y20.0010.0010.0760.0820.062IR × FA × G6<0.001<0.001<0.001<0.001<0.001IR × FA × G × Y30.8360.8560.2460.6360.994Fig. 4Impact of different exogenously sprayed substances on number of grains per panicle (A), fertility percentage (B), 1000-grain weight (C), panicle weight (D), and grain yield (E) in diverse rice genotypes under well-watered and water deficit conditions. The bars on the top positioned above the columns of treatments correspond to Tukey's HSD (p ≤ 0.05). When the difference between two treatments extends beyond the HSD bar, it signifies their significant difference.Fig. 43.3Relationship among the evaluated treatments and traitsPrincipal component analysis (PC) was applied to study the association between the studied treatments and evaluated characteristics. The first two PCs displayed the most variability (79.69%) presenting 65.43% by PC1 and 14.26% by PC2 (Fig. 5). The PC1 was correlated with irrigation regimes, water deficit conditions were situated on the negative side while the well-watered conditions were positioned on the positive side. The PCA2 seems to correspond with rice genotypes from bottom to top as IRRAT-112, IR64, and Giza-178. Moreover, the genotype Giza-178 treated with nano-silicon and proline exhibited the highest values of most studied physiological and agronomic traits under well-watered and drought stress. Likewise, the heatmap based on the evaluated agronomic and physiological characters separated irrigation treatments, foliar applications, and rice genotypes into different clusters (Fig. 6). The irrigation treatment was the primary separating factor of the principal clusters. The genotype Giza-178 treated with proline and nano-silicon displayed the maximum values for most evaluated characteristics under drought stress (represented in blue). The evaluated characteristics presented by parallel vectors revealed a robust positive association, though those assigned almost opposite displayed a substantially negative relationship. The studied traits could be separated into two groups one contained photosynthetic pigments, growth, and agronomic traits, whereas the other contained enzymatic antioxidants and proline content. A robust positive relationship was identified among the traits within each group, whereas a negative relationship was perceived between the two groups.Fig. 5Principal component biplot for the assessed rice genotypes; IRRAT, G178, and IR64 and exogenously applied substances untreated control (Con), nano-silicon (Nano), potassium sulfate (Potas), and proline (Prol) under water deficit (DS) and well-watered (WW) conditions. Cha: chlorophyll a, Chb: chlorophyll b, Carot: carotenoids, CAT: catalase, PORX: peroxidase, Proline: proline content, LArea: leaf area, PH: plant height, PanL: panicle length, NPanP: number of panicles/plant, NGP: number of grains/panicle, 1000-grain weight, Fertil: fertility percentage, PNW: panicle weight, GY: grain yield. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 5Fig. 6Heatmap and hierarchical clustering for the evaluated rice genotypes; IRRAT, G178, and IR64 and applied-foliar substances; untreated control (Con), nano-silicon (Nano), potassium sulfate (Potas), and proline (Prol) under water deficit (DS) and well-watered (WW) conditions. Cha: chlorophyll a, Chb: chlorophyll b, Carot: carotenoids, CAT: catalase, PORX: peroxidase, Proline: proline content, LArea: leaf area, PH: plant height, PanL: panicle length, NPanP: number of panicles/plant, NGP: number of grains/panicle, 1000-grain weight, Fertil: fertility percentage, PNW: panicle weight, GY: grain yield.Red and blue colors were designated to high and low values for the studied trait, in the same order. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 64DiscussionDrought is one of the main environmental stresses that pose great obstacles to rice production, especially in arid regions. Furthermore, it is projected to become more severe and frequent due to decreasing water supply and climate changes. Accordingly, it is decisive to recognize innovative attempts to boost drought tolerance, particularly under global climate fluctuations. In the present study, three safe and efficient substances, nano-silicon, potassium sulfate, and proline, were exogenously applied to explore and compare their influence on physio-biochemical, growth, and agronomic performance of three different rice genotypes grown under drought stress conditions in a 2-year field trial. Besides, to determine which tolerance inducer could be recommended for enhancing drought tolerance and boosting rice productivity under drought stress conditions in arid regions. The obtained results displayed that the studied irrigation treatments, foliar applications, assessed rice genotypes, and their interactions displayed substantial effects on most studied characteristics. In Egypt, the summer season is characterized by a hot and dry climate, with no occurrences of precipitation. Subsequently, the water deficit regime considerably reduced photosynthetic pigments, growth, and yield traits of all the evaluated rice genotypes. Otherwise, antioxidant enzymes and proline content were raised compared to non-stressed conditions. These results may be due to that proline and antioxidant enzymes are the most important osmoprotectants under stress conditions.Photosynthetic pigments are important indicators for assessing drought tolerance in rice . The obtained findings revealed that water deficit significantly decreased the contents of chlorophyll a and b as well as carotenoids of all tested rice genotypes compared with well-watered conditions. The reduction might be attributed to the detrimental effects of water deficit on ribulose-1,5-biphosphate activity, causing elevated chloroplast degradation and structural disarray. Consequently, this condition results in decreased chlorophyll content . Likewise, growth traits such as flag leaf area and plant height considerably declined under water deficit conditions. This adverse effect could stem from reduced water uptake, resulting in decreased plant cell division and expansion . Studies such as those conducted by Manickavelu et al. , Afroz and Akhtar , Yang et al. elucidated the detrimental impact of water scarcity on rice, affecting photosynthetic pigments, flag leaf area, and plant height.The exogenous application of nano-silicon, potassium sulfate, and proline significantly increased the content of chlorophyll a and b and carotenoids in the leaves of all tested genotypes under drought stress compared to untreated plants. These findings suggested an effective role for the applied substances in enhancing photosynthesis, plant growth, and productivity of rice under water deficit conditions. Similarly, previous studies of Elshayb et al. , Alharbi et al. , Hanif et al. , Ahmad et al. , Ali et al. disclosed the positive impact of nano-silicon, potassium sulfate, and proline on promoting photosynthetic pigments while reducing oxidative damages, consequently improving plant performance under drought stress. Moreover, Farooq et al. , Mathur and Roy deduced that these substances can aid in protecting and enhancing rice plants to counteract drought-induced oxidative damage. The uppermost enhancement was assigned for foliar-supplied with nano-silicon followed by proline. Rios et al. demonstrated that foliar application of nano-silicon is more rapidly absorbed by plants compared to bulk silicon, resulting in more beneficial effects under drought stress. Esmaili et al. , Alharbi et al. disclosed the application of nano-silicon promotes plant growth and development by expanding the leaf surface area for increased light absorption, regulating stomatal aperture, enhancing photosynthesis, and improving physiological processes like leaf water status, osmoregulation, and nutrient uptake. These beneficial effects significantly mitigated the deleterious effects of water deficit in rice. Likewise, the pivotal role of proline in increasing previous characteristics is attributed to its ability to protect the plasma membrane, and cytoplasmic enzymes, stabilize membranes, and substantially prevent chlorophyll degradation under water scarcity as demonstrated by Hosseinifard et al. , Bhaskara et al. .The assessed rice genotypes displayed highly significant variations in their responses to irrigation water treatments and foliar applications. The evaluated genotypes accumulated catalase, peroxidase, and proline content in different trends under drought stress. The genotype Giza-178 exhibited the highest content of catalase, peroxidase, and proline under drought stress conditions. The accumulation of proline is a critical response of plant cells under water deficit to promote osmotic adjustment and antioxidant system . Furthermore, proline has a decisive role in tolerance to drought stress due to its capability to detoxicate the generated detrimental free radical species . Furthermore, the foliar applications of the three substances enhanced the proline under drought conditions compared with untreated plants. Exogenously sprayed proline exhibited the uppermost contents compared with the other applications. These results indicated that the increased accumulation of proline under water deficit conditions may be associated with the exogenous application of proline. Similarly, Abdelaal et al. , El-Bauome et al. manifested that the exogenous foliar application of proline enhanced proline content and ameliorated tolerance to water deficit stress in rice. Besides, Farooq et al. , Ghaffari et al. and AlKahtani et al. proved an increase in levels of proline under drought stress following foliage proline treatment.Water deficiency conditions considerably increased antioxidant enzymatic CAT and POX activities in all the evaluated genotypes compared with the well-watered conditions. Drought stress induces oxidative stress, as well as elevated levels of reactive oxygen species (ROS). To combat ROS toxicity, a highly effective antioxidant defense mechanism is necessary. It has been suggested that the increase in antioxidant activity exerts an ameliorative effect on drought stress . The foliar application of nano-silicon, potassium sulfate, and proline significantly enhanced the activity of CAT and POX compared with stressed untreated plants. The enhanced activity of the antioxidant enzymes upon the application of these substances facilitated the conversion of H2O2 into non-toxic compounds (H2O and O2), thereby protecting the plants from the harmful impacts of drought stress . These findings demonstrated the valuable impacts of the foliar application of these substances in ameliorating tolerance to water deficit by altering the antioxidant activities and detoxifying ROS. In agreement with our findings, similar results were noted in various crops under drought stress conditions [21,53,67]. Drought-stressed rice genotypes showed a decline in grain and its related traits compared to those of normal conditions. Elshayb et al. , Sakran et al. depicted that the decrease in yield-contributing traits might resulted from reduced leaf area, decreased chlorophyll content, and disrupted carbohydrate metabolism, resulting in reduced assimilate transport and increased reproductive abortion. Additionally, this decline might be attributed to the adverse impact of water deficit on growth parameters, resulting from reduced water availability . This reduction in nutrient uptake and organic carbon likely led to a decline in reproductive tillers, fertility percentage, 1000-grain weight, and ultimately, grain yield . The exogenous application of nano-silicon, potassium sulfate, and proline significantly enhanced yield traits in treated plants under water deficit compared to untreated stressed plants. Notably, proline and nano-silicon exhibited greater effectiveness in improving the yield of the evaluated genotypes. These outcomes underscore the significant role of proline and nano-silicon in boosting grain yield under drought stress conditions. This enhancement was attributed to their impact on increasing chlorophyll content, antioxidant activity, proline content, number of panicles, plant growth, fertility percentage, and 1000-grain weight.The evaluated genotypes showed significant alterations in all the measured traits under normal and water-deficit conditions. However, deficit irrigation treatment altered the performance of the genotypes more than normal watering. From this perspective Hassan et al. , Yadav et al. , Sedeek et al. , and Hussain et al. elucidated highly significant variations among rice genotypes under normal and water-deficit conditions. The genotype Giza 178 displayed the uppermost physiological performance compared with other genotypes under drought stress conditions. Consequently, this genotype sustained to be tolerant under water deficit by stimulating photosynthetic efficiency, proline content, and enzymatic antioxidants. These improvements were exhibited in enhanced agronomic performance under drought stress conditions, in particular under the foliage application of nano-silicon, potassium sulfate, and proline. The interaction between irrigation level, foliar application, and assessed genotypes showed substantial impacts on most evaluated traits. Generally, under drought stress, Giza 178 genotype in combination with applied exogenously proline or nano-silicon exhibited superior enzymatic antioxidants, photosynthetic pigments, growth, and yield-related traits, compared to untreated treatment.The PC-biplot, heatmap, and hierarchical clustering are helpful methods for exploring relationships among studied variables and parameters [, , ]. The obtained results of the heatmap and PCA biplot reinforced the positive impacts of exogenously applied substances on all evaluated parameters. The heatmap and PCA biplot exhibited that photosynthetic pigments, growth, and agronomic traits were positively related to foliar-applied proline and nano-silicon, in particular with the genotype Giza 178 under drought stress. These findings confirmed that the foliar application of both substances ameliorated the physiological characteristics, growth, and yield traits. Subsequently, the exogenously applied proline and nano-silicon could be a beneficial attempt to stimulate rice productivity, particularly under drought stress conditions. Besides, the PCA biplot showed that growth traits and most physiological parameters were positively associated with yield traits. Subsequently, selection for these traits is efficient for increasing rice productivity under water deficit conditions [, , ].5ConclusionsWater deficit significantly reduced photosynthetic pigment contents, growth parameters, and grain yield traits. Conversely, antioxidant enzyme activities and osmoprotectants showed significant increases under drought stress compared to well-watered conditions. However, foliar applications of nano-silicon, potassium sulfate, or proline significantly alleviated the adverse effects of water deficit and considerably enhanced all studied characteristics. Proline and nano-silicon emerged as the most effective treatments, further enhancing rice performance and drought stress tolerance. The assessed rice genotypes exhibited diverse responses to irrigation treatments and exogenously applied substances. The genotype Giza 178 when treated with foliar proline or nano-silicon demonstrated superior growth, photosynthetic pigments, enzymatic antioxidants, and yield traits compared to untreated plants under water deficit conditions. Thus, foliar application of proline or nano-silicon stands as a promising approach to enhance drought tolerance in high-yielding rice genotypes facing water scarcity.Data availability statementThe data presented in this study are available upon request from the corresponding author.FundingPrincess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R402), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.CRediT authorship contribution statementMohamed S. Abd-El-Aty: Visualization, Investigation, Formal analysis, Data curation, Conceptualization. Mohamed M. Kamara: Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Walid H. Elgamal: Validation, Methodology, Investigation, Data curation. Mohamed I. Mesbah: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. ElSayed A. Abomarzoka: Visualization, Validation, Methodology, Investigation. Khairiah M. Alwutayd: Writing – review & editing, Writing – original draft, Software, Resources, Project administration, Formal analysis, Data curation. Elsayed Mansour: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Project administration, Investigation, Formal analysis, Conceptualization. Imen Ben Abdelmalek: Writing – review & editing, Writing – original draft, Software, Resources, Project administration, Funding acquisition, Formal analysis. Said I. Behiry: Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Formal analysis, Data curation. Ameina S. Almoshadak: Writing – review & editing, Writing – original draft, Software, Resources, Project administration, Funding acquisition, Formal analysis. Khaled Abdelaal: Writing – review & editing, Visualization, Validation, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.Declaration of competing interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Science Advances

PMC10917424

10.1126/sciadv.adl5234

Erratum for the Research Article: “Dissolved oxygen from microalgae-gel patch promotes chronic wound healing in diabetes”

In the Research Article “Dissolved oxygen from microalgae-gel patch promotes chronic wound healing in diabetes,” the authors note that Fig. 2 appeared incorrectly.In Fig. 2B, an incorrect image was used for Alga-gel (dark) group.In Fig. 2C, the value of the ordinate should have been multiplied by ten.Figure 2 has been replaced. The PDF and HTML (full text) have been corrected.

PMC

Schizophrenia Research: Cognition

38486790

PMC10937232

3-07-2024

10.1016/j.scog.2024.100305

Using virtual reality to improve verbal episodic memory in schizophrenia: A proof-of-concept trial

Bogie Bryce J.M., Noël Chelsea, Gu Feng, Nadeau Sébastien, Shvetz Cecelia, Khan Hassan, Rivard Marie-Christine, Bouchard Stéphane, Lepage Martin, Guimond Synthia

BackgroundSchizophrenia is associated with impairments in verbal episodic memory. Strategy for Semantic Association Memory (SESAME) training represents a promising cognitive remediation program to improve verbal episodic memory. Virtual reality (VR) may be a novel tool to increase the ecological validity and transfer of learned skills of traditional cognitive remediation programs. The present proof-of-concept study aimed to assess the feasibility, acceptability, and preliminary efficacy of a VR-based cognitive remediation module inspired by SESAME principles to improve the use of verbal episodic memory strategies in schizophrenia.MethodsThirty individuals with schizophrenia/schizoaffective disorder completed this study. Participants were randomized to either a VR-based verbal episodic memory training condition inspired by SESAME principles (intervention group) or an active control condition (control group). In the training condition, a coach taught semantic encoding strategies (active rehearsal and semantic clustering) to help participants remember restaurant orders in VR. In the active control condition, participants completed visuospatial puzzles in VR. Attrition rate, participant experience ratings, and cybersickness questionnaires were used to assess feasibility and acceptability. Trial 1 of the Hopkins Verbal Learning Test – Revised was administered pre- and post-intervention to assess preliminary efficacy.ResultsFeasibility was demonstrated by a low attrition rate (5.88 %), and acceptability was demonstrated by limited cybersickness and high levels of enjoyment. Although the increase in the number of semantic clusters used following the module did not reach conventional levels of statistical significance in the intervention group, it demonstrated a notable trend with a medium effect size (t = 1.48, p = 0.15, d = 0.54), in contrast to the control group where it remained stable (t = 0.36, p = 0.72, d = 0.13). These findings were similar for the semantic clustering ratio in the intervention (t = 1.61, p = 0.12, d = 0.59) and control (t = 0.36, p = 0.72, d = 0.13) groups. There was no significant change in the number of recalled words in either group following VR immersion.DiscussionThis VR intervention was feasible, acceptable, and may be useful for improving the use of semantic encoding strategies. These findings support the use of more ecological approaches for the treatment of cognitive impairments in schizophrenia, such as VR-based cognitive remediation. Highlights•Cognitive remediation in virtual reality (VR) is feasible and acceptable in schizophrenia.•The current VR-based module was associated with a low attrition rate.•Participants found the module enjoyable and reported little to no side effects.•Improvements in semantic encoding strategies after the VR module are promising.•Larger studies with longer and more frequent intervention sessions are needed.

1IntroductionCognitive performance significantly impacts functional outcomes in individuals with schizophrenia (SZ; Alptekin et al., 2005). One of the most common cognitive impairments implicated in SZ is verbal episodic memory (Bogie et al., 2023; Cirillo and Seidman, 2003; Guimond et al., 2016; Guo et al., 2019). Impairments in verbal episodic memory are a core feature of SZ during both the acute and non-acute phases of the illness, suggesting that this cognitive domain may represent an important target for treatment outcomes (Bogie et al., 2023; Molina and Tsuang, 2020). However, current pharmacological treatments have shown limited efficacy at improving the cognitive symptoms of SZ (Keefe et al., 2013; Tsapakis et al., 2015). There is therefore a need for improved non-pharmacological interventions to supplement current pharmacological treatments (Bowie et al., 2020; Harvey et al., 2019).Cognitive remediation (CR) is the preferred treatment method for improving cognitive functioning in SZ (Medalia and Erlich, 2017; Vita et al., 2021). Although CR can help improve some of the cognitive symptoms of SZ, these interventions have limited generalizability and require long durations of treatment (Lejeune et al., 2021; Seccomandi et al., 2020; McCleery and Nuechterlein, 2019; Vita et al., 2021). Moreover, traditional CR interventions are often associated with high attrition rates and variable levels of transfer to real-world situations, suggesting that current approaches may not meet patients' needs nor preferences (Dickinson et al., 2010; Gomar et al., 2015; Wykes et al., 2011).Virtual reality (VR) is an innovative tool that can help overcome some of the limitations of CR interventions (Park et al., 2019; Rus-Calafell et al., 2018; Schroeder et al., 2022). VR is an immersive experience which simulates three-dimensional environments, allowing for the development of novel treatments with increased ecological validity (Campbell et al., 2009; Freeman, 2008; O'Connor et al., 2016). Research has shown that immersive VR programs are superior to non-immersive approaches at promoting the transfer of learned skills to real-world situations (Dobrowolski et al., 2021; Wu et al., 2020). These findings highlight the potential therapeutic utility of immersive VR technology for the treatment of mental health conditions. Preliminary evidence has also demonstrated that CR interventions delivered using VR can lead to improvements in positive symptoms, attention, memory, spatial learning, and social skills in SZ (du Sert et al., 2018; Rus-Calafell et al., 2014; Spieker et al., 2012; Tsang and Man, 2013). Furthermore, research suggests that individuals with SZ perceive VR-based CR as enjoyable and engaging (Chan et al., 2010; Rus-Calafell et al., 2018; Schroeder et al., 2022). Hence, this technology may represent a more effective approach to the delivery of CR for individuals with SZ (Rothbaum et al., 1995; Rus-Calafell et al., 2018). Further investigation is needed to assess the utility of VR-based CR approaches for improving specific domains of cognition in individuals with SZ, such as verbal episodic memory.The current proof-of-concept study employed a randomized controlled design with intervention and active control conditions. The aim was to assess the feasibility, acceptability, and preliminary efficacy of a VR-based CR module designed to improve the use of verbal episodic memory strategies in SZ compared to a VR-based control condition. We hypothesized that the VR-based CR module would be associated with a low attrition rate, positive feedback from participants, and minimal adverse side effects, demonstrating the overall feasibility and acceptability of the intervention. We further hypothesized that the module would improve the use of semantic clustering strategies and verbal episodic memory performance at post- versus pre-intervention assessments.2MethodsThis research was approved by the Research Ethics Board at the Royal Ottawa Mental Health Centre (ROMHC; Ottawa, Canada) and was pre-registered on ClinicalTrials.gov (identifier: were recruited from outpatient services in the Ottawa region affiliated with the ROMHC. Inclusion criteria were: an established diagnosis of SZ or schizoaffective disorder (hereinafter considered together as “SZ”), confirmed by the Mini International Neuropsychiatric Interview (MINI; Sheehan et al., 1998); aged 20–60 years; a score ≤ 95 on the Positive and Negative Syndrome Scale (PANSS; Kay et al., 1987); a stable medication regimen for at least one month prior to study participation; and the ability to read and speak fluent English. Exclusion criteria were: uncorrected vision problems; a significant comorbid medical disorder that may produce cognitive impairment; history of alcohol and/or substance use disorder within the past three months; lifetime history of migraines, seizures, epilepsy, or cybersickness; and decisional incapacity requiring a guardian. All participants provided written informed consent prior to participation.2.2Study designThe study followed a single-blind (participants blind to condition), parallel groups, randomized controlled trial design. Participants were randomized in a 1:1 ratio to either the intervention or active control group according to a predetermined randomization sequence. Both the intervention and active control conditions were delivered in VR. We chose to deliver the active control condition in VR to ensure that: participants were blind to condition; and any differences observed following the VR session were not simply due to the experience of VR immersion alone.2.3Measures2.3.1Clinical assessments2.3.1.1Mini International Neuropsychiatric InterviewThe MINI was used to confirm current and past psychiatric diagnoses (Sheehan et al., 1998).2.3.1.2Positive and Negative Syndrome ScaleThe severity of psychosis-related symptoms was evaluated using the PANSS (Kay et al., 1987; see Supplementary Material 1).2.3.2VR feasibility assessmentsThe primary measure of feasibility was the attrition rate (proportion of enrolled participants who failed to complete the trial).2.3.2.1Exclusion VR Criteria QuestionnaireThe Exclusion VR Criteria Questionnaire (EVCQ), a 4-item questionnaire developed by our research team to assess participants' eligibility to use the VR equipment on the day of participation, was administered to examine the presence and severity of any physiological discomfort after trying the VR technology following a one-minute trial. Participants who experienced severe physiological discomfort following one-minute of VR immersion were terminated from the trial. The EVCQ was also used as a primary measure of feasibility. Further details about the EVCQ can be found in Supplementary Material 1.2.3.3VR acceptability assessmentsThe following two assessments were the primary measures of acceptability.2.3.3.1Simulator Sickness QuestionnaireThe Simulator Sickness Questionnaire (SSQ) was administered to both groups after the full VR session to assess their experience with VR immersion (Kennedy et al., 1993).2.3.3.2VR Experience QuestionnaireTo further assess the acceptability of the VR sessions, our research team designed a 5-item VR Experience Questionnaire (VEQ). The VEQ was administered to both groups after the full VR session.Further details about the VR acceptability assessments can be found in Supplementary Material 1.2.3.4Assessment of preliminary efficacyThe primary measure of the preliminary efficacy of the VR-based CR module was the Hopkins Verbal Learning Test – Revised (HVLT; Benedict et al., 1998). The HVLT was administered to both groups before and after the VR session (different version forms were used at each time point). Participants were assessed on one trial of immediate recall per version form to avoid interference. The post-intervention assessment was administered within five minutes of the VR session, allowing time for participants to readapt to reality following the VR immersion.The number and ratio of semantic clusters were used as primary outcome measures of preliminary efficacy. The raw total number of recalled words was used as a secondary outcome measure (see Supplementary Material 1).2.3.5Trial design outcomesParticipant enrollment rate was used as a general marker of trial design feasibility. This variable was defined as the proportion of invited participants who enrolled in the trial, all of whom previously expressed interest in participating in research and met basic pre-screening eligibility criteria.2.3.5.1Blinding questionAt the conclusion of the study, participants rated the likelihood with which they believed they were assigned to the intervention group (see Supplementary Material 1).2.4VR InterventionsBoth VR conditions were delivered using an Oculus Rift head mounted display, handheld controllers, and sensors.2.4.1Intervention groupThe CR training followed the Strategy for Semantic Association Memory (SESAME) training principles (Guimond et al., 2018). The training coached participants how to use semantic information to organize items (i.e., semantic clustering), followed by cues to apply these strategies within a restaurant order-taking memory task. The training encouraged participants to use the semantic clustering strategy in combination with active rehearsal strategies. The training manual is available in Supplementary Material 2.Following the CR training, participants in the intervention group completed a 15-minute verbal episodic memory task in a three-dimensional VR restaurant environment, developed by our research team (Fig. 1). Participants played the role of a waiter/waitress. While using the VR equipment, participants walked around the virtual restaurant environment and took “orders” from customers. The avatars at each table verbalized their orders aloud. Participants were instructed to remember the orders using semantic clustering and active rehearsal, walk to the cashier in the VR environment, and verbally recall the items. Outside of the VR environment, the experimenter interacted with the participant and tracked their progress. If the participant did not correctly recall all items from the order, the experimenter instructed them to return to the table and repeat the level. Participants proceeded to the next level once all items were correctly recalled.Fig. 1Images from the virtual reality environment delivered to the intervention group.Note. The participant took on the role of a waiter/waitress in this restaurant environment. Using the handheld controllers, the participant walked to a pre-determined table (e.g., top right image), listened to the customers' orders, and walked back to the cashier (bottom right image) to recall the orders from memory. Participants were encouraged to engage the semantic encoding strategies coached during the CR training throughout this task.Fig. 1The verbal episodic memory task included four levels (i.e., tables), including a total of ten customers. Each table increased in the level of difficulty (i.e., number of customers at the table, number of items ordered, number of possible semantic categories). The VR session was considered complete after 15 minutes had elapsed or once the participant correctly recalled the orders from all four levels.2.4.2Control groupParticipants in the control group played a 15-minute three-dimensional puzzle game in VR called “Cubism” ( This game was moderately cognitively challenging and did not require the use of verbal memory processes (Fig. 2). Instead, the game engaged spatial reasoning processes. Participants were tasked with assembling progressively more complex puzzles using colorful blocks (see Fig. 2). Participants manipulated the blocks using the handheld controllers. No CR was administered in this condition; instead, the experimenter provided active encouragement throughout the session (see Supplementary Material 2). The session was considered complete after 15 minutes had elapsed.Fig. 2Images from the virtual reality environment delivered to the control group.Note. In each level, the participant used the handheld controllers to manually retrieve and manipulate colorful geometric shapes to complete a puzzle board. Each level was progressively more difficult than the previous level. The black arrowheads represent the participant's left and right hands. Images reproduced with permission from the owner of Cubism.Fig. 22.5ProcedureSee Fig. 3 for a schematic summary of the study procedure. Participants were compensated CAD$30.00 for their participation.Fig. 3Schematic summary of study procedures.Note. The assessments surrounded by the gray boxes occurred outside of the VR environment.Abbreviations: HVLT: Hopkins Verbal Learning Test – Revised; MINI: Mini International Neuropsychiatric Interview; PANSS: Positive and Negative Syndrome Scale; VR: virtual reality.Fig. 32.6Statistical analysesDifferences in age, years of education, symptom severity, and antipsychotic dosage (chlorpromazine equivalents, in mg) were compared between groups using t-tests. Difference in the distribution of sex between groups was compared using a Chi-squared test.t-Tests were used to compare results from the SSQ, VEQ, and blinding question between groups. Changes in the use of semantic clustering strategies and overall verbal episodic memory performance were analyzed across time points within each group using t-tests due to the small sample size.Cohen's d effect sizes were calculated to characterize the magnitude of change in the use of semantic clustering strategies and overall verbal episodic memory performance across time points. Given the small sample size, Cohen's d effect sizes were considered when drawing conclusions about preliminary efficacy.Statistical significance was set at p < 0.05 and trending towards significance was set at p < 0.15. All analyses were performed in R (version 4.2.2).3Results3.1Demographic and clinical characteristicsThe final sample comprised 30 participants equally divided between the two groups. Table 1 summarizes the demographic and clinical characteristics of the participants. The groups did not significantly differ in age (range: intervention = 24–48; control = 20–51), sex, years of education, psychosis-related symptom severity, nor dosage of antipsychotic medications.Table 1Demographic and clinical characteristics of the intervention (n = 15) and control (n = 15) groups.Table 1Intervention GroupControl Groupt/X2pMeanSDMeanSDDemographics Age36.607.5834.0711.130.730.47 Sex (Male/Female)12M/3F13M/2F0.001.00 Years of Education15.203.9315.532.900.260.79Clinical Characteristics PANSS Total Score60.4011.6959.8712.450.120.90Current Medications Antipsychotic Dosea198.93543.78302.77844.240.400.69 Rangea0.00–2142.860.00–3333.33Co-morbid Diagnosesb Major Depressive Disorder (Recurrent)n = 4n = 1 Suicide Behaviour Disordern = 1 Panic Disordern = 1 Agoraphobian = 2n = 1 Social Anxiety Disordern = 2 Generalized Anxiety Disordern = 2 Obsessive Compulsive Disordern = 2n = 3 Alcohol Use Disorder (Past Year)n = 1 Substance Use Disorder (Past Year)n = 1Note. The two groups did not significantly differ in: age, sex, years of education, PANSS total score, nor antipsychotic dose.Abbreviations: PANSS: Positive and Negative Syndrome Scale.aChlorpromazine (CPZ) equivalent (in mg).bCo-morbid diagnoses confirmed through the Mini International Neuropsychiatric Interview.3.2FeasibilityA total of 34 participants were enrolled in this study, of which two were excluded due to reasons unrelated to the feasibility of the VR technology (current alcohol use disorder and distraction due to noise outside of the assessment room; both were assigned to the intervention group).Two additional participants were excluded due to difficulties experienced during VR immersion: discomfort following the VR practice session (i.e., according to the EVCQ; control group); and anxiety reported during the full VR session (intervention group). It is noted that the latter participant subsequently disclosed significant pre-appointment anxiety which almost caused them to postpone the appointment. This participant had also rescheduled this appointment once before due to significant anxiety. This corresponded to a low attrition rate of 5.88 %.Of the 30 participants who were included in the final sample, two provided positive responses to the EVCQ question, Are you feeling dizzy? One participant reported that the dizziness was a result of VR immersion, while the other reported that it was unrelated. Follow-up questioning revealed that the level of dizziness was minimal, and both participants wanted to continue. Both participants were assigned to the intervention group.3.3AcceptabilityThe results of the SSQ and VEQ are presented in Table 2. Generally, participants reported low levels of cybersickness. While the intervention group showed increased sub-scale and total SSQ scores compared to the control group, these differences were not statistically significant. Per the VEQ, both groups judged the VR experience as highly enjoyable and realistic. Both groups also endorsed wanting to try the VR activity again and that they would recommend it to a friend. The total VEQ scores did not significantly differ between groups. The qualitative feedback was also mostly positive, with many respondents commenting that the VR immersion was fun and realistic (see Supplementary Material 3).Table 2Acceptability of the virtual reality environments according to the intervention (n = 15) and control (n = 15) groups.Table 2Intervention GroupControl GrouptpMeanSDMeanSDSSQScaled Scores Sub-scale Scores Nausea Symptoms20.3524.1513.9912.420.910.38 Oculomotor Symptoms19.2020.0317.1816.570.300.77 Disorientation Symptoms32.4848.6920.4220.290.890.39 Total Score18.2022.7414.2110.210.620.54 Raw Scores Sub-scale Scores Nausea4.073.063.601.590.520.61 Nausea (−Anxiety)3.932.743.271.160.870.40 Oculomotor2.403.182.001.890.420.68 Oculomotor (−Anxiety)1.932.461.601.590.440.66 Total Scores Total Raw4.876.083.802.730.620.54 Total Raw (−Anxiety)4.275.113.072.370.830.42 VEQStatement Scores 1 – Enjoyment4.800.414.600.63–– 2 – Realistic4.270.703.671.00–– 3 – Try Again4.530.644.400.91–– 4 – Recommend4.330.724.330.90–– Total Score17.931.8717.002.671.110.28Note. The results of the SSQ are presented using two scoring methods: the traditional scaled scoring method (Kennedy et al., 1993) and the more recent method which accounts for anxiety symptoms (Bouchard et al., 2021; see Supplementary Material 1). Each item is rated on a 4-point Likert scale (anchors: 0 = Not At All, 1 = Mild, 2 = Moderate, 3 = Severe). Possible (unscaled) scores on the SSQ range from 0 to 48, with higher scores representing stronger perceptions of cybersickness (Bimberg et al., 2020). Possible total scores on the VEQ range from 4 to 20 (response anchors: 1 = Strongly Disagree, 3 = Neutral, 5 = Strongly Agree; see full individual statements in Supplementary Material 1).Abbreviations: SSQ: Simulator Sickness Questionnaire; VEQ: Virtual Reality Experience Questionnaire.3.4Preliminary efficacy3.4.1Number of semantic clustersThe mean number of semantic clusters used by the intervention and control groups across time points is presented in Fig. 4A. The intervention group showed a moderate increase in the mean number of semantic clusters used between time points. This change trended towards statistical significance (t = 1.48, p = 0.15, CI: −0.31, 1.91) and was associated with a medium effect size (d = 0.54). In comparison, participants in the control group did not show a significant nor trending significant change between time points (t = 0.36, p = 0.72, CI: −0.94, 1.34, d = 0.13).Fig. 4Pre- vs. post-intervention HVLT performance for the intervention (n = 15) and control (n = 15) groups.Note. A. Mean number of semantic clusters used across the pre-intervention (intervention group: M = 1.53, SD = 1.13; control group: M = 1.33, SD = 1.35) and post-intervention (intervention group: M = 2.33, SD = 1.76; control group: M = 1.53, SD = 1.68) time points. B. Mean semantic clustering ratios across the pre-intervention (intervention group: M = 0.26, SD = 0.16; control group: M = 0.19, SD = 0.18) and post-intervention (intervention group: M = 0.36, SD = 0.20; control group: M = 0.21, SD = 1.68) time points. C. Raw total HVLT scores across the pre-intervention (intervention group: M = 5.80, SD = 1.52; control group: M = 5.80, SD = 2.08) and post-intervention (intervention group: M = 6.00, SD = 1.56; control group: M = 5.67, SD = 2.13) time points. All data are presented as mean ± standard error. Cohen's d effect sizes for both groups are displayed on each figure.Abbreviations: HVLT: Hopkins Verbal Learning Test – Revised.Fig. 43.4.2Semantic clustering ratioThe mean semantic clustering ratios for the intervention and control groups across time points are presented in Fig. 4B. The intervention group showed a slight increase in the mean semantic clustering ratio between time points. Similar to the previous findings, this change trended towards statistical significance (t = 1.61, p = 0.12, CI: −0.03, 0.24) and corresponded to a medium effect size (d = 0.59). The control group similarly showed a slight increase in the mean semantic clustering ratio between time points, but this change was not statistically significant and was of small effect size (t = 0.36, p = 0.72, CI: = −0.12, 0.17, d = 0.13).3.4.3Raw total HVLT scoresThe raw total HVLT scores for the intervention and control groups across time points are presented in Fig. 4C. The intervention group showed a slight increase in the raw total HVLT score across time points. However, this change was not statistically significant (t = 0.36, p = 0.73, CI: −0.95, 1.35). This difference corresponded to a small effect size (d = 0.13). In comparison, the control group showed a slight decrease in the total HVLT score between the time points. This change was similarly not statistically significant and reflected a small effect size (t = −0.17, p = 0.86, CI: −1.71, 1.44, d = −0.06).3.5Trial design outcomesA total of 41 participants who met basic pre-screening eligibility criteria were invited to participate in the current trial. Of these, seven declined to participate, representing an enrollment rate of 82.93 %.In response to the blinding question, all participants in the intervention group thought it was likely that they were assigned to the intervention condition, while the majority of participants in the control group reported feeling neutral or likely that they were assigned to the intervention condition (see Supplementary Material 3).4DiscussionOverall, this single, brief VR-based CR module targeting verbal episodic memory in SZ was found to be feasible (attrition rate: 5.88 %), acceptable, and demonstrated medium effect sizes for the improvement of semantic clustering strategies. These findings add to emerging evidence suggesting that the use of VR-based interventions in SZ are feasible, acceptable, and efficacious at treating cognitive outcomes (Jespersen et al., 2023; Schroeder et al., 2022). The current research is the first to evaluate these outcomes for a VR-based CR module specifically targeting verbal episodic memory in SZ, which could be incorporated into future interventions.4.1FeasibilityOf the 34 enrolled participants, most completed the VR session with no cybersickness. Only two participants were excluded from the trial due to poor tolerability of the VR technology. The overall attrition rate (5.88 %) was therefore low, indicating that the use of the VR technology was feasible and well tolerated. It is important to acknowledge that the current intervention involved only a single session, so it is difficult to compare this attrition rate to that of longer, multi-session studies (Wykes et al., 2011). Nevertheless, only one of the excluded participants failed to pass the one-minute VR practice session (control condition), indicating poor tolerance of the VR technology irrespective of condition. The second excluded participant experienced anxiety during the full VR session (intervention group). However, at the end of the appointment, the participant disclosed that they considered postponing the appointment due to high anxiety. Thus, it is possible that this poor tolerance was a reflection of significant pre-existing clinical symptoms and not a direct result of the VR technology.4.2AcceptabilityThe VR-based intervention was well accepted, with both groups demonstrating minimal cybersickness and high enjoyment per the SSQ and VEQ, respectively (see Table 2). Importantly, the intervention and control groups did not significantly differ in their scores on the SSQ and VEQ, suggesting that perceptions of cybersickness and VR experience were similar between conditions. Given that the intervention condition was more immersive than the control condition (i.e., more movement and social interaction), these findings suggest that the increased level of immersiveness did not significantly contribute to increased levels of cybersickness.Overall, the measures of feasibility and acceptability used in the current study all suggest that the VR-based CR module was feasible and acceptable.4.3Preliminary efficacyThe intervention group showed an increase in the use of semantic clustering strategies following the VR-based CR module which trended towards statistical significance. These changes highlight the potential for this VR intervention to yield clinically meaningful increases in the use of semantic clustering. In comparison, the control group showed no statistically significant differences between the pre- and post-intervention assessments of verbal episodic memory nor the use of semantic clustering (see Fig. 4).In their original study, Guimond and Lepage evaluated the efficacy of the full SESAME training in participants with SZ with confirmed deficits in the self-initiation of semantic encoding strategies (n = 13). Following the training, participants displayed a significant increase in verbal episodic memory performance (d = 1.27) and the number of semantic clusters used (d = 1.20). In a more recent and slightly larger study (n = 15), Guimond et al. again showed that the full SESAME intervention led to significant improvements in the number of semantic clusters used by participants with SZ (d = 0.62). However, change in the number of recalled words only trended towards statistical significance (p = 0.09, d = 0.46).The adapted module used in the current study involved a single, much shorter version of the original full SESAME training intervention. Moreover, the participants in the current study did not have confirmed deficits in the self-initiation of semantic encoding strategies. Nevertheless, our results align with the behavioural findings from these two previous studies, showing a statistical trend towards significant improvements in the use of semantic clustering strategies.4.4Trial design outcomesThe majority of invited participants agreed to be enrolled in the current trial (34/41, 82.93 %). This high enrollment rate reflected a strong interest among individuals with SZ to participate in VR-based interventions. The seven individuals who declined to participate in the trial did not disclose the reason(s) for their decision. However, it is important to note that these individuals were invited because they previously disclosed a general interest in participating in research. These individuals only met basic pre-screening eligibility criteria; their full eligibility to participate, determined through the MINI and PANSS, was not yet established.Finally, both groups provided high likelihood ratings concerning their assignment to the intervention group, demonstrating that the blinding procedures employed in the current trial were effective (see Supplementary Material 3).4.5LimitationsThe main limitation of the current study was the relatively small sample size. However, given the pilot nature of this study, this sample size was sufficient to test the feasibility, acceptability, and preliminary efficacy of the module.Second, the intervention and control conditions were delivered in VR and both engaged cognitive processes. Given that there was no control group which did not include a VR-based intervention, it is difficult to isolate the potential effect of the VR component on verbal episodic memory performance. Future research should contrast a VR-based CR program with a more traditional, non-VR approach. Nevertheless, the protocol used in the current proof-of-concept trial was rigorous, allowing us to reject the hypothesis that any changes in behavioural performance following the intervention were due to the VR immersion alone or repeated HVLT assessments.Third, given the single-blind study design, the experimenter who conducted the cognitive assessments was unblinded to group assignment, introducing a potential risk of experimenter bias.Fourth, the observed and self-reported semantic encoding strategies used by participants in the intervention group were not documented. During the intervention, the experimenter frequently asked participants, “What strategies did you use, and do you think they helped you?”, while also encouraging the use of active rehearsal and semantic clustering strategies (see Supplementary Material 2). It is possible, however, that differences in the use of these strategies influenced subsequent performance on the HVLT. It is also possible that participants engaged semantic encoding strategies outside of those that were taught in the CR intervention. Future research should report the observed and self-reported use of the target encoding strategies among participants, as well as their influence on the primary outcome measure(s).Finally, considering the brief duration of VR immersion used in the current study, along with concerns that pre-intervention assessments might prime higher post-intervention ratings, the SSQ was administered at the post-intervention time point alone to assess acceptability (Brown et al., 2022). Future studies should consider pre- and post-immersion SSQ administrations per Bouchard et al. .5ConclusionThe VR-based CR module used in the current study was found to be feasible and acceptable by users with SZ. The results also suggest that the module may have a positive impact on semantic encoding strategies. These findings have implications for the conceptualization, development, and delivery of non-pharmacological treatments for cognition in SZ.VR-based CR offers a more realistic and ecological approach to treating cognitive impairments in SZ. The current module could be used as part of future CR interventions targeting improvements in verbal episodic memory for individuals with SZ. Further research in larger samples involving a greater number of sessions and extended durations is warranted.FundingThis work was supported, in part, by the 10.13039/501100000024Canadian Institutes of Health Research (Doctoral Award: Frederick Banting and Charles Best Canada Graduate Scholarship; BB); the Dr. Charles W. MacLeod Fellowship (BB); the Ontario eSports Scholarship Program (BB); the Canada Research Chairs Program (SB); the Emerging Research Innovators in Mental Health (eRIMh) Award from the Royal's Institute of Mental Health Research (SG); and the Chercheur Boursier Junior 1 from the Fonds de Recherche du Québec en Santé (FRQS; PI: SG).CRediT authorship contribution statementBryce J. M. Bogie: Writing – review & editing, Writing – original draft, Visualization, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Chelsea Noël: Writing – review & editing, Writing – original draft, Investigation, Conceptualization. Feng Gu: Writing – review & editing, Writing – original draft, Project administration, Investigation, Conceptualization. Sébastien Nadeau: Writing – review & editing, Writing – original draft, Software, Methodology, Investigation. Cecelia Shvetz: Writing – review & editing, Software, Investigation, Conceptualization. Hassan Khan: Writing – review & editing, Conceptualization. Marie-Christine Rivard: Writing – review & editing, Software, Conceptualization. Stéphane Bouchard: Writing – review & editing, Software, Methodology, Funding acquisition, Conceptualization. Martin Lepage: Writing – review & editing. Synthia Guimond: Writing – review & editing, Writing – original draft, Visualization, Supervision, Software, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.Declaration of competing interestML reports grants from Otsuka Lundbeck Alliance, personal fees from Otsuka Canada, personal fees from Lundbeck Canada, grants and personal fees from 10.13039/100014554Janssen, grants from 10.13039/100004337Roche, and personal fees from Boehringer-Ingelheim, all outside the submitted work. SB is President of, and owns equity in, Cliniques et Développement In Virtuo, a spin-off from the university that distributes virtual environments. The terms of these arrangements have been reviewed and approved by the Université du Québec en Outaouais in accordance with its ‘Conflict of Interest’ policies. SG has received financial compensation for consulting services from Boehringer Ingelheim, outside the submitted work.

PMC

Stroke and Vascular Neurology

36972920

PMC10648045

3-27-2023

10.1136/svn-2022-002075

Comparison of carotid endarterectomy and repeated carotid angioplasty and stenting for in-stent restenosis (CERCAS trial): a randomised study

Hrbáč Tomáš, Fiedler Jiří, Procházka Václav, Jonszta Tomáš, Roubec Martin, Pakizer David, Václavík Daniel, Netuka David, Heryán Tomáš, Školoudík David

Background and aimIn-stent restenosis (ISR) belongs to an infrequent but potentially serious complication after carotid angioplasty and stenting in patients with severe carotid stenosis. Some of these patients might be contraindicated to repeat percutaneous transluminal angioplasty with or without stenting (rePTA/S). The purpose of the study is to compare the safety and effectiveness of carotid endarterectomy with stent removal (CEASR) and rePTA/S in patients with carotid ISR.MethodsConsecutive patients with carotid ISR (≥80%) were randomly allocated to the CEASR or rePTA/S group. The incidence of restenosis after intervention, stroke, transient ischaemic attack myocardial infarction and death 30 days and 1 year after intervention and restenosis 1 year after intervention between patients in CEASR and rePTA/S groups were statistically evaluated.ResultsA total of 31 patients were included in the study; 14 patients (9 males; mean age 66.3±6.6 years) were allocated to CEASR and 17 patients (10 males; mean age 68.8±5.6 years) to the rePTA/S group. The implanted stent in carotid restenosis was successfully removed in all patients in the CEASR group. No clinical vascular event was recorded periproceduraly, 30 days and 1 year after intervention in both groups. Only one patient in the CEASR group had asymptomatic occlusion of the intervened carotid artery within 30 days and one patient died in the rePTA/S group within 1 year after intervention. Restenosis after intervention was significantly greater in the rePTA/S group (mean 20.9%) than in the CEASR group (mean 0%, p=0.04), but all stenoses were <50%. Incidence of 1-year restenosis that was ≥70% did not differ between the rePTA/S and CEASR groups (4 vs 1 patient; p=0.233).ConclusionCEASR seems to be effective and save procedures for patients with carotid ISR and might be considered as a treatment option.Trial registration number NCT05390983.

WHAT IS ALREADY KNOWN ON THIS TOPICIn-stent restenosis is relatively prevalent complication after carotid stenting.Optimal management of in-stent restenosis is controversial.WHAT THIS STUDY ADDSBoth carotid endarterectomy and repeat percutaneous transluminal angioplasty with or without stenting are safe procedures.Both interventions have comparable low risk of perioperative complications.HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYCarotid endarterectomy is second level option for in-stent restenosis.It might be considered after repeated angioplasty failure.IntroductionCarotid angioplasty and stenting (CAS) is a safe and effective alternative to carotid endarterectomy (CEA) that is widely used for primary and secondary stroke prevention in patients with severe symptomatic or asymptomatic carotid artery stenosis, especially in patients at high risk for complications during or after CEA because of co-morbidities, anatomic variations or contraindications.1–5 In-stent restenosis (ISR) is an infrequent but potentially serious complication after CAS that occurs in 3.5%–14% of patients and causes an ischaemic event in up to 2% of patients.3–11 Data from the largest randomised controlled trials in which the safety and efficacy of CAS and CEA were determined reported the incidence of ISR at the upper end of this range, that is, 11.1% in the SPACE trial3 and 12.2% in the CREST trial.4 Thus, ISR is one of factors limiting the long-term efficacy of CAS.Nevertheless, the optimal management of patients with carotid ISR is controversial due to a lack of consensus with respect to definitions, intervention indications, type of intervention and technical strategies.12–14 Usually, reintervention with repeated endovascular procedures is recommended for patients with ISR.13–15 Restenosis should be first treated with in-stent balloon inflation (repeated percutaneous transluminal angioplasty; rePTA), and if necessary, with the implantation of a new stent, that is, stent-in stent. However, both methods (especially rePTA) are sometimes associated with unsatisfactory long-term results due to high rates of repeated ISR.3–11 Moreover, rePTA with or without stenting (rePTA/S) might be contraindicated or technically problematic in some patients. Therefore, other treatment methods are being investigated. Carotid endarterectomy with stent removal (CEASR) is one of these methods but clinical randomised trials demonstrating its safety and efficacy compared with rePTA/S are still lacking.2 6 16–18 The aim of this randomised study was to compare the safety and effectiveness of CEASR and rePTA/S in patients with carotid ISR.MethodsAll consecutive patients with ISR after CAS treated at the University Hospital Ostrava between July 2017 and June 2021 were selected for the study. The inclusion criteria were as follows:ISR in the carotid artery (80%–99%).Indication for carotid reintervention.18–80 years of age.Functionally independent with a modified Rankin score value of 0–2 points.Signed informed consent.The exclusion criteria were as follows:Contraindication to general anaesthesia.Contraindication to angiography (eg, iodine allergy).Technically impossible to perform angioplasty with or without stenting.Participation in another clinical study within 60 days.Technically impossible to perform carotid endarterectomy (according to surgeon discretion, eg, high cervical position of ISR).Patients with ISR of the carotid artery and an indication for carotid intervention who met all inclusion and exclusion criteria were randomly allocated using computer-based randomisation system into the rePTA/S or CEASR groups.CEA with stent removalSurgery was performed using general anaesthesia using a cut at the front angle of the sternomastoid muscle by one experienced surgeon (˃700 CEA during 10 years). The common carotid artery (CCA), then the internal carotid artery (ICA) and external carotid artery (ECA) were isolated. The CCA, ICA and ECA were temporarily closed. Using a longitudinal cut to the CCA and ICA, a stent with an atherosclerotic plaque was visualised. The stent and plaque were withdrawn under microscopic control and a suture for the arteriotomy was placed using a monofilament, non-absorbent 6/0 fibre. Before completing the procedure, haemostasis was controlled and drainage was set. Surgery was completed by suturing the subcutis and cutis. Unfractionated heparin (100 IU/kg of body weight) was administered to all patients just before the arteriotomy. In cases with insufficient collateral flow into the middle cerebral artery after clipping of the CCA and ICA, a temporal shunt was used. Antiplatelet therapy (clopidogrel (75 mg/day) or acetylsalicylic acid (100 mg/day)) was used continuously in all patients (figure 1).Figure 1Carotid endarterectomy with stent removal in patients with carotid in-stent restenosis. Carotid in-stent restenosis on digital subtraction angiography before surgery (A), stent removal during carotid endarterectomy (B), removed stent with atherosclerotic plaque after carotid endarterectomy (B) and histology of atherosclerotic plaque with a stent (B).Repeated carotid percutaneous transluminal angioplasty with or without stentingEndovascular interventions were performed under local anaesthesia via femoral access in the department with 100–150 performed CAS per year by two experienced interventional radiologists. Unfractionated heparin (100 IU/kg of body weight) was administered to all patients. The procedure began with a diagnostic angiography. On verification of severity and morphology of the in-stent stenosis, a 90 cm 6F sheath was introduced into the CCA. The procedure was performed using distal filter protection (FilterWire EZ; Boston Scientific, Natick, Massachusetts, USA). ISR was treated preferentially with a 5 mm diameter drug-eluting balloon (Sequent Please OTW, B. Braun Melsungen AG, Melsungen, Germany; off label use). In patients with gracile arteries or more severe stenoses, predilation with a 3 mm or 4 mm diameter balloon (according to the decision of the radiologist) was carried out. Atropine (<1.0 mg) was administered intravenously to prevent serious bradycardia during dilation. The single-layer stent (Carotid Wallstent, Boston Scientific, Santa Clara, California, USA) was placed within the previous stent in patients with a suboptimal result after angioplasty at the discretion of the interventionist. Whenever possible, a double-layer technology stent (Roadsaver, Terumo, Tokyo, Japan) was used to ensure maximum wall coverage. The distal filter was removed, followed by completion of the angiogram, including the intracranial arteries. Dual-antiplatelet therapy (clopidogrel (75 mg/day) and acetylsalicylic acid (100 mg/day)) was administered for at least 6 weeks postprocedure. All patients underwent preprocedural laboratory testing to determine clopidogrel resistance. Clopidogrel was replaced by prasugrel or ticagrelor in clopidogrel non-responders (figure 2).Figure 2Repeat percutaneous transluminal angioplasty in patient with carotid in-stent restenosis. Carotid in-stent restenosis on digital subtraction angiography before angioplasty (A) and residual in-stent stenosis after angioplasty (B).Clinical examinationsStandard physical and neurological examinations using the National Institutes of Health Stroke Scale and modified Rankin scale were performed before intervention (CEASR or rePTA/S), and at 24 hours, after 30 days and 1 year after the CEA by blinded certified vascular neurologist.The primary end-point was defined as combined end-point of any vascular event (stroke, transient ischaemic attack, amaurosis fugax, retinal infarction, myocardial infarction or vascular death) within 30 days after intervention, restenosis ˃50% or occlusion of intervened artery within 1 year after intervention and local complications associated with CEASR or rePTA/S within 30 days after intervention.Neurosonology examinationA standard neurosonology examination was performed prior to intervention, 30±3 days and 1 year±14 days after intervention by certified neurosonologist. Specifically, the examination included duplex sonography of the carotid and vertebral arteries with evaluation of residual stenosis or restenosis in the intervened artery, and transcranial colour-coded duplex sonography with evaluation of blood flow in the main arteries of the circle of Willis. Criteria published by Setacci et al 19 and von Reutern et al 20 were used for evaluation of the severity of restenosis. Criteria for 80% restenosis were: average peak systolic velocity (PSV) 370 cm/s, poststenotic PSV 140 cm/s, present collateral flow and carotid ratio (ICA/CCA) >4.Statistical analysisAn estimate for the minimum sample size calculation to demonstrate non-inferiority was based on a 40% difference in composite end-point with an alpha level of 5% and a power of 80%. A prestudy statistical calculation determined that a minimum sample size of 30 patients was required to complete the study. Assuming that 25% of screened patients will not meet all inclusion criteria or will not complete the study, a minimum of 40 patients were needed to screen for study eligibility.All statistical tests were performed at the Centre for Health Research (Faculty of Medicine, University of blinded). The normality of the distribution of all data was checked using the Shapiro-Wilk test. Data with a normal distribution are reported as the mean±SD. Parameters not fitting a normal distribution are presented as the mean, median and IQR. Categorical variables in the two arms were compared using the Pearson χ2 or Fisher’s exact test. Continuous variables were compared by the Student’s t-test for normally distributed values or the two-sample Wilcoxon rank sum (Mann-Whitney) test. All tests were carried out at a 0.05 alpha level of significance using STATA V.17 (StataCorp).ResultsOf 40 screened patients (24 males and 16 females; mean age, 68.5±6.0 years) with carotid artery stenosis (˃80%), 31 (19 males and 12 females; mean age, 67.6±6.2 years) fulfilled all inclusion criteria. Fourteen and 17 patients were randomly allocated to the CEASR and rePTA/S groups, respectively. New stents were implanted in six patients in the rePTA/S group; the other patients underwent angioplasties. The demographic data are shown in table 1. There was no statistically significant difference between groups for any parameter. All patients in both groups who underwent ISR procedures were asymptomatic prior to randomisation (table 2).Table 1Demographic dataCEASR groupRePTA/S GroupP valueNo of patients; n1417NAMale gender; n (%)9 (64.3)10 (58.8)0.756Age at time of randomisation; mean±SD (years)66.3±6.668.8±5.60.295Age at first CAS; mean±SD (years)62.7±7.265.5±5.60.250Severity of carotid in-stent restenosis; mean±SD (%)86.1±6.082.6±5.70.131Severity of carotid stenosis prior to first CAS; mean±SD (%)84.3±4.981.2±7.40.187Right side of in-stent restenosis; n (%)8 (57.1)11 (64.7)0.667Stroke/TIA/amaurosis fugax/retinal infarction medical history; n (%)8 (57.1)13 (76.5)0.224Symptoms (stroke/TIA/amaurosis fugax/retinal infarction) in the territory of the intervened carotid artery in medical history; n (%)5 (35.7)4 (23.5)0.363Arterial hypertension; n (%)12 (85.7)15 (88.2)0.622Diabetes mellitus; n (%)10 (71.4)8 (47.1)0.171Hyperlipidaemia; n (%)12 (85.7)14 (82.4)0.597Atrial fibrillation; n (%)2 (14.3)2 (11.8)0.622Coronary heart disease; n (%)9 (64.3)11 (64.7)0.981Myocardial infarction in medical history; n (%)3 (21.4)5 (29.4)0.466Other vascular intervention; n (%)8 (57.1)9 (52.9)0.815Smoking; n (%)10 (71.4)8 (47.1)0.171Alcohol use; n (%)9 (64.3)10 (58.8)0.756Statin use; n (%)12 (85.7)15 (88.2)0.622Antiplatelet treatment; n (%)13 (92.9)16 (94.1)0.708 Acetysalicylic acid (100 mg); (%)4 (28.6)16 (94.1) Clopidogrel (75 mg); n (%)9 (64.3)16 (94.1)Anticoagulation; n (%)1 (7.1)1 (5.9)0.968CAS, carotid artery stenting; CEASR, carotid endarterectomy with stent retrieval; n, number; NA, not available; rePTA/S, repeated percutaneous transluminal angioplasty with or without stenting; TIA, transient ischaemic attack.Table 2Study resultsCEASR groupRePTA/S groupP valueNo of patients; n1417NASymptomatic in-stent restenosis; n (%)0 0 1.000Duration of procedure; mean±SD (min)86.9±23.433.5±13.4<0.001Residual stenosis after intervention; mean±SD (%)0±020.9±6.7<0.001Residual stenosis (≥30%) after intervention; n (%)0 4 (23.5)0.040Stroke/retinal infarction within 30 days after intervention; n (%)0 0 1.000TIA/amaurosis fugax within 30 days after intervention; n (%)0 0 1.000Myocardial infarction within 30 days after intervention; n (%)0 0 1.000Death within 30 days after intervention; n (%)0 0 1.000Stroke/retinal infarction within 1 year after intervention; n (%)0 0 1.000TIA/amaurosis fugax within 1 year after intervention; n (%)0 0 1.000Myocardial infarction within 1 year after intervention; n (%)0 0 1.000Death within 1 year after intervention; n (%)0 1 (5.9)0.548Restenosis (≥70%) 1 year after intervention; n (%)1 (7.1)4 (23.5)0.233Local complications associated with CEASR or rePTA/S within 30 days; n (%)2 (14.3)1 (5.9)0.704Combined end-point; n (%)3 (21.4)5 (29.4)0.719CAS, carotid artery stenting; CEASR, carotid endarterectomy with stent retrieval; n, number; NA, not available; rePTA/S, repeated percutaneous transluminal angioplasty with or without stenting; TIA, transient ischaemic attack.Both interventions were safe, no periprocedure vascular events were recorded 30 days and 1 year after intervention and combined end-point was recorded in only 21.4% patients in CEASR group and 29.4 % patients in rePTA/S group, respectively (table 2). The implanted stent in carotid restenosis was successfully removed in one peace with atherosclerotic plaque without vessel wall damage in all patients in the CEASR group. Only one patient in the CEASR group had asymptomatic occlusion of the intervened carotid artery within 30 days. One patient died due to COVID-19 infection in the rePTA/S group within 1 year after intervention. Residual stenosis after intervention was significantly greater in the rePTA/S group (mean, 20.9 %) than the CEASR group (mean 0 %, p=0.04). Nevertheless, residual stenosis ≥50% was not detected in any patient in either group. Restenosis (≥70 %) after 1 year was not significant more often in the rePTA/S group compared with the CEASR group (4 vs 1 patient; p=0.233; table 2). No intimal injuries were observed.DiscussionThe current randomised pilot study showed that both rePTA/S and CEASR are safe procedures for treating carotid ISR. No patient in any group had a stroke, transient ischaemic attack or myocardial infarction within 1 year after intervention.Although CEA is still the method of first choice in patients with severe symptomatic or asymptomatic stenosis, in recent years CAS has become a completely equivalent method to CEA in many such patients.1–5 In some cases, such patients at high surgical risk or in patients in whom a CEA cannot be performed for technical reasons, CAS is clearly the first choice.8 The advantages of CAS include the cosmetic effect, avoidance of cranial nerve palsy and a shorter hospitalisation time.8 21 22 ISR after CAS is one of the more serious complications associated with this procedure.3–11 The incidence of ISR is relatively rare and varies significantly between studies. One of the reasons for the variations in incidence is the inconsistent definition of ISR.12 The definition of ISR depends not only on the minimum percentage of restenosis, which is already evaluated as ISR, but also on the time of the evaluation, that is, the time since CEA, and the examination method.The most common method with which to evaluate ISR is duplex sonography. It is necessary, however, to take into account the change in hemodynamics of the stent and modify the criteria for evaluating the severity of stenosis according to flow rates.2 19 23–25 Digital subtraction angiography is the gold standard for evaluating DSA, but it is limited by invasiveness, while CT angiography and MR angiography are limited by stent artefacts.26 27 The risk of clinical manifestation of stenosis are as follows: the risk of an ischaemic cerebrovascular event, including transient ischaemic attack, ischaemic stroke, ischaemia of the eyeball (amaurosis fugax and retinal infarction) and the haemodynamic risk of injury (reduced local perfusion) to the brain, especially the risk of accelerating the progression of cognitive function decline with the risk of developing dementia.7 8 Currently, there are several treatment options for ISR. The best medical treatment is one of the intervention methods. Recently, cilostazol is tested for a reduction of ISR risk.28 29 At present, rePTA is used more often than rePTAS and different drug-coated baloons are tested. However, there is still no clear evidence of effectiveness of drug-coated balloons.13 30 31 CEASR is the next method of choice, especially in patients in whom rePTA/S has failed or this method cannot be performed due to technical obstacles.6 13 17 18 31 Rarely, carotid bypass or external beam radiotherapy can also be used.14 The results of published meta-analyses were similar to our results. Specifically, CEASR is a comparable method to rePTA/S with a low risk of perioperative vascular events. A recent meta-analysis with >1000 patients with intervention for ISR from 11 studies demonstrated that both rePTA/S (85% of the cohort) and CEASR (15% of the cohort) had similarly low rates of perioperative vascular events (1% vs 2 %), death (0% vs 3 %) and restenosis after the second intervention (0% vs 0 %); the procedures were equally feasible and effective for treating ISR.13 No statistically significant differences were found in the incidence of the other severe complications. Ten patients in the rePTAS group had a documented deformation or kinking of the stent as a shortcoming of CAS.13 These results are comparable with results of our randomised study where no vascular events or death were recorded.Although rePTA/S remains the method of first choice, CEASR can be considered in patients with repeated rePTA/S failure, in patients with contraindication or high risk of rePTA/S, for example, in patients with severely calcified plaques or preocclusive stenosis.6 17 18 Contrary, a relative contraindication to CEASR might be a patient with a high risk for open surgery or a technically difficult procedure, for example, if the stent is long enough or placed in a distal position of ISR beyond the surgeon’s control.27 There were limitations to this study that need to be considered. First, the number of included patients was relatively low. The sample size calculation was performed to show non-inferiority of tested methods in combined end-point. The incidence of ISR was low and a randomised clinical trial comparing rePTA/S and CEASR with a higher number of included patients is still missing. However, due to the positive results of this pilot study, a multicentre clinical randomised trial is being prepared. The second limitation was the impossibility of comparing rePTA with rePTAS because the stent was implanted according to technical possibilities at the discretion of the interventional radiologist and was not randomised or mandatory. The last limitation was the follow-up time. Patients were followed for 1 year, thus it was not possible to determine the long-term risk of restenosis.ConclusionsBoth CEASR and PTA/S appear to be safe and effective methods for the treatment of carotid ISR with a comparable low risk of perioperative complications and long-term risk of vascular events.

PMC

Autism

36597933

PMC10374993

8-01-2023

10.1177/13623613221143590

Development of stigma-related support for autistic adults: Insights from the autism community

Han Emeline, Scior Katrina, Heath Eric, Umagami Kana, Crane Laura

Many autistic adults experience public stigma and some internalise this stigma with negative effects on their mental health. While efforts to reduce public stigma are paramount, change can be slow, and interventions to prevent internalised stigma may also be needed. Using a mixed methods online survey, we gathered the views of 144 autistic adults and parents/caregivers of autistic people in the United Kingdom on whether a stigma-related support programme for autistic adults is needed and, if so, what it should ‘look’ like. Quantitative data (summarised descriptively) showed that most participants felt it was important for autistic adults to have support in managing stigma and revealed diverse preferences in terms of programme delivery, underscoring the need for flexibility. Using reflexive thematic analysis, four main themes were identified from the qualitative data: ‘We need to change society not autistic people’, ‘Stigma is difficult to manage alone’, ‘Focus on positive, practical support’, and ‘There is no one size fits all approach’. We discuss the important implications our findings have for how future interventions in this area are framed and delivered.Lay AbstractMany autistic adults experience public stigma, which refers to negative attitudes and treatment from others. Because of that, some autistic adults may also apply unhelpful beliefs to themselves, which is known as internalised stigma. There is some evidence that both public stigma and internalised stigma are linked to poorer mental health in autistic adults. Clearly, it is crucial to change how society thinks and acts towards autistic people. There are several programmes that are trying to do this. But as change can be slow, support may also be needed to help autistic people cope with and challenge stigma. Using an online survey, we gathered the views of 144 autistic adults and parents/caregivers of autistic people in the United Kingdom on whether a stigma support programme for autistic adults is needed and, if so, what it should ‘look’ like. Most participants felt it was important for autistic adults to have support in managing stigma because of the harmful effects that stigma has on mental health and the challenges that autistic adults face in disclosing their diagnosis. However, participants were also concerned that such a programme could convey the message that autistic people, rather than society, need to change. Participants suggested that the programme should be positive and practical, helping autistic adults to understand and accept themselves, as well as learn context-specific strategies for responding to stigma and/or disclosing their diagnosis. They also stressed that the programme should be flexible and inclusive, recognising that autistic adults have very different needs and preferences.

IntroductionStigma is a multi-level phenomenon. Public stigma refers to a process in which differences are labelled by society, and labels are associated with negative stereotypes, leading to discrimination and status loss for labelled individuals (Link & Phelan, 2001). In addition to the direct effects of social devaluation, labelled individuals may also internalise stigma with further harms to their self-esteem and self-efficacy (Corrigan et al., 2006). Internalised stigma is a process that involves becoming aware of public stereotypes, agreeing with those stereotypes, and applying those stereotypes to oneself (Corrigan & Rao, 2012). This process of internalisation is particularly insidious as, although it originates from social attitudes, it ‘can become self-generating and persist even when individuals are not experiencing direct external devaluation’ (Meyer & Dean, 1998, as described in Frost & Meyer, 2009, p. 2). In response to stigma, some may seek to dissociate themselves from the stigmatised group/identity, while others may choose to strongly associate themselves with the group/identity and advance social justice, akin to how the autistic self-advocacy and neurodiversity movements have reclaimed and reframed autism as a positive identity (Leadbitter et al., 2021).Autistic adults commonly report perceiving, anticipating and experiencing public stigma (Han et al., 2022). Many autistic adults are aware of negative stereotypes that society has of autistic people, and some apply these to themselves (Botha et al., 2020; Leedham et al., 2020; Punshon et al., 2009). There is evidence that both public stigma and internalised stigma are associated with poorer mental health in autistic adults. Less perceived autism acceptance from external sources and less personal autism acceptance has predicted greater levels of depression (Cage et al., 2018). Higher exposure to stigma-related stressors such as victimisation, everyday discrimination, expectation of rejection, internalised stigma, concealment, and disclosure has also been linked to lower levels of wellbeing and higher psychological distress (Botha & Frost, 2020). Interestingly, both concealment and disclosure have been negatively correlated to wellbeing, illustrating what has been called a ‘double bind’ by autistic adults where either option may have adverse outcomes (Botha et al., 2020).Clearly, it is paramount to address public stigma, as society is the locus of the problem and should be the locus of change. There is an emerging body of literature on interventions designed to reduce autism stigma at the interpersonal level, including education-based interventions that provide factual information about autism, and contact-based interventions that facilitate interaction between autistic and non-autistic people. Generally, interventions solely based on education have reported larger changes in knowledge of autism than stigma towards autistic people, suggesting that the latter is more resistant to change (Gillespie-Lynch et al., 2015; Obeid et al., 2015; Someki et al., 2018). Interventions combining education with high-quality contact have shown promise for improving attitudes and behavioural intentions towards autistic people, but it remains unknown if these effects will persist over time and extend to real-world environments (Dachez & Ndobo, 2018; Gillespie-Lynch et al., 2021; Jones et al., 2021). Thus, while public stigma still exists, some autistic individuals may also benefit from efforts to address stigma at the individual level to prevent internalised stigma. To the authors’ knowledge, there are currently no interventions that specifically provide stigma-related support for autistic adults, although such interventions exist for other stigmatised groups.In the mental health field, several types of internalised stigma interventions have been identified, including Healthy Self-Concept, Self-Stigma Reduction Programme, Ending Self-Stigma, Narrative Enhancement and Cognitive Therapy, Honest Open Proud (HOP), and Photo-Voice (Yanos et al., 2015). These interventions draw on some common techniques, such as using psychoeducation to correct myths, cognitive reframing to counter negative self-beliefs, and narrative approaches to make meaning out of personal experiences. However, HOP differs from the other interventions by focusing on supporting individuals to reach careful decisions around disclosing their diagnosis, rather than directly targeting internalised stigma (Corrigan et al., 2013; Scior et al., 2020). HOP was originally designed as a peer-led group programme involving three weekly sessions that guide participants in weighing the pros and cons of disclosure in different contexts and crafting an empowering personal narrative. A recent meta-analysis found that HOP had significant positive effects on stigma stress as well as smaller, statistically non-significant effects on self-stigma and depression in individuals with mental health problems (Rüsch & Kösters, 2021).Within the disability field, research on stigma interventions at the individual level is still in its infancy. A pilot study adapting HOP for people with dementia and their carers found that the intervention was feasible in community settings in central London but not in healthcare settings in outer London, possibly due to organisational factors and transportation barriers (Bhatt et al., 2020). Nonetheless, qualitative results suggested that participants who attended the adapted HOP intervention found it acceptable and felt they benefitted from peer support. A novel programme, Standing Up For Myself (STORM), has also been developed to empower adults with intellectual disabilities to manage and resist stigma (Scior et al., 2022). STORM was delivered in a peer-led group format with four weekly sessions and a follow-up session, during which participants discussed experiences of stigma and planned their responses to stigma. Preliminary evaluation showed that participants valued the opportunity to process difficult events and emotions, strengthen connection with others, and enhance self-advocacy and self-efficacy. At present, it is unknown whether it may be appropriate to adapt such programmes for an autistic population, or whether there is a need to develop a new stigma support programme for autistic adults.The Medical Research Council (MRC) guidance for developing and evaluating complex interventions has emphasised the importance of engaging stakeholders to maximise the potential of producing an intervention that is acceptable and effective (Skivington et al., 2021). In the development of an intervention for autistic adults, autistic adults are the primary stakeholders and their perspectives should be prioritised as they are experts on their own experience. In addition, we also considered caregivers as secondary stakeholders as some autistic adults may require their support to participate in an intervention, and some studies on interventions for autistic adults have reported caregiver involvement as a facilitator to success (Laugeson et al., 2015; Mandelberg et al., 2014). Furthermore, it is well-documented that parents also experience public and internalised stigma in association with their autistic children, which means that they may be able to share relevant insights on autism-related stigma (Mitter et al., 2019; Papadopoulos et al., 2019). From here on, autistic adults and their parents/caregivers are collectively referred to as ‘the autism community’ (as per Pellicano et al., 2014). It is well recognised that meaningful involvement of the autism community can improve the quality of autism research and contextualise findings in a real-world setting, thereby facilitating the translation of research into practice (Fletcher-Watson et al., 2019; Keating, 2021). Thus, the aim of this study was to conduct a survey to examine the views of the autism community on whether a stigma support intervention for autistic adults is needed and, if so, what it should ‘look’ like, in order to guide future research and intervention development.MethodsMaterialsAn online consultation survey was developed, which comprised a mix of multiple choice and open-ended questions. The survey began with a question asking participants to select the capacity in which they were completing the survey (i.e. as an autistic person, as a parent/caregiver of an autistic person, or both). Autistic adult respondents were additionally asked to state whether they had a formal diagnosis of autism, and the extent to which they were open about their diagnosis/identity (e.g. not open, selectively open, open, or very open). The survey was then organised into three sections. Section 1 collected participants’ demographic information (gender, age, ethnicity), and views on whether it was important for autistic people to have support in managing stigma (on a 5-point scale from ‘definitely not’ to ‘definitely yes’). Section 2 presented a short video describing the HOP and STORM programmes to facilitate discussion on the topic, followed by questions asking which (if either) programme would be appropriate for autistic adults and, if so, how to make the programme more suitable for autistic adults. The third and final section of the survey sought to identify design preferences, including delivery format (e.g. small group, one-to-one, guided self-help; online, in person, or both). Perceived barriers and facilitators to participation were also asked about (e.g. whether having a trained autistic facilitator might help autistic adults to take part; whether not feeling comfortable/ready to talk about their diagnosis might prevent participation). A copy of the survey is available as supplemental material.The survey materials were developed by the first author, with input from autistic members of the team who made suggestions to improve accessibility, such as including a preview of the full survey in the participant information sheet and a transcript of the video in the survey. The autistic team members also emphasised the importance of providing a clear justification for why a stigma intervention would target the stigmatised group rather than the stigmatisers. As such, clarifications were made to the rationale for the research. Specifically, it was emphasised that ongoing efforts to reduce public stigma are indeed crucial, but as change can be slow, programmes that empower autistic people to cope with and challenge stigma may also be needed in parallel.ProcedureEthical approval was obtained from the Department of Psychology and Human Development at IOE, UCL’s Faculty of Education and Society. The survey was set up on the Qualtrics online platform, with a link to the survey disseminated through the Cambridge Autism Research Database (CARD). 1 CARD is an established database of autistic adults and parents of autistic people, predominantly based in the United Kingdom, who volunteer to take part in autism research ( Applications to recruit participants via CARD are considered and approved by a scientific committee before studies are advertised in a monthly mailout to their database of volunteers. All participants read the participant information sheet and provided consent before proceeding to the survey. Data collection occurred during November and December 2021.ParticipantsA total of 208 participants consented to taking part in the survey, of which 64 did not complete the survey. Participants were told that they could skip any questions and could also withdraw at any point by closing their web browser. Thus, partial responders (i.e. participants who exited the survey before the end point) were excluded from the final sample. Complete responders were defined as those who reached the end of the survey and submitted their responses, even if they had not answered all the questions. A total of 144 autistic adults and caregivers/parents of autistic individuals living in the United Kingdom completed the survey. Table 1 contains a breakdown of the participants’ characteristics.Table 1.Participant characteristics.Participant characteristicsN = 144 (100%)aParent/caregiver of an autistic person38 (26.39%)Autistic adult124 (86.11%) Formal diagnosis of autism Yes115 (92.74%) No8 (6.45%) Degree of openness about autism diagnosis/identity Not open (does not tell anyone)3 (2.42%) Selectively open (only tells selected people)39 (31.45%) Open (neither hides nor actively tells others)39 (31.45%) Very open (actively tells and educate others)33 (26.61%)Gender identity Man50 (34.72%) Woman75 (52.08%) Non-binary/other18 (13.19%)Age 18–249 (6.25%) 25–3421 (14.58%) 35–4430 (20.83%) 45–5438 (26.39%) 55–6432 (22.22%) 65–7414 (9.72%)Ethnicity White-British116 (80.56%) Other white background16 (11.11%) Asian4 (2.78%) Middle Eastern1 (0.69%) Mixed background5 (3.47%) Other ethnic group (unspecified)2 (1.39%)aThe total number of parents/caregivers of an autistic person and autistic adults exceeds 100% because 18 participants belonged to both categories.Data analysisTable 2 shows the questions included for data analysis. For quantitative data, descriptive summaries of the responses to each multiple-choice question were produced. For qualitative data, thematic patterns were developed from responses across all open-ended questions. This approach followed Braun et al. , who recommend treating qualitative survey data as one cohesive dataset, coding and developing analytic patterns across the entire dataset rather than summarising responses to each question.Table 2.Questions used for data analysis.QuestionAnalysisDo you think it is important for autistic adults to have support in managing stigma?QuantitativePlease explain your answer to the previous question.QualitativeBased on the information in the video and table, do you think it would be more suitable to adapt the HOP programme or STORM programme for autistic adults?QuantitativePlease explain your answer to the previous question.QualitativeWhat thoughts or advice do you have on making the stigma support programme more relevant to and helpful for autistic adults?QualitativeWhat do you think would be the best way of delivering this programme?QuantitativeIf this programme is delivered in small groups of autistic adults, do you think it should be held online or in-person or a mix of the two?QuantitativeWhat do you think would help autistic adults to take part in this programme?QuantitativeWhat do you think would prevent autistic adults from taking part in this programme?QuantitativeHOP: Honest Open Proud; STORM: Standing Up For Myself.We adopted a reflexive approach to thematic analysis (TA), which involved six phases: familiarising with the data, coding the data, generating initial themes, reviewing and developing themes, refining, defining and naming themes, and writing up (Braun & Clarke, 2019). Reflexive TA involves later theme development, whereby themes are developed from codes and conceptualised as patterns of shared meaning underpinned by a central organising concept. This stands in contrast to coding reliability TA, where themes are often developed early based on data collection questions and conceptualised as domains. Coding reliability TA typically requires multiple researchers to use a coding frame to correctly identify evidence falling within each domain, whereas reflexive TA is not about ‘accurate’ coding but about the researcher’s thoughtful engagement with their data. Here, coding is understood as an inherently subjective process that requires a reflexive researcher who strives to reflect on their assumptions and how these might shape their analysis (Braun & Clarke, 2021).Regarding positionality, the author who led the analysis (EH) does not identify as autistic. All the authors endorse a neurodiversity-affirmative approach to autism research: we believe that there is no one ‘correct’ way of thinking, learning or behaving, and that divergence from the norm should not be pathologised (e.g. Walker, 2021). We also align with social models of disability (e.g. Oliver, 1986), which explains that autistic people are disabled by societal barriers (which systemically exclude/discriminate against them) and not by within-person impairments or deficits. As the purpose of this survey was to inform the potential development of a stigma support programme, we were more likely to identify data that align with these beliefs. However, EH was particularly conscious of her own beliefs about the need for such a programme, and the importance of remaining open to differing views. The researcher was guided by participatory research values throughout the analysis and write-up process, trying to ensure that autistic voices were genuinely heard and represented.To enhance the trustworthiness of our analysis, we followed the 15-point checklist of criteria for good thematic analysis by Braun and Clarke . We ensured that our themes were not developed from a few examples but instead from a comprehensive range of examples across the dataset after thorough review and re-review of the data. In addition to selected quotes embedded within our analytic narrative in the Results section, we have collated a longer table of data extracts corresponding to each theme and sub-theme in the supplemental material to demonstrate a good ‘fit’ between our analytic claims and the raw data. All quotes and extracts are also accompanied by a unique identifier code to show that a variety of participants have been represented. To enhance the quality of our reporting, we applied the 20 questions for evaluating thematic analysis manuscripts by Braun and Clarke . This included clearly specifying which type of TA we used and ensuring that our procedures reflected this form of TA. As explained in the preceding paragraphs, we used reflexive TA rather than coding reliability TA, and as such did not seek to include multiple coders to establish inter-rater reliability; rather, a single researcher led the analysis (in discussion with the other researchers) and strived to reflect on her own positioning.Community involvement statementThis study was conducted by a team that included both autistic and non-autistic members, who had research and/or lived expertise in relation to autism and/or stigma. Autistic team members were involved in developing the research questions and data collection materials. While they were not directly involved in data analysis, they reviewed and discussed the findings with the first author, which informed the write-up of the paper.ResultsQuantitative resultsMost respondents (n = 114, 79%) thought it was important for autistic adults to have support in managing stigma, with 52% indicating ‘definitely yes’ and 27% indicating ‘probably yes’. Seventeen percent felt that autistic adults may or may not need support in managing stigma, and the remaining 4% felt that autistic adults would ‘probably not’ or ‘definitely not’ need such support. When presented with two stigma-related support programmes designed for individuals with mental health problems (HOP) or intellectual disabilities (STORM), 44% thought that it would be suitable to adapt either programme for autistic adults, 25% thought HOP seemed more suitable, 16% thought STORM seemed more suitable, and 15% felt neither programme would be suitable for autistic adults.In terms of delivery method, 32% preferred a small group of autistic adults with a trained facilitator, 18% preferred one-to-one with a trained facilitator, and 13% preferred a guided self-help approach (e.g. completing a workbook on their own with the option to contact a trained facilitator). ‘Other’ responses (37%) commonly stated that the programme should use a combination of these approaches or make all three options available for participants to choose, depending on their needs. If the programme were to be delivered in a small group, 20% preferred it to be conducted online, 22% in-person, and 58% a mixture of the two.The key facilitators to participation endorsed by respondents were providing clear and detailed information so participants knew what to expect beforehand (83% of respondents), and conducting it online with flexible ways of participating (70%), followed by having a trained facilitator who is autistic (52%) and involving the autistic adult’s caregiver/family member (41%). ‘Other’ potential factors (29%) that could help autistic adults to take part included allowing more processing time, providing visual content, adopting a very logical format and structure, as well as coordinating with existing local adult autism support groups.The main barriers to participation endorsed by respondents were travelling and sensory environment (77%), uncertainty about what the programme involves (75% of respondents), and not feeling comfortable or ready to talk about their diagnosis (64%). These were followed by not wanting to be in a group with other autistic people (51%), shame or embarrassment (41%), and not needing help to cope with stigma (38%). ‘Other’ potential factors (32%) that could prevent autistic adults from taking part included not realising that they are experiencing stigma or that stigma is affecting them, not seeing how the programme will help them or change stigma, communication difficulties, emotional difficulties, social anxiety, struggling with group situations, struggling with technology, privacy concerns, time commitment concerns, and pandemic-related safety concerns. Table 3 delineates the quantitative responses given by autistic adults and parents/caregivers.Table 3.Breakdown of quantitative responses.Response categoriesAutistic adultsParents/caregivers Autistic adults’ need for stigma support 124 (100%) 38 (100%) Definitely yes64 (51.61%)23 (60.53%) Probably yes34 (27.42%)6 (15.79%) Might or might not21 (16.94%)6 (15.79%) Probably not3 (2.42%)3 (7.89%) Definitely not2 (1.61%)0 (0.00%) Suitability of HOP and STORM programmes 122 (98.39%) 38 (100%) Both HOP and STORM seem equally suitable54 (43.55%)15 (39.47%) HOP seems more suitable30 (24.19%)13 (34.21%) STORM seems more suitable19 (15.32%)4 (10.53%) Neither HOP nor STORM seem suitable19 (15.32%)6 (15.79%) Preferred format of delivery 124 (100%) 38 (100%) Small group39 (31.45%)12 (31.58%) One-to-one23 (18.55%)5 (13.16%) Guided self-help18 (14.52%)3 (7.89%) Other44 (35.48%)18 (47.37%) Preferred mode of delivery 122 (98.39%) 35 (92.11%) Online25 (20.16%)6 (15.79%) In-person25 (20.16%)8 (21.05%) Mix of online and in-person72 (58.06%)21 (55.26%) Facilitators to participation 123 (99.19%) 37 (97.37%) Providing clear and detailed information beforehand104 (83.87%)28 (73.68%) Conducting it online with flexible ways of participating91 (73.39%)23 (60.53%) Being led by a trained facilitator who is autistic69 (55.65%)17 (44.74%) Involving the autistic adult’s carer/family member44 (35.48%)25 (65.79%) Other37 (29.83%)9 (23.68%) Barriers to participation 123 (99.19%) 37 (97.37%) Concerns about travelling and sensory environment96 (77.42%)26 (68.42%) Uncertainty about what the programme involves93 (75.00%)27 (71.05%) Not comfortable or ready to talk about their diagnosis80 (64.51%)21 (55.25%) Not wanting to be in a group with other autistic people63 (50.81%)22 (57.89%) Shame or embarrassment51 (41.13%)12 (31.58%) Not needing help to cope with stigma50 (40.32%)14 (36.84%) Other41 (33.06%)10 (26.32%)HOP: Honest Open Proud; STORM: Standing Up For Myself.Qualitative resultsFour main themes were identified in the qualitative data: ‘We need to change society not autistic people’, ‘Stigma is difficult to manage alone’, ‘Focus on positive, practical support’, and ‘There is no one size fits all approach’. Figure 1 displays these themes and their sub-themes. All quotes below are accompanied by a participant number and code, with ‘AA’ referring to an autistic adult, ‘PC’ to a parent/caregiver of an autistic individual, and ‘AA-PC’ to a participant who identified as both an autistic adult and a parent/caregiver of an autistic individual.Figure 1.Themes and sub-themes.Theme 1: ‘We need to change society not autistic people’Participants emphasised that the problem of stigma lies with society and not autistic people. Therefore, the primary onus should be on non-autistic people to change their attitudes and behaviours, rather than on autistic people to cope with stigma: ‘I believe that it is important to be clear that prejudice and ignorance stem from society and individuals not from autistic people–it is not our responsibility to change others, but we can help society to change!’ (P122, AA).Sub-theme: ‘Autism is not widely understood’Participants felt that the general population has little to no understanding of autism: ‘I think the real problem is that there is widespread ignorance in the general population about autism and neurodiversity’ (P41, AA). They highlighted that most public knowledge about autism appears to be stereotypical, as fostered by media portrayals of autistic people: ‘I think the majority of the population [still] thinks that autism looks like “Rainman”, so are intolerant and sometimes resentful of people [whose] autism [don’t] present like “Rainman”, which then further alienates and isolates them’ (P106, AA). Autistic adults experienced being met with disbelief if they did not fit the stereotypes others had in mind: ‘When I disclose my autism very frequently people either think I have learning disabilities or, if they realise that I am quite capable academically and professionally, they dismiss my autism and think I’m lying or have a fake diagnosis’ (P126, AA). They described how broad and harmful assumptions regarding their abilities and personalities caused significant problems for them both at work and in their day-to-day life, as others failed to make necessary accommodations or judged them based on their label rather than on merit.Sub-theme: ‘The main solution for stigma is education of wider society’Given that stigma was perceived to stem from ignorance, participants suggested that the main solution was to increase public awareness and understanding of autism: ‘An ideal way to dispel the stigma surrounding autism would be to educate non-autistic people about autism’. (P47, AA). This led some participants to be opposed to the idea of a stigma support programme for autistic people: ‘I do not believe that such programmes [should] exist at all. [It] is up to employers, society and everyone to understand and appreciate autism, not for autistic people to [be] told it is normal to expect negative responses when telling people you are autistic’ (P92, PC). Meanwhile, other participants felt that both efforts targeting autistic and non-autistic people are needed: ‘I think it is important that we support autistic adults to manage stresses and strains of modern society. I also think we should educate people more about autism and learn more about the condition while discouraging discrimination towards autistic people’ (P96, AA). A few participants also recognised that it can be empowering for autistic people to educate those around them: ‘Involvement in the retraining of society empowers those being ostracised–it gives people a sense of power to elicit change, rather than being powerless. A lot of stigma is about an imbalance of power’ (P122, AA).Theme 2: ‘Stigma is difficult to manage alone’Those who saw value in a stigma support programme for autistic adults reported that stigma is a pervasive reality in many autistic people’s lives, and it can be challenging to deal with judgement and negativity on a daily basis on their own: ‘I find stigma is difficult to manage alone because when people react in strange ways, I take it wholly on board and it makes me feel really uncomfortable. I also start to see myself through their eyes. I was diagnosed extremely late and I encountered a lot of scepticism from people I thought I could trust with the information’ (P100, AA-PC).Sub-theme: ‘Stigma can affect mental health and self-worth’Participants shared that ‘lack of understanding, information and the stigma attached to autism can cause autistic adults to feel ashamed, embarrassed or misinformed about their condition’ (P74, AA). They also explained that ‘there are various different unhelpful beliefs about autistic people [that] can be damaging to self-worth’ (P133, PC), causing low self-esteem, self-doubt and trauma to those who are subjected to it. This was perceived to have an extensive negative psychological impact: ‘Stigma is something that can hurt people very much and has long-term consequences for mental health and wellbeing’ (P97, AA). Hence, these participants recognised that it was important for autistic people who experience stigma and are affected by stigma to be protected from internalising it: ‘It would be good to have an outlet instead of internalising the effects of stigma’ (P77, AA). They lamented the general lack of support available for autistic adults, which was felt to breed ‘insecurity and confidence issues’ (P27, AA). They called for more post-diagnostic support to promote autistic adults’ mental health and wellbeing, part of which would include support to deal with the stigma surrounding autism.Sub-theme: ‘It is often difficult to know who to tell’Another common sub-theme under the difficulty of managing stigma related to disclosure. Autistic adults highlighted the struggle of ascertaining who it was safe to disclose their diagnosis to, and how to go about it in a way that would allow them to avoid bias and discrimination: ‘The question is–what do I do as an autistic person–I want to explain how the world looks and feels to me and why I have difficulties with certain environments but, at the same time, I don’t want people to see my diagnosis as a negative thing that they have to “deal” with or that is an inconvenience’ (P111, AA). Some described facing negative outcomes of disclosure, including judgmental responses, hostility and rejection, which then made them extremely cautious about telling people that they are autistic. Others recognised that internalised stigma prevented them from disclosing, even if others may not necessarily stigmatise them: ‘I think I hold a lot of self-stigma around autism, and that motivates me to not tell other people, who may not hold such prejudices’ (P140, AA).Both autistic participants and parents of autistic individuals mentioned that many autistic adults mask (hide their autistic traits, whether by a conscious decision or a behaviour which becomes so ingrained that the person is no longer aware of doing so): ‘My adult son with a diagnosis spends most of his time masking in public because of the stigma attached to autism’ (P49, AA-PC). Masking was perceived to have additional negative effects on mental health: ‘This stigma is reinforced into us as the more that we mask the more people treat us better and so reward us for treating ourselves badly’ (P35, AA). Therefore, some participants saw the need for support on how to unmask: ‘There is a lot on the Internet of people’s experiences of “unmasking”, but no one actually helps with this. No one supports you to navigate how people’s perceptions of you change, how you change what you think about yourself. There are lots of layers of internalised ableism that prevent people [from] being who they are and feeling better about themselves, but you have to work it all out alone’ (P50, AA).Theme 3: ‘Focus on positive, practical support’In order for support to be beneficial, participants stated that it should be positive and practical, rather than focusing on negative experiences of stigma that may increase feelings of victimisation: ‘Stigma is bad and hurtful; I know from experience. However sometimes it is better not to remind people they are victims’ (P89, AA). In view of that, a few participants felt that the concept of ‘stigma’ should be left out altogether, including one caregiver who expressed, ‘I wouldn’t want to introduce the idea of stigma to my son for whom we try to make autism as positive as possible’ (P92, PC). Others, however, felt that it would be helpful for autistic adults to learn how to identify stigma: ‘Autistic adults may not recognise stigma when it occurs, as it can be subtle and often conveyed through things other than words’ (P143, PC). Nonetheless, there was general consensus that the programme should revolve around empowerment and not ‘mutual sympathy’.Sub-theme: ‘Understanding and accepting themselves’Participants stressed that autistic adults should be equipped with knowledge of their own autism, and that this understanding should be grounded in the neurodiversity paradigm or social model of disability: ‘Participants should receive helpful information that challenges the predominant views of autism, especially medicalized views, as well as the disability justice resources’. (P118, AA). Accordingly, autistic adults should be encouraged to see autism as ‘positive or neutral, rather than negative’ (P110, AA) and ‘less of a disability and more of a difference’ (P63, AA). Some participants also suggested that the programme should enable autistic adults to identify advantages of autism and how to make use of them or ‘sell themselves’, which can help to bust myths and stereotypes. Others preferred a balanced approach that would help autistic people to understand and accept both their strengths and limitations: ‘I think it is key to educate autistic people on the strengths of their condition and promote confidence in being upfront and honest about any difficulties they anticipate’ (P21, PC).Sub-theme: ‘Strategies need to be context specific’The second aspect of providing positive and practical support pertained to developing context-specific strategies for responding to stigma and/or disclosing one’s diagnosis. One autistic adult explained, ‘generalised ideas will be too complicated to apply in the moment as you need a great deal of social skill and self-awareness to alter them to each situation’ (P23, AA). One parent also stated, ‘Discussing the different types of people they might meet is important. For example, they might want/need to behave differently if they are talking to an employer as opposed to police authorities’ (P15, PC). The context most commonly mentioned by autistic adults in our sample was employment: ‘Perhaps someone is about to apply for a job and [wants] to educate themselves on discrimination law and also to access practical information to support them through the recruitment process e.g. a list of phrases to help them disclose or even challenge something being said in a way that is respectful and appropriate to the context of recruitment’ (P134, AA). A few mentioned that they would like to learn scripts and planned behaviour, or suggested that scenarios and role-play could be used to practise the skills taught.Theme 4: ‘There is no one size fits all approach’A recurring theme throughout the data was that there is no single approach that would be suitable for every autistic individual. Given the heterogeneity of the autistic population, participants acknowledged the difficulty of designing a support programme that would be relevant to all: ‘The relevance would need to take into account the continuum that is autism, and be matched to the cohort, which, as we know, varies considerably . . . What is then helpful to one person is patronising to another, and so on’ (P76, AA-PC).Sub-theme: ‘There is a huge spectrum and range of abilities’Participants highlighted the wide range of intellectual abilities among autistic adults and importance of ensuring that support is pitched at the right level: ‘not too patronising if it’s for autistic adults without intellectual disabilities, but also not too complicated for those with’ (P4, AA). They also emphasised the need to accommodate different communicative abilities: ‘Non-speakers, who face some of the most severe stigma and are a population whose views are routinely ignore[d], should be actively recruited and not segregated from the speaking groups’ (P118, AA). One caregiver expressed that her autistic son would need her support to participate: ‘my son would not interact or understand if I were not there’ (P83, PC).Following on from differing abilities, participants recognised that autistic adults may differ in their level of awareness of stigma: ‘Some may be unaware of other people’s attitudes towards them or towards autism in general. Others may be acutely aware. It would need to be a flexible programme’ (P116, AA). They may also vary in their level of confidence in managing stigma: some ‘may prefer not to have support in this situation, as they feel that they can manage’ (P109, PC). The type and level of support needed could depend on time of diagnosis, with potential differences between autistic people diagnosed as adults versus those diagnosed as children, and individuals who are newly diagnosed versus those who have had a diagnosis for some time. Linked to this are potential disparities in prior knowledge of autism and involvement in the autism community: ‘Some people will have read a lot about autism, have already done a lot of self-discovery, or be heavily involved in communities, so might be in a better position to reflect than those who are perhaps still adjusting to seeing themselves as/admitting to themselves that they are autistic’ (P108, AA).Sub-theme: ‘Groups could be a major barrier for some people’Many autistic adults mentioned that they would not personally attend a group programme: ‘group sessions . . . would dis-incentivise me from taking part and hinder my learning–taking turns, filtering out the noise of other voices and presenting our own thoughts are problems most autistic people experience’ (P136, AA). In particular, they felt that discussing sensitive topics such as identity and stigma would be anxiety-inducing in a group setting: ‘discussing something as deeply personal as how I feel about being autistic in the presence of a group, and not knowing what people are thinking, would be too stressful’ (P81, AA). Some proposed that one-to-one support would be more appropriate: ‘individual support mixed with informative videos explaining about what autism [is] and how as an autistic adult the individual has very positive things to give to the community they live in would be more helpful’ (P98, AA). Others preferred a self-help guide they could complete in their own private space and time: ‘I would go through an online workbook or PowerPoint as I’d be able to remain anonymous’ (P134, AA).However, a few participants felt that ‘it would be helpful to hear the experience of others’ (P46, AA) and ‘one of the key benefits might be enjoying time spent in the company of other autistic people face-to-face, regardless of learning about disclosure strategies’ (P139, AA). To make a group format work, they recommended keeping group sizes small and group members as similar to each other as possible. Many mentioned that allowing virtual attendance would make the programme more accessible: ‘The way you could do this is through Zoom [and other video communication platforms]. Everyone has their cameras and mics off, no one has to interact at all, but they can turn on their mics and cameras if they like. They get to actively choose their role in the group this way, making it less intimidating to autistic people (P74, AA)’.DiscussionUsing a mixed methods survey, we examined the views of autistic adults and parents/caregivers of autistic people on whether a stigma support programme for autistic adults is needed and what it should ‘look’ like. Quantitative data showed that most participants felt it was important for autistic adults to have support in managing stigma. Qualitative data revealed some of the reasons behind this, including the negative impact of stigma on mental health and the challenges that autistic people face in navigating disclosure. However, concerns were also raised that such a programme would be placing the burden on autistic people to cope with stigma rather than on society to remove stigma. For support to be beneficial, respondents suggested that it should have a positive and practical focus on building self-understanding and self-acceptance, as well as developing context-specific strategies for responding to stigma and/or disclosing one’s diagnosis. Regarding programme delivery, both quantitative and qualitative data revealed diverse preferences and underscored the importance of flexibility (i.e. offering different ways of participating and taking into account a wide range of needs).First, it is crucial to consider the valid concerns raised by participants that such a programme could convey the message that stigma is a normal and acceptable part of being autistic. It should be noted that such comments were not only made by those who were against the idea of a stigma support programme, but also by those who were in favour of or unsure about such a programme. In other words, some participants felt that programmes to manage stigma could be helpful, but that programmes to reduce stigma towards autistic people were more important. Some participants also emphasised that it is not autistic people who need to change, but support can empower autistic people to educate others and elicit change in society. Meanwhile, a minority stated that such support should not exist at all, and that society needs to educate itself. These sentiments are perhaps unsurprising given the persistent onus placed on autistic individuals to change and ‘fit in’, with many interventions being designed to reduce atypical behaviours and/or teach normative behaviours without clear benefits for the autistic individual (Bottema-Beutel et al., 2018; Mottron, 2017). For years, the autistic self-advocacy and neurodiversity movements have opposed these attempts to ‘cure’ or ‘normalise’ autistic people, instead calling for more interventions that can improve mental health and quality of life for the autistic population (Leadbitter et al., 2021; Pellicano et al., 2014). We echo these sentiments.Our findings have important implications for how stigma-related interventions for autistic people are framed. Compared to an intervention that focuses directly on stigma (like STORM), an intervention that empowers individuals to make strategic disclosure decisions (like HOP) may be more acceptable and relevant to the autistic community. It is noteworthy that many respondents brought up issues of disclosure even before HOP was presented in the survey, including how public and internalised stigma led to concealing or masking, which in turn reinforced stigma. Such experiences have also been well-documented in previous research (Punshon et al., 2009; Schneid & Raz, 2020) and points to how a disclosure-focused programme like HOP could be useful to autistic adults in several ways. For those who decide to disclose, HOP may reduce the risk of internalised stigma by promoting positive group identification, community pride and active engagement in advocating for social change (Corrigan et al., 2013). By supporting effective disclosure (i.e. sharing the right information with the right person at the right time), HOP also has the potential to indirectly reduce public stigma via positive contact experiences, although this has not been empirically tested. For those who decide not to disclose, HOP may still be beneficial by ensuring that non-disclosure is empowered and not driven by internalised stigma or shame (Scior et al., 2020). Regardless of the individual’s decision(s), support in the decision-making process can help to increase self-efficacy in managing stigma and decrease stigma-related stress. Nonetheless, it should be made clear that stigma is a societal and systemic problem, and that interventions targeting those who are stigmatised only represent a small part of the much wider efforts needed to address stigma at multiple levels. Relatedly, such interventions should be developed and evaluated with the goal of contributing towards broader support for the mental health and wellbeing of autistic people, alongside other programmes that have been created to help them understand and accept their diagnosis.Our findings also highlight several considerations regarding how stigma-related interventions for autistic adults are delivered. It was apparent from the qualitative data that a group approach would be helpful for some, but challenging to the point of being inaccessible for others. While a group format was preferred by about a third of participants in the quantitative data, about half of participants also endorsed ‘not wanting to be in a group with other autistic people’ as a potential intervention barrier. Dissociation from the stigmatised group may be indicative of internalised stigma, and it is arguably these individuals who need a stigma support programme the most. Other research has shown that autistic adults can benefit from being in a group with other autistic people, including gaining a sense of belonging, feeling less socially isolated, meeting positive role models and learning from each other’s experiences (Crane, Hearst, et al., 2021; Crompton et al., 2020; Leedham et al., 2020). However, autistic adults who are struggling with internalised stigma and disclosure may be less likely to attend a group programme. Recruitment in past HOP studies has been difficult for the same reason, which led to the recent adaptation of HOP in a guided self-help format combined with an optional online peer forum (Scior et al., 2021). Further research would be needed to determine whether a similar format combining both individual and group options (as our participants suggested) may be feasible and acceptable to autistic adults, and we have reported our plans to test an autism-specific version of HOP elsewhere (Han et al., 2022). Existing studies do indicate that online forums may be particularly well-suited for autistic people (albeit not all), as they can accommodate different modes of communication, alleviate sensory concerns, and provide a safe space for autistic individuals to explore and express a positive collective identity (Brownlow & O’Dell, 2006; Parsloe, 2015).A major limitation of our study was the lack of attention paid to intersectionality, a term coined by Crenshaw to describe how people with multiple stigmatised identities experience compounded forms of discrimination. According to Crenshaw, understanding differences within stigmatised groups is needed to effectively engage in collective action that transforms a stigmatised identity into an empowered one (Crenshaw, 1991, as described in Botha & Gillespie-Lynch, 2022). In relation to disclosure, intersectionality also introduces additional layers of complications, as autistic people may not only be concerned about disclosing their autistic identity but also other stigmatised identities, and the concealability of these identities may also vary. However, our sample was predominantly White, and we did not collect extensive demographic information (e.g. on sexuality, socio-economic status, or co-occurring disabilities) that would give us insights into intersectionality. Autistic people from racial, ethnic, gender, sexual minorities and/or low-income backgrounds are typically underserved and underrepresented in autism research and practice (Steinbrenner et al., 2022; Strang et al., 2020). Moving forward, it is crucial that stigma-related research and support for autistic people appreciate and account for their multifaceted identities, ensuring that such efforts do not ignore or even reproduce other forms of stigmatisation. In addition to the lack of ethnic diversity, it should be noted that as we only recruited participants from one database, our sample may not be representative of the larger autism community in the United Kingdom (an issue that we have expanded on in a footnote within the Methods section). Nonetheless, it was encouraging to see that our survey garnered rich and nuanced data from participants with varying degrees of openness about their autism diagnosis/identity, across different age groups, and with diverse opinions on the proposed programme.Although our sample comprised many more autistic adults than parents/caregivers, we do not perceive this as a limitation as we believe that autistic voices should be centred in research and development of support that directly concerns them. At the same time, including input from parents/caregivers provided additional, valuable perspectives. For example, there were a few parents who commented that they were participating precisely because their autistic grown-up child was conscious of stigma and reluctant to identify as autistic. However, it must be acknowledged that there may be disparities between the views of autistic adults and parents/caregivers, especially those who are not autistic themselves. The quantitative data suggest that autistic adults and parents/caregivers may value different things in an intervention, as autistic adults were more likely to endorse having a trained autistic facilitator, while parents/caregivers were more likely to endorse caregiver involvement. In the qualitative data, parents’ comments that stigma-support programmes should not exist may also stem from a more idealistic position that is possible because they are not the main targets of stigma. Moreover, parents’ reluctance to introduce the concept of stigma to their children align with research on autism diagnosis showing that non-autistic parents may withhold information from their autistic child to protect them from stigma, while autistic parents may prefer honest discussions that facilitate better self-understanding and stronger autistic identification (Crane, Lui, Davies, & Pellicano, 2021; Smith et al., 2018). This links to intersectionality, as parents from racial and ethnic minorities tend to prepare their children for stigma and impart community pride, whereas parents of autistic individuals who do not share the same minority identity may respond to stigma differently (Botha & Gillespie-Lynch, 2022). Nonetheless, as there were a few autistic adults/autistic parents who also expressed a more idealistic or protective stance (e.g. ‘autistic people aren’t the ones who need this’, ‘leave out the word stigma’), these differences are not conclusive.To conclude, we gathered insights from the autism community on whether stigma-related support is needed for autistic adults and if so, what it should ‘look’ like. Our results suggest that it may be worthwhile to pursue interventions that focus on empowerment, self-acceptance and disclosure strategies within a multi-level framework of stigma reduction efforts. However, intervention researchers and practitioners need to be extremely careful that they do not inadvertently reinforce stigma by suggesting that stigma is the fault of the individual, or that internalised stigma is a flaw that needs correcting (Corrigan & Rao, 2012). While this is applicable to interventions that target the stigmatised individual generally, its importance here cannot be overstated given the deficit narrative that has characterised much autism intervention research (see Leadbitter et al., 2021 for a discussion). In trying to help autistic people mitigate the negative effects of stigma, researchers and practitioners must continue to engage in participatory practices, critically reflect on their positions and privileges, and seek to redress the power differences that underpin stigma.Supplemental Materialsj-docx-1-aut-10.1177_13623613221143590 – Supplemental material for Development of stigma-related support for autistic adults: Insights from the autism communityClick here for additional data file.Supplemental material, sj-docx-1-aut-10.1177_13623613221143590 for Development of stigma-related support for autistic adults: Insights from the autism community by Emeline Han, Katrina Scior, Eric Heath, Kana Umagami and Laura Crane in Autismsj-docx-2-aut-10.1177_13623613221143590 – Supplemental material for Development of stigma-related support for autistic adults: Insights from the autism communityClick here for additional data file.Supplemental material, sj-docx-2-aut-10.1177_13623613221143590 for Development of stigma-related support for autistic adults: Insights from the autism community by Emeline Han, Katrina Scior, Eric Heath, Kana Umagami and Laura Crane in Autism

PMC

Ear and Hearing

37171375

PMC10583909

5-11-2023

10.1097/AUD.0000000000001382

Typewriter Tinnitus: Value of ABR as a Diagnostic and Prognostic Indicator

Sun Huiying, Yang Ruizhe, Jiang Hong, Tian Xu, Zhao Yang, Gao Zhiqiang, Wu Haiyan

Introduction:Typewriter tinnitus refers to a special kind of staccato tinnitus, which is mostly described by patients as Morse code, popcorn, or machine-gun. It has been accepted that the mechanism of typewriter tinnitus is caused by the neurovascular compression of the cochleovestibular nerve. Patients who suffered from typewriter tinnitus have exhibited a good response to carbamazepine or oxcarbazepine, but there is a risk of recurrence after treatment cessation. The present study aims to determine the value of auditory brainstem response (ABR) in diagnosing typewriter tinnitus and predicting relapse after drug withdrawal.Methods:Patients who presented with typewriter tinnitus from March 2019 to March 2022 were included for the present retrospective study. The auditory and vestibular test results and drug treatment effects were collected and analyzed. Patients with idiopathic unilateral subjective tinnitus, who were matched by age to patients with typewriter tinnitus at a ratio of 2:1, were consecutively recruited for the control group.Results:Eighteen patients with typewriter tinnitus and 38 controls were included. Ears with typewriter tinnitus had longer interpeak latency (IPL) I-III, and wave III and V latencies, and a higher ratio of IPL I-III ≥2.3 ms based on ABR, when compared to the unaffected side and controls (p 0.05). Ramsay Hunt syndrome and neuromyelitis optica spectrum disorders were identified in two cases.Conclusion:Prolonged IPL I-III based on ABR can help in the diagnosis of typewriter tinnitus and its prognosis after treatment cessation. Patients with IPL I-III greater than 2.4 ms, older age and poorer hearing are more likely to relapse. In addition to the neurovascular conflict of the cochleovestibular nerve, the etiologies of neuroinflammation and demyelinating diseases are also possible for typewriter tinnitus.

INTRODUCTIONTypewriter tinnitus refers to a special kind of staccato tinnitus, which is mostly described by patients as Morse code, popcorn, or machine-gun. This was first reported by Mardini . Typewriter tinnitus shares several clinical characteristics with neurovascular compression of the cranial nerves or NVCC (trigeminal neuralgia, vestibular paroxysmia [VP], and hemifacial spasm), including unilateral, staccato and intermittent attacks, and effective response to medications, such as carbamazepine or oxcarbazepine (Levine 2006; Sunwoo et al. 2017; Huh et al. 2020; Koo et al. 2021). Typewriter tinnitus has been classified as a type of cochleovestibular compression syndrome (CVCS), which is caused by the compression of a loop of intracranial vessels, such as the anterior-inferior cerebellar artery, posterior inferior cerebellar artery, or dilated vertebrobasilar artery (De Ridder et al. 2007; Huh et al. 2020). The cisternal segment of the eightth cranial nerve has been reported to range within 14.2-19.2 mm (Guclu et al. 2012), which is particularly longer than other cranial nerves, making it more vulnerable to compression by the neighboring blood vessels. This neurovascular conflict may lead to demyelination (Devor et al. 2002) and endoneurial fibrosis (Schwaber & Whetsell 1992), creating foci of ectopic excitation. Typewriter tinnitus should be differentially diagnosed from objective tinnitus (crackling or buzzing objective tinnitus due to muscle contraction), including palatal myoclonus and middle ear myoclonus. The observation of tympanic membrane movement or palatal tremor, and good response to medication (i.e., muscle relaxants, sedatives and anticonvulsants) or surgery (i.e., tensor tympani, tensor veli palatini, or stapedius muscle tenotomy) provides strong evidence for the diagnosis of objective tinnitus. Carbamazepine and oxcarbazepine have been used in NVCC to release symptoms by inhibiting the voltage-gated sodium channel and suppressing the aberrant ephaptic axonal transmission of the cranial nerve. These drugs have also been used to treat VP, which has been accepted to be a type of CVCS, as described by the Bárány Society (Strupp et al. 2016). Although not licensed for use for typewriter tinnitus at present, most patients with typewriter tinnitus have a good response to carbamazepine or oxcarbazepine, as reported in the literature, suggesting that these two drugs have good potential for the treatment of typewriter tinnitus. However, typewriter tinnitus may recur after drug withdrawal (Sunwoo et al. 2017). At present, there are no instructions or guidelines for treatment using carbamazepine and oxcarbazepine in typewriter tinnitus, and no indicator has been reported as a possible predictor of relapse after drug cessation.Auditory brainstem response (ABR) has been described as a useful tool to diagnose CVCS, and screen patients for microvascular decompression surgery (Møller 1990; De Ridder et al. 2007; Sun et al. 2022). To our knowledge, few studies have reported the characteristics of ABR in typewriter tinnitus. Han et al. reported that for patients with typewriter tinnitus, a decrease in wave II amplitude was more likely, when compared to patients with tinnitus, due to middle ear myoclonus. Furthermore, although no clear difference was observed, patients with typewriter tinnitus presented with a longer interpeak latency (IPL) of wave I-III and a higher rate of IPL I-III ≥2.3 ms, when compared to patients with middle ear myoclonus (Han et al. 2020). Brantberg reported that two of four patients had ABR abnormalities in a study conducted for paroxysmal staccato tinnitus, while the other two patients had normal ABR results. Interestingly, the ABR results were normal in cases reported by Mathiesen and Brantberg and Reynard et al. . To date, there is no consensus on the clinical values for ABR in the diagnosis or prediction of relapse of patients with typewriter tinnitus. Thus, the present study aimed to investigate the application of ABR in the diagnosis of typewriter tinnitus, and its prognosis after medical treatment is stopped.MATERIALS AND METHODSThe present study was exempted from the Institutional Review Board review by the Medical Ethics Committee of Peking Union Medical College Hospital due to the retrospective design of the study (No. I-22PJ211).The medical information of patients who presented to the Department of Otolaryngology-Head and Neck Surgery of Peking Union Medical College Hospital from March 2019 to March 2022, and were diagnosed with unilateral typewriter tinnitus, was retrospectively analyzed. Typewriter tinnitus was defined as unilateral staccato sounds, such as Morse code, popcorn, or machine-gun. To exclude objective tinnitus (palatal myoclonus and middle ear myoclonus), physical examination was performed to exclude the rhythmic movement of the tympanic membrane and the tremor of the soft palate, and long-time-based tympanometry was conducted to exclude those with rhythmic changes in middle ear compliance. Pulsatile tinnitus consistent with the heartbeat was also excluded. The other exclusion criteria were otitis media, internal acoustic canal (IAC) or cerebellopontine angle (CPA) tumor, and head or temporal bone trauma. Age-matched control subjects with idiopathic unilateral subjective tinnitus were consecutively recruited from the same hospital during the same enrollment period as patients with typewriter tinnitus at a ratio of 2:1. The same exclusion criteria were applied to the control subjects.The demographic characteristics of all the patients were recorded. Vertigo that met the criteria for VP (≥10 attacks of spontaneous spinning or non-spinning vertigo, duration <1 minute, stereotyped phenomenology, response to treatment with carbamazepine/oxcarbazepine, and exclusion of other diagnoses), as suggested by the Bárány Society (Strupp et al. 2016), was documented as VP. Otherwise, it was documented as non-VP. Pure-tone audiometry (PTA) was used to evaluate the hearing level, and the average hearing threshold was calculated at 0.5, 1.0, 2.0, and 4.0 kHz. For patients with typewriter tinnitus, auditory and vestibular electrophysiological tests, including ABR, ocular vestibular evoked myogenic potential (oVEMP), and cervical vestibular evoked myogenic potential (cVEMP), were performed. The relative location of the vessel and eighth cranial nerve at the CPA and IAC on magnetic resonance imaging (MRI) was recorded. Neurovascular contact was defined as the disappearance of the cerebrospinal fluid signal gap between the vessel and nerve (Best et al. 2013). This was classified into three types, according to the Chavda system (McDermott et al. 2003): type I, the vascular loop lies at the CPA, but does not enter the IAC; type II, the vascular loop lies within the medial half of the IAC; type III, the vascular loop extends to the lateral half of the IAC. The PTA and ABR results were recorded for the control group.Carbamazepine (400–800 mg/day) or oxcarbazepine (300–600 mg/day) was administered for patients with typewriter tinnitus for a period of 3 months, along with the active monitoring of side effects, such as tiredness, dizziness, ataxia, and nausea. The therapeutic effectiveness was recorded and classified into three categories: nonresponse (NR), no reduction of typewriter tinnitus; partial remission (PR), partial relief of typewriter tinnitus, including lower frequency and shorter duration of attacks; complete remission (CR), complete elimination of typewriter tinnitus (Sunwoo et al. 2017). If a relapse of typewriter tinnitus occurred after drug cessation, the same medication was prescribed again at the effective dose. Follow-up was achieved via subsequent on-site visit or telephone interview, up to July 2022. Patients who responded positively to the medication (PR and CR) were further divided into two groups, based on the recurrence or exacerbation of typewriter tinnitus after discontinuation of medication: relapse group and nonrelapse group. Then, the clinical characteristics were compared between these two groups, and analyzed for risk of recurrence.Statistical AnalysisThe data were analyzed using SPSS v.23.0 (Chicago, IL, USA). Normally distributed continuous variables were reported as mean ± standard deviation, and analyzed using Student’s t-test, while non-normally distributed continuous variables were reported in median (interquartile range), and calculated using the Mann–Whitney U-test. Categorical data were reported in absolute and relative frequencies, and calculated using chi-squared test or Fisher’s exact test. A two-tailed p value of <0.05 was considered statistically significant. Receiver operating characteristic (ROC) curves were drawn for the positive results in the univariate analysis between the relapse and nonrelapse groups. The area under the curve (AUC) and best cutoff values were determined by maximizing the Youden index for sensitivity and specificity optimization.RESULTSDemographic and Testing Characteristics of the Typewriter Tinnitus GroupEighteen patients with typewriter tinnitus (7 male and 11 female patients) were enrolled for the present study. For patients with typewriter tinnitus, the average age was 49.4 ± 13.6 years, and the average duration of typewriter tinnitus was 1.0 ± 1.1 years. No patient was lost to follow up, and the mean follow-up time was 13.6 ± 7.9 months. Furthermore, the left ear was affected in eight patients, and the right ear was affected in 10 patients. Moreover, six patients merely had typewriter tinnitus, while 12 patients had accompanying dizziness/vertigo. Among these 12 patients, 11 patients met the criteria for VP (Strupp et al. 2016). In addition, 16 patients had CR, one patient had PR, and one patient had NR after medication. Oxcarbazepine, which is generally administered by our hospital as a first-line therapy for this condition, was prescribed for 14 patients who had no obvious side effects from the medication, while carbamazepine was prescribed for three patients who presented with nausea and vomiting after taking oxcarbazepine. In addition, 10 patients had no relapse after treatment cessation, while seven patients had a relapse (Table 1 and File in Supplemental Digital Content 1, 1.Demographic characteristics of all patients with typewriter tinnitus.No.GenderAge (y)LateralPossible TriggerSymptomNeurovascular Contact TypeDuration (y)Pharmacological Treatment SchemeFollow-up Time (mo)Therapeutic ResponseRelapse1F56L/TT+non-VPI0.2Oxcarbazepine, 600 mg/d16CRRelapse2F43L/TTNone0.6Oxcarbazepine, 600 mg/d13CRNo relapse3M52L/TT+VPI1.0Oxcarbazepine, 600 mg/d12PRNo relapse4F56LNeuromyelitis optica spectrum disordersTT+VPII0.1Oxcarbazepine, 600 mg/d12CRRelapse5F31R/TT+VPII2.0Oxcarbazepine, 600 mg/d4CRNo relapse6M35R/TT+VPII1.0Oxcarbazepine, 600 mg/d24CRNo relapse7M61L/TT+VPIII1.5Carbamazepine, 800 mg/d34CRRelapse8F73R/TT+VPII1.0Oxcarbazepine, 600 mg/d14CRRelapse9F74R/TT+VPII1.0Carbamazepine, 400 mg/d25CRRelapse10M33L/TT+VPI0.1Oxcarbazepine, 600 mg/d17CRNo relapse11M51L/TTI0.6Oxcarbazepine, 600 mg/d7CRNo relapse12M44R/TTI4.0Oxcarbazepine, 600 mg/d16CRRelapse13F56RRamsay Hunt SyndromeTT+VPI0.4Oxcarbazepine, 600 mg/d6CRNo relapse14F65R/TTI0.5Carbamazepine, 400 mg/d13CRNo relapse15F38L/TTI0.8Oxcarbazepine, 600 mg/d6CRNo relapse16F37R/TTNone0.1Oxcarbazepine, 600 mg/d, and carbamazepine, 400 mg/d6NR/17F33R/TT+ VPII3.0Oxcarbazepine, 600 mg/d14CRNo relapse18M52R/TT+VPI0.3Oxcarbazepine, 600 mg/d6CRRelapseCR = complete remission; F = female; ; L = left; M = male; PR = partial remission; R = right; TT = typewriter tinnitus; VP = vestibular paroxysmia.published online ahead of print May 11, 2023.In the ABR analysis, the wave III and V latencies on the affected side were more prolonged, when compared to the unaffected side (wave III, p = 0.002; wave V, p = 0.030). Furthermore, IPL I-III was notably longer in the affected side, when compared to the unaffected side (p < 0.001, Table 2). However, no difference was observed in wave I latency or IPL III-V between the affected and unaffected sides (wave I latency, p = 0.321; IPL III-V, p = 0.572). In addition, IPL I-III ≥2.3 ms was noted for all affected ears, and this was consistent with Møller’s criteria for NVCC, in which IPL I-III ≥2.3 ms is an important criterion for diagnosing the microvascular conflict of the eighth cranial nerve (Møller 1990).TABLE 2.Comparison of auditory and neuro-electrophysiological results between the affected side (with typewriter tinnitus) and unaffected side (without typewriter tinnitus) of all patients (n = 18)ValuablesUnaffected Side (n = 18)Affected Side (n = 18) p PTA (median [IQR], dB HL)15.6 (10.0–27.8)16.3 (10.9–27.8)0.815ABR I wave latency (median [IQR], ms)1.6 (1.6–1.7)1.7 (1.6–1.7)0.321 III wave latency (mean ± SD, ms)3.9 (3.8–4.1)4.1 (4.0–4.3) 0.002 * V wave latency (median [IQR], ms)5.7 (5.6–5.8)5.9 (5.8–6.1) 0.030 * IPL I-III (median [IQR], ms)2.3 (2.2–2.3)2.4 (2.3–2.5) <0.001 * IPL III-V (mean ± SD, ms)1.8 ± 0.11.8 ± 0.10.572 IPL I-III ≥2.3 ms (n, %) No14 (77.8)0 (0.0) <0.001 * Yes3 (16.7)18 (100.0) Absent response1 (5.5)0 (0.0)*P 0.05). Based on the ABR, the wave III and V latencies, and IPL I-III of the affected side were significantly prolonged in the typewriter tinnitus group, when compared to the control group (wave III latency, p < 0.001; wave V latency, p = 0.003; IPL I-III, p < 0.001). Furthermore, the ratio for IPL I-III ≥2.3 ms of the affected side was significantly higher in patients who suffered from typewriter tinnitus, when compared to the controls (p < 0.001, Table 3).TABLE 3.Comparison of auditory and neuro-electrophysiological results between patients with typewriter tinnitus and controls with idiopathic unilateral tinnitusValuablesSensorineural Tinnitus (n = 38)Typewriter Tinnitus (n = 18) p Age (mean ± SD, y)45.2 ± 13.849.4 ± 13.60.282Gender (n, %) Male19 (50.0)7 (38.9)0.436 Female19 (50.0)11 (61.1)Duration of tinnitus1.0 (0.4–5.0)0.7 (0.3–1.1)0.122Lateral (n, %) Left23 (60.5)8 (44.4)0.258 Right15 (39.5)10 (55.6)PTA (median[IQR], dB HL)17.5 (11.3–28.8)16.3 (10.9–27.8)0.752ABR I wave latency (median[IQR], ms)1.7 (1.6–1.7)1.7 (1.6–1.7)0.209 III wave latency (median[IQR], ms)3.8 (3.7–3.9)4.1 (4.0–4.3) <0.001 * V wave latency (median[IQR], ms)5.7 (5.6–5.8)5.9 (5.8–6.1) 0.003 * IPL I-III (median[IQR], ms)2.2 (2.1–2.3)2.4 (2.3–2.5) <0.001 * IPL III-V (mean ± SD, ms)1.9 ± 0.11.8 ± 0.10.055 IPL I-III ≥2.3 ms (n, %) No35 (92.1)0 (0.0) <0.001 * Yes2 (5.3)18 (100.0) Absent response1 (2.6)0 (0.0)*P 0.05, Table 4).TABLE 4.Comparison of relapsed and nonrelapsed patients after medicationValuablesNonrelapse (n = 10)Relapse (n = 7) p Gender (n, %)1.000 Male4 (40.0)3 (42.9) Female6 (60.0)4 (57.1)Age (mean ± SD, y)43.7 ± 11.759.4 ± 10.9 0.013 * Duration of typewriter tinnitus (median [IQR], y)0.7 (0.5–1.3)1.0 (0.2–1.5)0.922Follow-up period (mean ± SD, mo)11.6 ± 6.117.6 ± 9.20.127Vertigo (n, %)0.304 No5 (50.0)1 (14.3) Yes5 (50.0)6 (85.7)Neurovascular contact type (n, %) Type I6 (60.0)3 (42.9)0.579 Type II3 (30.0)3 (42.9) Type III0 (0.0)1 (14.2) None1 (10.0)0 (0.0)PTA (mean ± SD, dB HL)15.5 ± 6.032.5 ± 16.7 0.009 * OVEMP (n, %)1.000 Abnormal5 (50.0)3 (42.8) Normal3 (30.0)2 (28.6) Absent response2 (20.0)2 (28.6)CVEMP (n, %)1.000 Abnormal5 (50.0)4 (57.1) Normal3 (30.0)1 (14.3) Absent response2 (20.0)2 (28.6)ABR of affected side I wave latency (median [IQR], ms)1.7 (1.6–1.8)1.7 (1.6–1.7)0.784 III wave latency (mean ± SD, ms)4.1 ± 0.24.3 ± 0.30.140 V wave latency (mean ± SD, ms)5.8 ± 0.26.2 ± 0.2 0.018 * IPL I-III (median [IQR], ms)2.3 (2.3–2.4)2.5 (2.4–2.8) 0.004 * IPL III-V (mean ± SD, ms)1.8 ± 0.21.9 ± 0.10.144 IPL I-III ≥2.3 ms (n, %)/ No0 (0.0)0 (0.0) Yes10 (100.0)7 (100.0)*P<0.05.ABR, auditory brainstem response; cVEMP, cervical vestibular evoked myogenic potential; IPL, interpeak latency; IQR, interquartile range; oVEMP, ocular vestibular evoked myogenic potential; PTA, pure-tone audiometry; SD, standard deviation.Fig. 1.Receiver operating characteristic curves for IPL I-III, age, PTA, and wave V latency. The optimal cutoff point was calculated according to the max Youden Index (Youden Index = Sensitivity + Specificity − 1).DISCUSSIONThe present retrospective study compared the clinical characteristics of 18 patients with typewriter tinnitus and 38 controls, and determined the value of ABR in diagnosing typewriter tinnitus and predicting relapse after drug cessation. Among the patients with typewriter tinnitus, 16 patients had CR, one patient had PR, and one patient had no response to medical therapy. As a preliminary study, these results suggest that ABR can be used as a diagnostic indicator for typewriter tinnitus, and might serve as an objective prognostic indicator for relapse after treatment cessation, with relatively high sensitivity and specificity.To date, the diagnosis of typewriter tinnitus is mainly based on symptoms and the degree of response to carbamazepine (Sunwoo et al. 2017), and there is a lack of diagnostic indicators. The present study compared the ABR results between the side affected with typewriter tinnitus and the unaffected side, with reference to the characteristics of the ABR findings in NVCC or CVCS. The results revealed that the affected side had significantly longer IPL I-III, and wave III and V latencies, and a higher ratio of IPL I-III ≥2.3 ms, but had similar IPL III-V and wave I latency. Furthermore, the ABR results for patients with typewriter tinnitus were compared with those for the controls who had idiopathic unilateral subjective tinnitus. More prolonged IPL I-III, and wave III and V latencies, and a higher ratio of IPL I-III ≥2.3 ms were observed, but there were no differences in wave I latency and IPL III-V between the two groups. Since IPL III-V exhibited no differences, the elongation of wave V latency possibly resulted from the prolonged IPL I-III. These outcomes suggest that extended IPL I-III (especially ≥2.3 ms) and wave III latency are the characteristic findings for typewriter tinnitus, as supported by the ABR. These are the possible diagnostic indicators that concur with the clinical values of ABR in NVCC and CVCS.The present study also explored the applicable value of ABR in predicting the relapse of typewriter tinnitus after drug cessation. The results indicated that the IPL I-III and wave V latency were significantly longer in the relapse group, when compared to the nonrelapse group. In the ROC analysis, patients with IPL I-III greater than 2.4 ms had a higher risk of recurrence, when compared to patients with IPL I-III of less than 2.4 ms, and this was highly sensitive. To our knowledge, no previous study has analyzed the efficacy of ABR in screening patients who have a high risk of relapse of typewriter tinnitus after discontinuation of medication. The present study was the first to discuss this issue. As explored in the literature, IPL I-III prolongation has a significantly positive correlation with the severity of auditory nerve impairment and tinnitus caused by CVCS (De Ridder et al. 2007). Substantially in line with this early conclusion, the present study suggests that the more prolonged the IPL I-III, the more serious the damage to the auditory nerve, and the more chances of relapse after drug withdrawal.In the present study, 58.8% of patients had a limited course of medical therapy that appeared to have a lasting effect. This phenomenon was also observed in patients with VP. Chen et al. reported that 20.7% of patients with VP became attack-free after a follow-up period that ranged within 24 to 43 months, and Steinmetz et al. reported that 74% of patients became attack-free after a mean follow-up period of 4.8 years. The underlying mechanism for this may be that some abnormal discharges caused by the neurovascular compression of the cranial nerve disappeared after medication, or the neurovascular conflict was resolved with changes in the hemodynamic status. Another possible aspect is that the neuroinflammation or demyelination observed in typewriter tinnitus may be cured when the cause is removed over time. The present research did not arrive at a definite conclusion for this issue. Thus, dynamic and long-term observations are suggested in the future to answer this intriguing question.Patients in the relapse group were older, and had poorer hearing, when compared to patients in the nonrelapse group. A possible explanation is that older age increases the risk for hypertension and atherosclerosis of the affected vessel, leading to more severe damage to the eighth cranial nerve. Furthermore, the poorer hearing levels further support the damage to the auditory nerve fibers caused by the neurovascular conflict. The review conducted by Nash et al. revealed that among tinnitus-only cases, surgical decompression for neurovascular conflict is only effective when performed early in the course (<5 years), indicating that the longer the duration of the disease, the more severe the injury to the nerve. The present limited data did not reveal any relationship between the course of symptoms and its recurrence. This difference may be attributed to the short course of the included cases, among which merely one case had symptoms with a duration of more than 5 years.It has been considered that typewriter tinnitus arises from the neurovascular compression of the eighth cranial nerve. However, in the present study, two patients were found to have no neurovascular contact in either the CPA or the IAC on the MRI. It has been reported that some patients with typewriter tinnitus does not present with radiological evidence of neurovascular conflict (Bae et al. 2017), which is similar to the findings in NVCC (i.e., VP, hemispasmus facialis, and trigeminal neuralgia) (Girard et al. 1997; Lee et al. 2014; Steinmetz et al. 2022). IAC osteoma and basilar invagination have been reported to be associated with the incidence of typewriter tinnitus (Nam et al. 2010; Reynard et al. 2020). Apart from CVCS, some possible predisposing factors for typewriter tinnitus were found in two patients in the present study. One female patient developed typewriter tinnitus and VP at three months after Ramsay Hunt syndrome. The other female patient had neuromyelitis optica spectrum disorder, and she gradually developed optic neuritis, extremity numbness, and Sjogren’s syndrome by the age of 37. As previously reported in the literature, neuroinflammatory and demyelinating disorders may induce ectopic neural electrical activity (Smith & McDonald 1982; Schwaber & Whetsell 1992; Ori et al. 2018; Laakso et al. 2020). The present study results suggest that the neurovascular conflict was responsible for the majority of, but not all, cases of typewriter tinnitus. Furthermore, the ectopic neural electrical activity of the cochleovestibular nerve, which was caused by inflammation, autoimmune disease, neurovascular conflict, etc., was the main mode of pathogenesis of typewriter tinnitus.There were some limitations in the present study. The present study was retrospective in nature, and carried some of the disadvantages typically observed in a retrospective study, such as selection and observer bias. In addition, due to the rare incidence of typewriter tinnitus, the sample size of the present study was small, and the individual differences could not be ignored. Furthermore, the cutoff value for IPL I-III based on ABR needs to be verified in future randomized controlled trials with a larger sample size.CONCLUSIONABR is a useful tool for the diagnosis of typewriter tinnitus. The IPL I-III of most ears with typewriter tinnitus was longer than 2.3 ms. Furthermore, almost half of the patients with typewriter tinnitus had a risk of relapse after the cessation of medical treatment, and patients with IPL I-III greater than 2.4 ms were more likely to have recurrence. In addition to the neurovascular conflict of the cochleovestibular nerve, neuroinflammation and demyelinating diseases that involve the eighth cranial nerve were the other possible etiologies for the occurrence of typewriter tinnitus.ACKNOWLEDGMENTSWe would like to thank all the patient for allowing us to review their medical findings and records.Supplementary Material

PMC

Preventive Medicine Reports

PMC10300395

6-09-2023

10.1016/j.pmedr.2023.102278

Regional French evolution of tobacco and e-cigarette experimentation and use among adolescents aged 15–16 years: A cross-sectional observational study conducted in the Loire department from 2018 to 2020

Wamba André, Nekaa Mabrouk, Leclerc Lara, Denis-Vatant Christine, Masson Julien, Pourchez Jérémie

BackgroundWe assessed/compared the evolution of tobacco and e-cigarette experimentation and use among French adolescents of the Loire department aged 15–16 years.MethodsA descriptive, cross-sectional/observational study conducted in 2018–2020 among 7,950 Year 11 pupils attending 27 public secondary schools of the Loire department, France.ResultsFrom 2018 to 2020, 66.18% of adolescents were “non-vapers and non-smokers”, 19.76% were “vapers and smokers”, 7.90% were “non-vapers and smokers” and 6.15% were “vapers and non-smokers”. E-cigarette experimentation was more prevalent than tobacco experimentation (44.92% vs 41.67%), and daily vaping was less prevalent than daily smoking (5.40% vs 10.24%). More boys than girls were daily vapers or daily smokers. A decrease was observed in tobacco experimentation (from 41.22% in 2018 to 39.73% in 2020) and e-cigarette experimentation (from 50.28% in 2018 to 41.25% in 2020). Current vaping remained stable, with an increase in daily vaping. French adolescent vapers frequently use e-liquids with little or no nicotine or with fruit or sweet flavours.ConclusionsAdolescents used e-cigarettes mainly for experimental and/or recreational purposes, with no intention of progression to daily smoking. Although the design of this study is not longitudinal and caution must be exercised, from our cross-sectional observational study data, it appears that the proportion of “non-vapers and non-smokers” tended to increase. “Smokers” tended to progress to the dual use of vaping and smoked tobacco, with the likely intention to reduce or quit smoking.

1Introduction1.1Scientific controversies on e-cigarettes perceptionsA major scientific controversy is currently ongoing based on contrasting perceptions of e-cigarettes. On one side of the debate are those who consider that vaping helps smokers reduce or quit smoking. Some authors note that e-cigarettes are perceived as a smoking cessation tool (Hanafin and Clancy, 2020), with smokers themselves highlighting their therapeutic potential (Berlin, 2015, Hardie and Green, 2022, Kinouani et al., 2020, Notley et al., 2021). Three Cochrane reviews find that nicotine e-cigarettes are more effective than nicotine substitutes in helping young adult smokers wean themselves off smoking (Grabovac et al., 2020, Hartmann-Boyce et al., 2021, McRobbie et al., 2014). The data from these studies (McRobbie et al., 2014) also highlights that a large proportion of smokers who switch to vaping continue to vape, failing to achieve the primary goal of quitting. The Cochrane review also features data on the growing number of dual-users as a result of experimentation or use of electronic cigarettes as a cessation tool. In the UK, the 2021 Public Health England report (McNeill et al., 2021)concludes that e-cigarettes are an effective aid to smoking cessation and reduction, while Levy et al. (Levy et al., 2020) suggest that the observed reduction in smoking prevalence in the adult population is explained by the increased use of nicotine e-cigarettes. In France, Legleye et al. (Legleye et al., 2020) report a 42% reduction in the risk of becoming a daily smoker in adolescents aged 17–18.5 years who experimented with e-cigarettes first: the proportion of subsequent tobacco experimenters decreased continuously with age at exposure from 95% a age 11 to 25.3% at 17 years, and within the French population, Chyderiotis et al. (Chyderiotis et al., 2020) confirm the absence of an increased risk of progression from vaping to daily smoking among adolescents aged 17 years.On the other side of the debate are those who view vaping as a gateway to smoking. A detailed report of the National Academies of Sciences, Engineering and Medicine (Daynard, 2018); National Academies of Sciences, 2018, Stratton et al., 2018) describes e-cigarettes as a “gateway” object and concludes that they are associated with the risk of progression to smoking, especially when they contain nicotine. Likewise, a meta-analysis of 30 longitudinal studies from 22 different cohorts states that e-cigarette use can be considered a predictor of subsequent smoking (Adermark et al., 2021). Several other studies (Martinelli et al., 2021, Soneji et al., 2017, Zhong et al., 2016) highlight an association between e-cigarette use and subsequent smoking initiation among adolescents and young adults. More generally, Brown et al. (Brown et al., 2020)and Kinnunen et al. (Kinnunen et al., 2019)recommend caution over nicotine e-cigarettes, arguing that nicotine exposure can cause dependence and, consequently, the progression to smoking among young vapers.The contrasting perceptions of e-cigarettes have raised concerns among public health agencies about the uncertain impact of vaping in the general population, and especially in adolescents (Chyderiotis et al., 2019). For public health authorities to make fully informed decisions, it is crucial to better understand both the relationship between smoking and vaping in adolescence and the motivations of adolescents for tobacco and e-cigarette experimentation and use. As previously discussed, in general adolescent data are sorely lacking. Thus, we chose to work on 15–16-year-olds, although it will be of course essential to expand into any adolescent population. Thus, with a view to reinforcing French observational systems, we conducted a cross-sectional study to complement national data with regional data on smoking and vaping behaviour in the specific population of adolescents aged 15–16 years. Our main objective was to assess and compare the evolution of tobacco and e-cigarette experimentation and use among thousands of Year 11 pupils attending public secondary school in the Loire department. The point was to closely examine the regional evolution of smoking and vaping behaviour at the specific age of 15–16 years. Our secondary objective was to identify the motivations of some French adolescents from the Loire department for vaping and smoking and to describe the characteristics of their preferred e-liquids (nicotine content, flavour). Detailed information is available in the supplementary material to this article regarding the most recent French data on the prevalence of e-cigarette experimentation and smoking (see section 1 of Appendix A).2Materials and methodsA descriptive, cross-sectional, observational study was conducted from 2018 to 2020 in a population of 7,950 French adolescents aged 15–16 years and attending Year 11 in 27 public secondary schools of the Loire department, France. This study was approved by the ethics committee of Saint-Etienne University Hospital (CHU) and referenced under the number IRBN372018/CHUSTE. The study obtained written informed consent from the French ethics committee on behalf of all the research participants. It has received approval from the French Data Protection Authority (Commission Nationale de l'informatique et des Libertés) (CNIL). It was carried out in partnership between the Departmental Services of National Education of the Loire, Saint-Etienne University Hospital and the Ecole Nationale Supérieure des Mines de Saint-Etienne.The 27 high schools eligible to participate in the surveys were included in the sample on the basis of voluntary participation. To recruit participants, an information note was sent through the management of each high school to students and their parents before the questionnaire was administered. The note specified that participation in the survey was voluntary, and that to complete the questionnaire the students had to be present in a room reserved by the school management. Data was collected using self-administered questionnaires drawn up by a multidisciplinary team composed of a tobacco specialist, scientist specialized in adolescence, a researcher in public health and educational sciences as well as aerosol experts and toxicologists specialized in smoking and vaping. Adolescents were asked to answer a number of questions on their experimentation and use of tobacco and e-cigarettes allowing to determine their vaping or smoking status as previously described in Denis-Vatant et al. (Denis-Vatant et al., 2019) study. The status of “smoker” or “vaper” corresponds to adolescents who report, respectively, smoking tobacco or vaping electronic cigarettes, daily (every day) or occasionally (only on weekends, during festive events). The status of “non-smoker” or “non-vaper” corresponds to adolescents who declare at the time of the study, respectively, that they do not smoke tobacco or vape electronic cigarettes. Preliminary reliability and validity tests were conducted with some of the adolescents during the design phase of the questionnaires to ensure proper understanding of the questions.The analysis sought to determine whether the prevalence of vaping and smoking increased, decreased or remained stable from 2018 to 2020. The motivations of adolescents for vaping and smoking and their e-liquid preferences were also analyzed, as was the evolution of these motivations and preferences over time. Questionnaires were processed anonymously using Excel® software. Statistical analyses were carried out using IBM SPSS Statistics 21® software. Percentages were compared in univariate analysis using the Chi-square test. Univariate analysis was carried out on all the variables. The statistical tests used are: χ2 test, Fisher’s exact test and Wilcoxon test. Using SPSS statistical software, the data collected was weighted, which made it possible to correct the under- and over-representation of the sample of study participants.Additional information is available in the supplementary material to this article regarding the protocol for administering the questionnaire and the data collection (see section 2 of Appendix A).3ResultsA total of 7,950 adolescents aged 15–16 years were included in the study, namely 1,435 (18%) in 2018, 4,937 (62%) in 2019 and 1,578 (20%) in 2020. Overall, 4,112 (51.70%) girls and 3,838 (48.30%) boys responded to the questionnaire. While the sex ratio was similar in 2018 and 2019, there was a significant female predominance in 2020 (Table 1). Thus, the fact that the same sex ratio was observed in 2018 and 2019 is reassuring that there is no bias despite the difference in sample size for these two years. In contrast, the different sex ratio in the population included in 2020 during the COVID-19 pandemic could induce a potential bias (excepted if we compare use by sex) even if in terms of inclusion the sample size is globally very similar to that in 2018. However, overall, the sex ratio for the total population included over the three years is very close to that observed in 2018 and 2019, despite the difference observed in 2020, which does not seem to induce a particular bias with respect to the overall population (p < 0.001).Table 1Evolution of cross-sectional sample according to sex among French adolescents of the Loire department aged 15–16 years from 2018 to 2020.YearsNumber of adolescents includedSex%Girls%Boys%2018143518%71649.18%71950.82%2019493762%249150.46%244649.54%2020157820%90557.35%67342.65%Total7950100%4,11251.70%3,83848.30%3.1Evolution of tobacco experimentation and useOverall, 41.67% (n = 3,241) of adolescents reported having experimented with at least one tobacco product. The prevalence of current smoking was 27.50% (n = 2,170) (for 2018/2019/2020, p = 0.442), with more adolescents reporting occasional smoking (17.27%, n = 1,351) than daily smoking (10.24%, n = 805). The prevalence of use and experimentation of smoking for the 7,950 adolescents included in the study between 2018 and 2020 is shown in the Table 2.Table 2Prevalence (of use and experimentation) of tobacco and vaping products for the 7,950 French adolescents of the Loire department included in the study between 2018 and 2020.SmokingVaping productsPrevalence of use (n = 7,950)Daily use10.24%5.40%Occasional use17.27%20.44%Non-user72.49%74.16% Prevalence of experimentation (n = 7,950)Experimenter41.67%44.92%Non-experimenter58.33%55.08%As the analysis by year indicates, the prevalence of tobacco experimentation remained broadly stable between 2018 (39.7%) and 2019 (42.4%) and then decreased in 2020 (41.2%). There was also a slight decrease in the prevalence of daily smoking (from 10.86% in 2018 to 9.44% in 2020; p = 0,3) and that of occasional smoking (from 17.11% in 2018 to 16.86% in 2020; p = 0,48). The percentage of non-smokers remained stable between 2018 (72.02%) and 2019 (72.23%) and then increased by one point in 2020 (73.70%; p = 0,6) (Fig. 1).Fig. 1Evolution of tobacco experimentation and use among French adolescents of the Loire department aged 15–16 years from 2018 to 2020.3.2Evolution of e-cigarette experimentation and useOverall, 44.9% (n = 3,531) of adolescents reported having experimented with e-cigarettes. Moreover, 25.9% (n = 2,027) reported being current vapers. The prevalence of occasional vaping was 20.44% (n = 1,592) and that of daily vaping was 5.40% (n = 385). The prevalence of use and experimentation of vaping products for the 7,950 adolescents included in the study between 2018 and 2020 is shown in the Table 2. The analysis by year shows an important decrease in the prevalence of e-cigarette experimentation: from 50.28% in 2018 to only 44.60% in 2019 and 41.25% in 2020. The prevalence of occasional vaping remained stable between 2018 (19.96%) and 2020 (19.90%), despite a one-point increase in 2019 (20.74%; p = 0,459). However, the prevalence of daily vaping increased significantly from 3.50% in 2018 to 5.13% in 2020 (p = 0,045) (Fig. 2).Fig. 2Evolution of e-cigarette experimentation and use among French adolescents of the Loire department aged 15–16 years from 2018 to 2020.3.3Evolution of vaping and smoking statusOverall, 66.18% (n = 5,221) of adolescents were “non-vapers and non-smokers”, 19.76% (n = 1,506) were “vapers and smokers”, 7.90% (n = 673) were “non-vapers and smokers” and 6.15% (n = 483) were “vapers and non-smokers”. The analysis of vaping and smoking status by year shows a significant increase in the prevalence of “non-vapers and non-smokers”, from 64.85% (n = 887) in 2018 to 66.03% (n = 3260) in 2019 and 67.81% (n = 1,070) in 2020 (p = 0,003). The prevalence of “vapers and smokers” in 2020 (19.14%) was 1.42 points higher than in 2019 (20.56%) and 1.54 points lower than in 2018 (17.60%), but almost identical to the average prevalence of “vapers and smokers” for the entire study period (19.76% vs 19.14%). The prevalence of “non-vapers and smokers” fell significantly from 11.25% (n = 154) in 2018 to only 7.21% (n = 356) in 2019 and 7.16% (n = 113) in 2020 (p = 0,297). As for the prevalence of “vapers and non-smokers”, it remained stable between 2018 (6.30%, n = 86) and 2019 (6.20%, n = 306) and then fell slightly in 2020 (5.89%, n = 93) (p = 0,444). In contrast, the prevalence of “vapers and smokers” increased significantly from 17.60% (n = 241) in 2018 to 20.56% (n = 1,015) in 2019 and fell to 19.14% (n = 302) in 2020 (p = 0,001) (Fig. 3).Fig. 3Evolution of vaping and smoking behaviour among French adolescents of the Loire department aged 15–16 years from 2018 to 2020.Additional results are available in the supplementary material to this article regarding, the evolution of tobacco experimentation and use by sex, the evolution of e-cigarette experimentation and use by sex, what do French Adolescents like in terms of Motivations for vaping and smoking, and finally the nicotine content and flavour of e-liquids used and preferred types of vaping products (see section 3 of Appendix A).4DiscussionIn France, a sharp decline in smoking prevalence has been observed among adults (Pasquereau et al., 2021) and adolescents aged 17 years since 2014. In this context, our study aimed to assess and compare the evolution of smoking and vaping behaviour among French adolescents aged 15–16 years. Specifically, we sought to determine whether the prevalence of tobacco and e-cigarette experimentation and use increased, decreased or remained stable among Year 11 pupils attending public secondary school in the Loire department between 2018 and 2020.On the one hand, we can compare the prevalence of tobacco use and experimentation obtained in our study to some results described in the literature. In our sample of adolescents aged 15–16 years, the average prevalence of tobacco experimentation over the study period was 41.67%. This figure is almost identical to the European average (41%) and slightly lower than the French average (45%) reported in the 2019 ESPAD report for adolescents of the same age (15–16 years) (Mokinaro et al., 2020). The average prevalence of current smoking was 27.51%, which is nearly identical to the average prevalence reported by the OFDT (27.58%) for all French departments for the year 2021 (Vuolo et al., 2021) but lower than the European (30%) and French (34%) averages reported in the 2019 ESPAD report (Mokinaro et al., 2020). The average prevalence of daily smoking (10.24%) was very close to the European average (10.0%) and slightly lower than the French average (12%), while that of occasional smoking (17.27%) was lower than both the European (20%) and French (22%) averages (Mokinaro et al., 2020). Between 2018 and 2020, a continuous decline was observed in tobacco experimentation in our sample. A similar downwards trend was observed in the study by Chyderiotis et al. (Chyderiotis et al., 2020) and in the 2017 ESCAPAD survey (Le Nézet et al., 2018) for adolescents aged 17 years. Likewise, the prevalence of daily smoking decreased in our sample, which is perfectly consistent with the results of the 2015 and 2019 ESPAD reports for French adolescents aged 15–16 years (Kraus and Nociar, 2016, Mokinaro et al., 2020). This decline is also in line with the findings of the 2017 (Pasquereau et al., 2018) and 2018 Baromètre Santé surveys (Andler et al., 2019) for the adult population and those of the 2017 ESCAPAD survey (Spilka et al., 2018). In the US, the study by Jarvis et al. (Jarvis et al., 2020) observed a similar decrease (20.4%) in smoking prevalence among adolescents (from 28.5% in 1999 to 8.1% in 2018), a trend that was associated with the increased market availability of e-cigarettes in the study by Sokol et al. (Sokol and Feldman, 2021).On the other hand, we can compare the prevalence of vaping use and experimentation obtained in our study to some results described in the literature. The average prevalence of e-cigarette experimentation over the study period was 44.92%. This figure is higher than the European average (40%) and almost identical to the French average (46%) reported in the 2019 ESPAD report for adolescents of the same age (Mokinaro et al., 2020). By contrast, the average prevalence of daily vaping was low at 5.40%. The prevalence of e-cigarette experimentation in our sample decreased from 50.28% to 41.25% between 2018 and 2020. However, if we compare the prevalence of e-cigarette experimentation for the year 2020 with that reported in the 2015 ESPAD report (35.1%) (Kraus and Nociar, 2016), we observe a considerable increase in this indicator over a period of 5 years. Conversely, the prevalence reported in the 2018 EnCLASS report (52.1%) (Spilka et al., 2019) suggests a decrease in this indicator between 2018 and 2020. It should be noted that in the study by Hammond et al. (Hammond et al., 2020), the prevalence of e-cigarette experimentation increased from 29.3% in 2017 to 40.6% in 2019 among adolescents aged 16–19 years. Over the same period, the study by Jarvis et al. (Jarvis et al., 2020) observed a statistically significant increase of 15.8% (from 11.7% to 27.5%) in the prevalence of past-30-day e-cigarette use among high school students in the US. In our sample of French adolescents of the Loire department, there was a moderate increase in daily vaping (from 3.50% in 2018 to 5.13% in 2020), but this finding needs to be confirmed in future studies.Over the study period, the prevalence of current smoking was higher in boys than in girls, as was that of current vaping. These findings are in line with published data showing a male predominance in the prevalence of smoking and vaping in adolescents, young adults and the general population in France and beyond (Denis-Vatant et al., 2019, Spilka et al., 2018).In our study, approximately one in five (19.76%) adolescents were dual users of vaping and smoked tobacco. The exclusive use of either type of products was less frequent, as only 7.90% of adolescents were ‘non-vapers and smokers’ and 6.15% were ‘vapers and non-smokers.’ These results are consistent with other French studies conducted among adolescents aged 17 years (Chyderiotis et al., 2019) and with studies from the UK (Aladeokin and Haighton, 2019). Between 2018 and 2020, the proportion of “non-vapers and non-smokers” increased from 64.85% to 67.81%, while that of “vapers and non-smokers” remained stable with a slight decrease from 6.30% to 5.89%. Our analysis could not provide reliable results on the proportion of “non-vapers and smokers” who became “vapers and non-smokers” or “non-vapers and non-smokers”. However, we did find that 2–3% of “non-vapers and smokers” seems to progress to the dual use of vaping and smoked tobacco. This progression may be explained by the fact that exclusive smokers initiate vaping with the intent to reduce or quit smoking in the future. In this regard, the study by 2021 Eurobarometer survey (Eurobarometer, 2021) found dual users to be more likely to attempt to quit smoking than exclusive smokers. Yet, the progression from exclusive tobacco use to dual use could also be explained by a loss of interest in smoking and a concomitant desire to adopt exclusive vaping. The motivations for this progression have so far been studied by means of quantitative approaches (Khouja et al., 2021, McCabe et al., 2019) and should be clarified in future studies using qualitative methods.Our most relevant finding for public health was the temporal coincidence between an increase in the prevalence of daily vaping and a decrease in that of daily smoking. This finding may be explained by the fact that adolescents who first experiment with e-cigarettes are more likely not to initiate smoking or to delay their entry into smoking. Thus, the study by Chyderiotis et al. (Chyderiotis et al., 2019) found that French adolescents aged 17 years who initially experiment with e-cigarettes have a very low risk of becoming daily smokers. Likewise, in the study by Friedman et al. (Friedman and Xu, 2020), adolescent dual users who had experimented with e-cigarettes first were less likely to become daily smokers than those who had experimented with tobacco first. In the study by Legleye et al. (Legleye et al., 2020), experimenting with e-cigarettes before tobacco cigarettes was associated with a 42% reduction in the risk of daily smoking among youth aged 18 to 21 years. Kalhoran et al. (Kalkhoran et al., 2020)found early e-cigarette use to be associated with higher odds of prolonged smoking abstinence, suggesting that e-cigarettes play a role in delaying smoking initiation (Etter, 2018). Lastly, the study by Coleman et al. (Coleman et al., 2019), conducted in the same sample of US adults, noted that dual users who had initially experimented with e-cigarettes were more likely to quit smoking than those who had initially experimented with tobacco. Future studies are needed to determine the association between vaping initiation and smoking prevalence at the specific age of 15–16 years.Additional discussion points are available in the supplementary material for this article regarding the motivations for e-cigarette and tobacco experimentation and use among French adolescents of the Loire department aged 15–16 years, and the study limitations (see section 4 of Appendix A).5ConclusionsOur study shows that e-cigarette experimentation is significantly more prevalent than tobacco experimentation among French adolescents of the Loire department aged 15–16 years. Between 2018 and 2020 a clear increase is observed in the prevalence of daily vaping. Their motivations for experimenting with e-cigarettes seem to be mainly associated with recreational leisure, much less so with the desire to reduce or quit smoking. No increase was observed in the progression from vaping to smoking in our sample. French adolescent vapers of the Loire department frequently use e-liquids with little or no nicotine or with fruit or sweet flavours. Besides, some “non-vapers and smokers” seems to switch to dual use, maybe with the likely intention to reduce or quit smoking. E-liquids without or with low nicotine content and flavours with fruity and sweet tastes are frequently used in electronic cigarettes, which would explain, in part, the lack of increased risk of switching from vaping to smoking. Given the increase in the proportion of “non-vapers and non-smokers” and the stabilization of e-cigarette use, the health situation of French adolescents of the Loire department aged 15–16 years can be said to have globally improved between 2018 and 2020.Authors contributionsA. Wamba drafted and reviewed the manuscript; J. Pourchez co-drafted the manuscript, coordinated the project; M. Nekaa co-coordinated the project and conducted the statistical analyses; J. Masson co-coordinated the project and reviewed the literature; L. Leclerc read and reviewed the manuscript; C. Denis-Vatant read and reviewed the manuscript; All authors contributed to substantial editing of the manuscript and approved the final submitted version. All authors agreed to be personally accountable for their contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which they were not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.FundingThis work was supported by a grant from the French National Cancer Institute (INCa) and French Public Health Research Institute. Both funding agencies have no role in the study design, collection, analysis, or interpreting of the data, writing the manuscript, or the decision to submit the paper for publication. Authors declared no funding and connection to tobacco/vaping industry.Institutional Review Board StatementThe study was approved by the ethics committee of Saint-Etienne University Hospital (CHU) and referenced under the number IRBN372018/CHUSTE. The methods were carried out in accordance with the relevant guidelines and regulations set out in the Declaration of Helsinki.Informed Consent StatementThe study obtained written informed consent from the French ethics committee on behalf of all the research participants. It has received approval from the French Data Protection Authority (Commission Nationale de l'informatique et des Libertés) (CNIL).Data Availability StatementThe datasets accessed in this research are owned and administered by the UMR INSERM U 1059 Sainbiose, “Écoles des Mines de Saint-Etienne” (Université Jean Monnet), Saint-Etienne, France. Availability is at the discretion of Écoles des Mines de Saint-Etienne (Université Jean Monnet), Saint-Étienne, France. The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.Declaration of Competing InterestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Orthopaedic Journal of Sports Medicine

PMC10102946

4-11-2023

10.1177/23259671231162864

Hidden Pitches in Major League Baseball: What Are the Injury Implications of These Often Overlooked Pitches?

Erickson Brandon J., Buchheit Paul, Rauch Joseph, Segedin Rob, Ciccotti Michael G., Cohen Steven B.

Background:Pitch counts are tightly monitored in Major League Baseball (MLB). Hidden pitches—including warm-up pitches before and between innings and pitches before the starting/relieving pitcher’s appearance in a game—are not as closely monitored.Purpose/Hypothesis:To report the number of hidden pitches thrown per game and over the course of a season for a single organization. We hypothesized that players who threw more hidden pitches would be at an increased risk of injury compared with those who threw fewer hidden pitches.Study Design:Case-control study; Level of evidence, 3.Methods:All pitchers who played for a single MLB organization in the 2021 season were included. Hidden pitches, in-game pitches, and total pitches thrown during all games in the season were recorded. Injuries to these pitchers were also recorded. Players were defined as having an injury if they spent any time on the injured list.Results:Overall, 137 pitchers were included, 66 (48%) of whom sustained an injury and were placed on the IL during the 2021 season (mean time on IL, 53.6 ± 45.6 days). Of the 66 players who sustained an injury, 18 (27.3%) sustained an elbow injury, while 12 (18.2%) sustained a shoulder injury. Only 1 player sustained an ulnar collateral ligament tear. When comparing hidden pitches, in-game pitches, and total pitches between pitchers who sustained an injury and those who did not, there were no significant differences between groups (P = .150; P = .830; and P = .377, respectively). On average, hidden pitches made up 45.4% of the total number of pitches thrown during the course of the season. When evaluating the number of hidden pitches as a percentage of the total number of pitches thrown in a season, there was no significant difference between pitchers who sustained an injury and those who did not (P = .654).Conclusion:MLB pitchers who sustained an injury did not throw more hidden pitches than those who did not sustain an injury. Larger scale studies are needed to confirm the results of this single-team study.

The number of injuries in Major League Baseball (MLB) pitchers has been on the rise over the past 10 years.1,4,7 These injuries include everything from oblique strains and ulnar nerve issues to shoulder impingement and ulnar collateral ligament (UCL) tears.1,8,10,11,13 Many risk factors for injury in baseball pitchers have been identified—including loss of shoulder motion, loss of hip motion, fastball velocity, and fatigue.2,3,5,14,21,22 One of the major risk factors that has been correlated with injury is pitch count.6,15,17,18 However, most attention surrounding pitches has involved the pitches that are thrown only during gameplay. While in-game pitches are likely the highest-stress pitches thrown, there are many other pitches that are thrown during a game but are often forgotten about. These pitches are commonly referred to as hidden pitches, as they are not accounted for in the pitch count for the game. 23 These hidden pitches are as follows: those thrown by the starting pitcher before the first live pitch of the game; pitches thrown between innings; pitches thrown after a mound visit for injury; pitches thrown after a rain delay; and itches thrown by relievers who are getting ready to enter a game; and warm-up pitches from the mound by relievers when they enter the game. If pitch counts are indeed a risk for injury, a high volume of hidden pitches may also increase a player’s risk for injury.The purpose of this study was to report the number of hidden pitches thrown per game and over the course of a single season for a single professional baseball organization and to determine whether hidden pitches correlate with injury risk. We hypothesized that players who threw more hidden pitches during warm-up and between innings would be at an increased risk of injury than pitchers who threw fewer hidden pitches.MethodsPitchers were included if they pitched for the Phillies baseball organization during the 2021 season and had their hidden pitches recorded. All pitches thrown by pitchers in a single MLB organization over the course of the 2021 baseball season were recorded by the pitching staff. Pitches were divided into in-game pitches and hidden pitches: in-game pitches included those thrown during the course of the game while the game was in play; and hidden pitches included those that were thrown before the first inning of a game by the starting pitcher, pitches thrown between innings by the pitcher during the course of a game, pitches thrown in the bullpen by any reliever before he entered the game, and warm-up pitches thrown by relievers from the mound when they were about to enter the game. These pitches were counted by the coaching staff. All player data were provided in a deidentified manner by the professional baseball team; therefore, institutional review board approval was not required.Injuries sustained by these pitchers were tracked over the course of the entire 2021 season. Players who were injured were then compared with players who were not injured, as an injury was related to each category of pitches thrown. Players were defined as having an “injury” if they spent time on the injured list (IL). Days on the IL were also recorded for injured pitchers.Statistical AnalysisContinuous data are presented as mean ± SD. The number of hidden pitches, in-game pitches, and total pitches were compared between the injured and uninjured players using t tests, and the corresponding P values were considered significant at P < .05.ResultsOverall, 137 pitchers were included in this study, of whom 66 pitchers (48%) sustained an injury and were placed on the IL during the 2021 season. The mean time spent on the IL was 53.6 ± 45.6 days. Of the 66 players who sustained an injury, 18 (27.3%) sustained an elbow injury, 12 (18.2%) a shoulder injury, and 36 (54.5%) an injury to another body part. As such, 30 of the 137 pitchers (21.9%) included in the study sustained an injury to their shoulder or elbow in 2021. With respect to elbow injuries, only 1 player sustained a UCL tear.The results of the comparison between injured and uninjured pitchers are shown in Table 1. There were no significant differences between the groups when comparing the total number of hidden pitches, in-game pitches, or overall total pitches thrown over the course of the 2021 season.Table 1Comparison of Hidden Pitches, In-Game Pitches, and Total Pitches Between Injured and Uninjured Pitchers During the 2021 Season a All(N = 137)Injured(n = 66)Uninjured(n = 71) P Hidden pitches678.3 ± 584.2604.7 ± 452.5746.7 ± 680.5.15In-game pitches771.0 ± 693.6757.8 ± 714.2783.4 ± 678.7.83Hidden + in-game pitches1449.4 ± 1112.71362.5 ± 1006.51530.1 ± 1204.6.377 a Values are presented as mean ± SD.On average, hidden pitches made up 45.4% of the total number of pitches thrown during the course of the season (Table 2). When evaluating the number of hidden pitches as a percentage of the total number of pitches thrown in a season, there was no significant difference between pitchers who sustained an injury and those who did not (Table 2).Table 2Comparison of Hidden Pitches as a Percentage of Total Pitches Between Injured and Uninjured Pitchers During the 2021 Season a All(N = 137)Injured(n = 66)Uninjured(n = 71) P Hidden pitches, %45.4 ± 13.345.9 ± 12.344.8 ± 14.4.654 a Values are presented as mean ± SD.DiscussionThe results of this study did not confirm our hypothesis, as players who sustained an injury during the course of a single season did not throw significantly more hidden pitches during the season compared with pitchers who did not sustain an injury.Pitch counts are one of the many components that make up a pitcher’s workload and have been tracked for quite some time.5,16 To reduce injury risk in adolescents, MLB created the Pitch Smart Program, 20 which provides recommendations on the maximum number of pitches per game, as well as the number of rest days that players should have based on their age and the number of pitches they threw in a game. The institution of pitch count limits rather than inning limits in youth baseball was started in 2007 as a way to more tightly monitor workload.15,18,19 Erickson et al 9 attempted to evaluate the effectiveness of these pitch count limits when compared with inning limits in reducing the risk of future UCL injury if these players made it to professional baseball. 9 The authors compared players who pitched in the Little League World Series (LLWS) before 2007 to those who pitched after 2007. They divided up the players who pitched before 2007 based on whether the number of pitches they threw in the LLWS would have followed the current pitch count recommendations. Despite the small number of pitchers included, 50% of LLWS pitchers who exceeded the current pitch count recommendations and went on to pitch professionally required UCL reconstruction (UCLR), while only 1.7% (1/58) of those who went on to play professionally and did not exceed pitch count recommendations required UCLR. This study did not take hidden pitches into account. Thus, while pitch count limits may be effective, it is unclear whether the hidden pitches played a role in injury risk.As this is the first study to evaluate hidden pitches as a risk factor for injury in professional baseball players, there has been limited work on hidden pitches thus far in the literature. Hidden pitches are often thrown at a lower intensity and with less stress than in-game pitches, and as such, it is not clear whether they pose the same injury risk as in-game pitches. Zaremski et al 23 evaluated the total number of pitches accumulated during a typical high school varsity baseball game, including bullpen, between-innings warm-up, and in-game pitches, with 105 pitches as the upper limit of a “safe” pitch count based on the current Pitch Smart guidelines. They reported 13,769 total pitches during 115 starting pitcher outings and found that no pitcher exceeded the recommended maximum of 105 in-game pitches. However, when assessing all pitches—including the hidden pitches—the mean number of pitches was 120 per game, thereby exceeding the maximum pitch count recommendations. They found that the mean number of total pitches thrown during a pitcher outing was ≥42.4% than that documented during the game alone. Furthermore, 70.4% of pitcher outings (81/115 outings) resulted in >105 game-day pitches thrown. However, the authors did not report injury rates in these players.The results of the present study mirror those of Zaremski et al, 23 as hidden pitches made up approximately 45% of the total number of pitches over the course of a season. It is interesting that the percentage of hidden pitches thrown does not vary between high school and professional baseball players. However, the number of hidden pitches thrown did not appear to play a significant role in injury risk to the study pitchers.It is not clear whether hidden pitches increase a player’s risk for injury, as they increase workload and resistive stress, or whether they are protective to pitchers, as they allow the pitchers to warm up. Taking the proper time to warm up allows muscles to increase pliability and may help to reduce the risk of injury when the players enter the game and increase their throwing intensity. However, this warming up comes at the cost of increased use of the arm. The results of the present study indicate that hidden pitches are more protective than detrimental.The present study did not find the number of in-game pitches thrown over the course of a season to be a risk factor for injury. While many have included pitch counts as a part of the workload equation, most studies on professional players have failed to show a correlation between high pitch counts during the season and the risk of injury. Erickson et al 12 reviewed all players who underwent UCLR and evaluated whether total in-game pitches over the course of a season after UCLR was a risk factor for needing a revision UCLR. The authors found that a player’s pitch count in the first full season back from UCLR was not a risk factor for requiring subsequent UCLR. Chalmers et al 5 evaluated the number of innings pitched as a risk factor for sustaining a latissimus dorsi injury in professional baseball pitchers and found that players who pitched more innings had a higher risk of injury; however, they did not evaluate pitch counts specifically. While the present study did not find the total number of hidden pitches or the total number of in-game pitches to be a risk factor for injury, this study only evaluated 1 professional baseball club over the course of 1 season. Further work regarding hidden pitches is needed to include multiple seasons across multiple teams to confirm these results.LimitationsThis study only evaluated professional baseball players in a single MLB organization, and therefore, the results may not be generalizable to pitchers in other organizations or to college/youth pitchers. Furthermore, this study only looked at 1 baseball season. Thus, the number of hidden pitches may have a cumulative effect on injury risk over the course of a player’s career. Days on the IL were not factored into the calculations in this study. Finally, although all pitchers for this organization were included, it is possible that the study was underpowered to detect a difference in injury rates. The authors plan to repeat this study each year, adding in the subsequent year’s pitch counts (in-game and hidden) to the current data to continually reassess these findings.ConclusionProfessional baseball pitchers who sustained an injury did not throw more hidden pitches than those who did not sustain an injury. Larger scale studies are needed to confirm the results of this single-team study.

PMC

World Journal of Gastroenterology

PMC10835541

1-28-2024

10.3748/wjg.v30.i4.346

Gastrointestinal manifestations of critical ill heatstroke patients and their associations with outcomes: A multicentre, retrospective, observational study

Wang Yu-Cong, Jin Xin-Yang, Lei Zheng, Liu Xiao-Jiao, Liu Yu, Zhang Bang-Guo, Gong Jian, Wang Lie-Tao, Shi Lv-Yuan, Wan Ding-Yuan, Fu Xin, Wang Lu-Ping, Ma Ai-Jia, Cheng Yi-Song, Yang Jing, He Min, Jin Xiao-Dong, Kang Yan, Wang Bo, Zhang Zhong-Wei, Wu Qin

BACKGROUNDExtreme heat exposure is a growing health problem, and the effects of heat on the gastrointestinal (GI) tract is unknown. This study aimed to assess the incidence of GI symptoms associated with heatstroke and its impact on outcomes.AIMTo assess the incidence of GI symptoms associated with heatstroke and its impact on outcomes.METHODSPatients admitted to the intensive care unit (ICU) due to heatstroke were included from 83 centres. Patient history, laboratory results, and clinically relevant outcomes were recorded at ICU admission and daily until up to day 15, ICU discharge, or death. GI symptoms, including nausea/vomiting, diarrhoea, flatulence, and bloody stools, were recorded. The characteristics of patients with heatstroke concomitant with GI symptoms were described. Multivariable regression analyses were performed to determine significant predictors of GI symptoms.RESULTSA total of 713 patients were included in the final analysis, of whom 132 (18.5%) patients had at least one GI symptom during their ICU stay, while 26 (3.6%) suffered from more than one symptom. Patients with GI symptoms had a significantly higher ICU stay compared with those without. The mortality of patients who had two or more GI symptoms simultaneously was significantly higher than that in those with one GI symptom. Multivariable logistic regression analysis revealed that older patients with a lower GCS score on admission were more likely to experience GI symptoms.CONCLUSIONThe GI manifestations of heatstroke are common and appear to impact clinically relevant hospitalization outcomes.

Core Tip: This study aimed to assess the incidence of gastrointestinal (GI) symptoms associated with heatstroke and its impact on outcomes. This was a retrospective, multi-center, observational cohort study that involved patients admitted to 83 intensive care unit located in 16 cities in the Sichuan Province, China between June 1 and October 31, 2022. Results showed older heatstroke patients with a lower Glasgow coma scale score on admission were more likely to experience GI symptoms, which had statistical difference. Clinicians should pay attention to the time at which heatstroke patients started manifesting GI symptoms, as well as the duration of said symptoms, to ensure that patients are timely treated with the proper enteral therapy and have the best prognosis possible.INTRODUCTIONOwing to the effects of climate change, extreme heat is rapidly becoming a global public health concern. Direct exposure to extreme heat can cause dysregulation of body temperature, leading to heatstroke. Over the past two decades, there has been a 50% increase in heat-related mortality among adults aged 65 and older. As an acute life-threatening condition manifesting an uncontrolled rise in core body temperature, heatstroke presents clinically as a systemic disorder and comprises the following symptoms: Encephalopathy, hypotension, respiratory failure, liver, muscle, coagulopathy and kidney damage. In recent years, some studies have indicated that sustained high body temperature can cause structural and functional damage to the gastrointestinal (GI) tract, resulting in vomiting, diarrhoea, or intolerance to enteral nutrition (EN), which can exacerbate patients' condition[3,4]. Nevertheless, the impact of heatstroke on the GI tract remains to be elucidated.Located in southwestern part of China, the Sichuan Province is the second largest Chinese province, with a permanent population of more than 80 million. According to the records of the Sichuan meteorological administration, as of May 2022, summer temperatures have reached a historical high since 1961, with two consecutive strong high-temperature periods. One of the most notable consequences of this phenomenon is the significant increase in the number of cases of heatstroke. Accordingly, we conducted a retrospective, multi-center study to examine the demographic characteristics of heatstroke patients admitted to the intensive care unit (ICU) in 2022. Our study primarily aimed to determine the incidence of GI disturbances among patients experiencing heatstroke from various medical centres in the Sichuan Province, with a secondary objective of identifying the risk factors for GI symptoms after heatstroke.MATERIALS AND METHODSThis was a retrospective, multi-center, observational cohort study that involved patients admitted to 83 ICUs located in 16 cities in the Sichuan Province, China between June 1 and October 31, 2022. Ethical approval for this study was obtained from the Biomedical Ethics Review Committee of the West China Hospital of Sichuan University (approval No. SCU-2022-1542), in accordance with the principles outlined in the Declaration of Helsinki. Given the retrospective nature of this study, the requirement for informed consent was waived.Patients and examinationInclusion criteria comprised: an age > 18 years; and hospitalization in any type of ICU due to heatstroke or heatstroke-related complications. Patients with heatstroke were diagnosed by front-line medical staff in each center, and the diagnosis was made according to the corresponding clinical manifestations, as well as clinical history. The exclusion criteria included an age < 18; burns; death within 24 h following ICU admission; palliative care; and simultaneous participation in any other nutrition-related interventional studies. Patients whose data is unsuitable for the analysis performed in this study were also excluded. Demographic characteristics were recorded at ICU admission, and clinical variables were recorded daily until up to day 15 of ICU stay or ICU discharge or death. Patients included in the study were managed by physicians in their respective ICUs. The treatment plan for each patient was determined by the attending physician based on the patient's individual condition.Data collection and definitionsAn electronic data capture system (Sichuan Zhikang Technology Co., Ltd, China) was implemented to gather information on heatstroke patients. Data collectors, who were primarily front-line physicians in each centre’s ICU, recorded information on each patient. The data collected was determined after extensive discussion based on expert opinions and in combination with literature review. The trial filling of data was conducted twice through two extensive online meetings with experts from each center to finalize all forms. A training in which the use of the electronic data capture system and heatstroke-related knowledge are explained was conducted in each center before initiating data collection. An online meeting would be hosted every week to check the quality of the data collected during said week. The data underwent a two-step verification process to ensure its completeness and accuracy, and unqualified data was to be collected again. Four researchers (Yu-Cong Wang, Lie-Tao Wang, Lv-Yuan Shi, and Ding-Yuan Wan) reviewed all data independently for completeness and accuracy, and the data management team (Min He, Jing Yang and Qin Wu) conducted a thorough cleaning of the data, identifying any missing information.Data comprising baseline information, laboratory test results, treatment plan, GI symptoms, nutrition support, and patient outcome were collected in the electronic data capture system. As demographic information, age, sex, body mass index (BMI), and concomitant diseases were collected. Patients’ body temperature at hospital admission, including duration of exposure to heat, first symptoms according to chief complain, nutrition risk screening 2002 (NRS-2002), and Glasgow coma scale (GCS) score were also recorded. Moreover, the average, maximum, and minimum temperature data for the months of May, June, and July in 2022, which was publicly available on the website of the Sichuan meteorological administration, was also collected. The definition of fever in this study was set as a body temperature greater than 37.3 °C as determined through anal temperature measurement. High environment temperature was defined as when the maximum environment temperature reaches or exceeds 35 °C. If the high temperature lasts for more than 3 d, it was defined as a high temperature heat wave. Treatment received during the observation period, including organ support technical, antibiotics, and steroids, were recorded.GI symptoms were defined as the presence of nausea/vomiting, diarrhoea, flatulence, or bloody stools that do not resolve with medical therapy[6-8]. Specifically, nausea/vomiting in non-intubated patients was defined as the self-reporting of epigastric discomfort followed by vomiting, self-reporting of nausea alone without vomiting, or vomiting alone without nausea. As for intubated patients, nausea/vomiting was defined as the presence of reflux or aspiration for abnormal causes. Diarrhoea was defined as frequent exclusion of loose thin faeces or even watery stools for more than 3 times daily of more than 200 mL each time. Flatulence was defined as awake patients feeling fullness in part or all of the abdomen or partial or total abdominal distention as determined by physical examination in non-awake patients. Bloody stool was defined as having a positive faecal occult blood test more than twice or dark red or black stool. Information on GI symptoms comes from the hourly nursing observation records or daily disease course records.For patients’ outcome, we collected complications during the observational period, mortality at 15 d, and length of stay in the ICU. The complications in this study included disturbance of water and electrolyte, rhabdomyolysis, myocardial damage, acute kidney injury, acute liver function impairment, and central nervous system impairment. More specifically, disturbance of water and electrolyte was defined as dehydration, oedema, hyperkalaemia, hypokalemia, hypercalcemia, hypocalcaemia, hypermagnesemia or hypomagnesemia, as determined by clinicians. Rhabdomyolysis was defined as muscle pain, tenderness, swelling, weakness, and other muscle involvement and serum creatine kinase levels being significantly elevated more than 5 times the upper limit of normal. Myocardial damage was defined as elevated myocardial enzymes with a normal electrocardiogram. Acute kidney injury was defined according to the kidney disease: Improving Global Outcomes criteria after high temperature exposure. Acute liver function impairment was defined as elevated serum aminotransferase and bilirubin levels above the normal limit after exposure to high temperature with the absence of chronic liver disease, liver failure, coagulation dysfunction, and hepatic encephalopathy. Central nervous system impairment was defined as the occurrence of seizures, motor dysfunction, or sensory dysfunction, including limb hemiplegia, immobility, numbness of the hemi limb, or spontaneous pain in a patient with no history of central nervous system disease.The primary outcome of the study was the incidence of post-heatstroke GI symptoms, as determined through data collection by trained data collectors. Secondary outcomes included the identification of risk factors for GI dysfunction following heatstroke.Statistical analysisStatistical analysis was performed using descriptive statistics. Continuous variables were reported as median and quartile ranges or simple ranges, while categorical variables were summarized as counts and percentages. Items missing more than 10% of their data will be excluded from the analysis, and no imputation was made for missing data. All data were analysed using SPSS Statistics version 25 software (IBM Corp., Armonk, NY, United States). Descriptive statistical analyses reflected the distribution of characteristics of the sample population across case and control groups in the form of counts and proportions. T tests and χ2 tests were applied to test the association between case and control group variables. The incidence of confirmed cases was visually represented using a map created with Dychart.com (Wuhan Dysprosium Metadata Technology Co., Ltd, Wuhan, Hubei, China). We developed a logistic regression model to assess the association between the rates of GI dysfunction after heatstroke and several high-risk indicators, including age, initial temperature, initial symptoms, and comorbidities using Graphpad Prism 9 XML project (Graphpad Software Inc., San Diego, CA, United States).RESULTSStudy populationBetween June 1, 2022 and October 31, 2022, a total of 873 patients admitted from 83 ICUs across 16 cities due to heatstroke were collected. Of these patients, 160 were excluded as follows: 11 patients were excluded as they were under 18 of age; 2 for heatstroke caused by burn; 16 for mortality within 24 h after ICU admission; 11 for palliative care after ICU admission; and 120 for incomplete data (Figure 1). A total of 713 patients were enrolled in the final analysis. The number of patients enrolled each day during the trial period and daily change in average and maximum temperature in the Sichuan Province are displayed in Supplementary Figure 1. The number of centres from different cities participating in the trial and corresponding total number of patients enrolled are shown in Supplementary Figure 2.Figure 1 Flow chart of the study. Patients were grouped according to whether gastrointestinal (GI) symptoms occurred on intensive care unit (ICU) admission, then whether GI symptoms occurred during ICU hospitalization and the onset of patients’ GI symptoms. ICU: Intensive care unit; GI: Gastrointestinal; w: With; o: Without.Of the 713 analysed patients, 46.6% were female, and the median age was 72 years [interquartile range (IQR): 64–80; Table 1]. The median body temperature of patients at hospital admission was 40.7 (IQR: 40.0 to 41.3). Around 50% of patients (343/713, 48.10%) were admitted with altered mental states or behaviours. Part of the cohort had at least one underlying illness, such as hypertension (187/713, 26.20%) or diabetes (87/713, 12.20%). Upon admission, the median level of C-reactive protein was elevated (5.0 mg/L, IQR: 1.0–11.8). The same was true for the median levels of procalcitonin (2.7 mg/mL, IQR: 0.5–13.1), and median D-dimer (4.6 mg/L, IQR: 1.8–12.9). A total of 439 patients (61.7%) underwent endotracheal intubation upon ICU admission. At day 15, 349 patients (48.9%) were discharged from the hospital, while 144 (20.2%) died, 187 (26.2%) were still hospitalized, and 33 (4.6%) transferred to another hospital. During hospitalization, acute liver dysfunction was observed in 42.8% (305/713) patients, 41.9% (299/713) experienced acute kidney injury, 39.4% (281/713) experienced myocardial damage, and 35.9% (256/713) experienced central nervous system damage.Table 1Clinical characteristics, laboratory findings at admission, gastrointestinal symptoms findings, complications, treatments, and clinical outcomes of the study patients, according to developed gastrointestinal symptoms or not1 All Patients, (n = 713) GI Symptoms whether2 P value Yes (n = 132) No (n = 581) Characteristic Age, median (IQR), yr72.0 (64.0-80.0)70.0 (59.0-76.0)73.0 (64.0-81.0)0.076Distribution, n (%) 0-39 yr13 (1.8)1 (0.01)12 (2.1)— 40-59 yr132 (18.5)32 (24.2)100 (17.2)— 60-79 yr371 (52.0)73 (55.3)298 (51.2)— ≥ 80 yr197 (27.6)26 (19.6)171 (29.4)— Female sex332 (46.6)60 (45.4)272 (46.8)0.561 BMI, median (IQR), kg/m222.1 (20.3-24.2)22.5 (20.0-24.6)22.0 (20.3-24.1)0.875 GCS score at ICU admission, median (IQR)6.0 (4.0-9.0)5.0 (3.0-7.0)6.0 (4.0-9.0)0.018 NRS-2002 score at ICU admission, median (IQR)4.0 (3.0-5.0)3.0 (3.0-5.0)4.0 (3.0-5.0)0.014Body temperature on admission Patients, n (%)710 (99.1)132 (100.0)578 (98.8)0.374 Temperature, median (IQR), °C40.7 (40.0-41.3)41.0 (40.1-42.0)40.5 (40.0-41.2) 40.0 °C379 (60.0)77 (64.7)302 (58.9)—Number of complaints and symptoms on admission, n (%) 3155/672 (23.1)52/126 (41.3)103/546 (18.9) 3317 (44.5)92 (69.7)225 (41.4)< 0.001 Disturbance of water and electrolyte412 (57.8)95 (72.0)317 (54.6)0.013 Rhabdomyolysis102 (14.3)34 (25.8)68 (11.7)0.004 Myocardial damage281 (39.4)70 (53.0)211 (36.3)< 0.001 Disseminated intravascular coagulation221 (31.0)62 (46.9)159 (27.4)0.006 Acute respiratory distress syndrome256 (35.9)66 (50.0)190 (32.7)0.001 Acute kidney injury299 (41.9)83 (62.9)216 (37.2)0.003 Acute liver function impairment305 (42.8)81 (61.4)224 (38.6)< 0.001 Central nervous system damage256 (35.9)74 (56.1)182 (31.9)0.003Treatments, n (%) Mechanical ventilation Invasive439 (61.7)108 (82.6)331 (57.1)< 0.001 Noninvasive10 (1.4)0 (0.0)10 (1.6)0.271 Use of continuous renal-replacement therapy24 (3.4)7 (9.2)17 (2.7)0.003 Length of ICU stay, median (IQR), d2.0 (1.0-4.0)4.0 (2.0-7.0)2.0 (1.0-3.0)0.001Clinical outcomes at data cutoff, n (%) Hospital discharge349 (48.9)64 (48.5)285 (49.1)0.906 Death144 (20.2)26 (19.7)118 (20.3)0.874 Still hospitalization187 (26.2)33 (25.0)154 (26.5)0.723 Transferred to another hospital33 (4.6)9 (6.8)24 (4.1)< 0.0011The denominators of patients who were included in the analysis are provided if they differed from the overall numbers in the group. Percentages may not total 100 because of rounding.2Nausea/vomiting, diarrhea, flatulence, or bloody stools are defined as gastrointestinal symptoms.3Included in this category is any type of cancer.IQR: Interquartile range; GCS: Glasgow coma scale; NRS: Nutrition risk screening; GI: Gastrointestinal; BMI: Body mass index; ICU: Intensive care unit; CK-Mb: Creatine kinase.Patient characteristics and outcomes according to whether gastrointestinal symptoms are presentOur study results showed that 18.5% (132/713) of heatstroke patients experienced at least one episode of GI symptoms during ICU stay. Of these patients, 8 (6.1%) experienced bloody stools, 21 (15.9%) experienced nausea/vomiting, 36 (27.3%) experienced flatulence, and 99 (75.0%) experienced diarrhoea (Table 1 and Figure 2). Patients with heatstroke were subsequently categorized into two groups: Those who experienced GI symptoms (n = 132) and those who did not (n = 581) during their ICU stay. There was no difference in the median age of patients between both groups (Table 1). Patients with GI symptoms had significantly lower GCS scores (5.0 vs 6.0, aP = 0.018) and lower NRS-2002 scores (3.0 vs 4.0, bP = 0.014) on admission. There was also no significant difference in the presence of comorbidities upon admission between the groups during the study period, except for a prior history of cancer (2.5% vs 0.5%, cP = 0.033; Table 1). Laboratory results on admission revealed that patients with GI symptoms had significantly lower levels of albumin (33.4 vs 37.0, dP = 0.014) and hemoglobin (115.0 vs 124.0, eP = 0.014) and a higher level of blood lactate (3.1 vs 3.4, eP = 0.036) and C-reactive protein (11.9 vs 5.0, fP = 0.043). It was observed that patients presenting with GI symptoms had an increased likelihood of developing multiple complications, including acute kidney injury (62.9%, gP = 0.003), acute liver function impairment (61.4%, hP < 0.001), and central nervous system damage (56.1%, iP = 0.003). However, the presence of GI symptoms did not have a significant impact on patient mortality. Multivariate logistic regression showed that heatstroke patients who were older than the average year of the cohort were more likely to develop GI symptoms (jP = 0.001; Figure 3A). Moreover, patients with a lower GCS score were prone to have GI symptoms (kP = 0.006; Figure 3A). This positive correlation of GCS score with GI symptoms persisted when we adjusted for complications (Figure 3B) and laboratory results (Figure 3C).Figure 2Number of patients with gastrointestinal symptoms and total number of heatstroke patients still in the intensive care unit per day.Figure 3 Multivariable-adjusted logistic regression of risk factors with gastrointestinal symptoms. A: It showed multivariable logistic regression between heatstroke patients’ gastrointestinal (GI) symptoms with age, sex, body mass index (BMI) Glasgow coma scale (GCS) score, nutrition risk screening 2002 (NRS-2002) score, high temperature exposed duration and patients’ temperature; B: It is adjusted for coexisting disorder and index in Figure 3A; C: It is adjusted for laboratory results at intensive care unit admission and index in Figure 3A. Age is a categorical variable bounded by the median age of the patient, with less than the median age being compared. Sex is the categorical variable, with women being compared. BMI, GCS score, NRS-2002, exposed duration and temperature are continuous variables. Diabetes, hypertension, chronic obstructive disease, chronic cardiac insufficiency, abnormal white cell count, PaO2/FiO2 ratio and hemoglobin are categorical variable. BMI: Body mass index; GCS score: Glasgow coma scale score; NRS-2002 score: Nutrition risk screening-2002 score; OR: Odds ratio; PF ratio: PaO2/FiO2 ratio; HB: Hemoglobin.Relationship between GI symptoms and enteral nutrition therapyConsidering that the predominant GI symptom is diarrhoea, a total of 439 heatstroke patients with endotracheal intubation shortly after ICU admission were analysed to explore the relationship between GI symptoms and EN therapy. We found that the presence of GI symptoms was not associated with EN therapy (Table 2). There was no statistical difference in the proportion of EN support, amount of calories and proteins, and total volume received on admission between the patients who underwent EN therapy. Of note, EN therapy was initiated in only a small proportion (139/439, 31.7%) of intubated patients within 48 h after ICU admission, as shown in Table 3. Patients who did not start EN within 48 h of ICU admission had a significantly lower GCS score (5.0 vs 4.0, lP = 0.002), experienced more GI symptoms after ICU admission (22.3% vs 12.9%, mP = 0.021), and had a longer ICU stay (3.0 vs 2.0, nP < 0.001). Logistic regression analysis showed that GI symptoms were an independent risk factor for not initiating early EN (oP = 0.037; Supplementary Figure 3). During the observational period, 266 (60.6%) patients with endotracheal intubation at admission failed to establish full EN (Table 4). Patients who do not receive full EN experienced more GI symptoms (22.6% vs 14.5%, pP = 0.036). Moreover, the mortality of patients who did not receive full EN was significantly higher than those who did (35.3% vs 16.8%, qP < 0.001). Moreover, rhabdomyolysis (20.7% vs 11.6%, rP = 0.013) and acute kidney injury (57.9% vs 45.7%, sP = 0.012) were more common, and ICU stay was longer in the former population (3.0 vs 2.0, tP < 0.001).Table 2Clinical characteristics of the heatstroke patients with endotracheal intubation, according to developed gastrointestinal symptoms or not1 All intubated patients (n = 439) GI symptoms whether2 P value Yes (n = 108) No (n = 331) Characteristic Age, median (IQR), yr71.0 (63.0-80.0)72.0 (64.0-80.0)69.5 (59.8-78.0)0.202 Female sex, n (%)197 (44.9)49 (45.4)148 (44.7)0.905 GCS score at ICU admission, median (IQR)4.0 (3.0-5.0)4.0 (3.0-5.0)4.0 (4.0-5.0)0.204 NRS score at ICU admission, median (IQR)4.0 (3.0-5.0)3.0 (3.0-5.0)4.0 (3.0-5.0)0.057Body temperature on admission Patients, n (%)439 (100.0)108 (100.0)331 (100.0)0.348 Temperature, median (IQR), °C41.0 (40.0-41.8)41.0 (40.0-42.0)41.0 (40.0-41.6)0.497Complaints and symptoms on admission, n (%) Fever296 (67.4)76 (70.4)220 (66.5)0.452 Altered mental state or behavior205 (46.7)41 (38.0)164 (49.5)0.036 Dry skin or excessive sweating45 (10.320 (18.5)25 (7.6)0.001 Rubefaction17 (3.9)9 (8.3)8 (2.4)0.006 Fast pulse99 (22.6)34 (31.5)65 (19.6)0.105 Polypnea122 (27.8)31 (28.7)91 (27.5)0.807 Headache7 (1.6)2 (18.5)5 (1.5)0.805 Syncope207 (47.2)70 (64.8)137 (41.4)< 0.001 Other61 (13.9)71 (65.7)40 (12.1)< 0.001Laboratory findings, median (IQR) PaO2/FiO2 ratio225.0 (135.5-306.5)221.0 (148.7-308.0)226.0 (155.0-305.5)0.999 White-cell count, 109/L11.7 (8.1-15.6)11.8 (8.0-15.8)11.7 (8.1-15.6)0.405 Lymphocyte count, 109/L1.7 (0.8-2.8)1.6 (0.8-2.8)1.8 (0.8-28)0.279 Platelet count, 109/L92.0 (56.0-138.0)83.5 (55.5-114.5)97.0 (59.0-141.0)0.143 Hemoglobin, g/L124.0 (109.0-139.0)121.0 (108.3-135.8)125.0 (110.0-139.0)0.053 Albumin, g/L35.3 (32.5-38.9)34.5 (31.8-38.0)35.3 (32.5-38.9)0.028EN support Early (< 48 h) EN, n (%)68 (15.5)13 (12.0)55 (16.6)0.253 Average EN calorie, median (IQR), kcal/d1000.0 (750.0-1500.0)1000.0 (750.0-1500.0)1000.0 (750.0-1500.0)0.559 Average EN protein, median (IQR), g/d28.0 (17.0-56.0)30.0 (17.0-56.0)28.0 (20.0-56.0)0.867 Average EN volume, median (IQR), mL/d600.0 (150.0-1150.0)625.0 (142.5-1200.0)600.0 (150.0-1082.5)0.31Complications, n (%) Disturbance of water and electrolyte271 (61.7)89 (82.4)187 (56.5)< 0.001 Rhabdomyolysis81 (18.5)31 (28.7)50 (15.1)0.002 Myocardial damage216 (49.2)63 (58.0)153 (46.2)0.029 Disseminated intravascular coagulation172 (39.2)54 (50.0)118 (35.6)0.001 Acute respiratory distress syndrome226 (51.5)62 (57.4)164 (49.5)0.156 Acute kidney injury233 (53.1)77 (71.3)156 (47.1)< 0.001 Acute liver function impairment332 (75.6)73 (65.6)159 (48.0)< 0.001 Central nervous system damage206 (46.9)65 (60.2)141 (42.6)0.001Clinical outcomes at data cutoff, n (%) Hospital discharge210 (47.8)51 (47.2)159 (48.0)0.234 Death123 (28.0)24 (22.2)99 (29.9)0.122 Still hospitalization78 (17.8)24 (22.2)54 (16.3)0.163 Transferred to another hospital28 (6.4)9 (8.3)19 (5.7)0.3381The denominators of patients who were included in the analysis are provided if they differed from the overall numbers in the group. Percentages may not total 100 because of rounding.2Nausea/vomiting, diarrhea, flatulence, or bloody stools are defined as gastrointestinal symptoms.IQR: Interquartile range; GCS: Glasgow coma scale; NRS: Nutrition risk screening; GI: Gastrointestinal; EN: Enteral nutrition; ICU: Intensive care unit.Table 3Characteristics of the heatstroke patients with endotracheal intubation, according to received enteral nutrition within 48 h after intensive care unit admission or not1 All intubated patients (n = 439) EN therapy whether ≤ 48 h P value Yes (n = 139) No (n = 300) Characteristic Age, median (IQR), yr71.0 (63.0-80.0)72.0 (63.0-80.0)71.0 (63.0-79.0)0.801 Female sex, n (%)197 (44.9)62 (46.8)135 (43.6)0.938 GCS score at ICU admission, median (IQR)4.0 (3.0-5.0)5.0 (3.0-7.0)4.0 (3.0-5 .0)0.002 NRS score at ICU admission, median (IQR)4.0 (3.0-5.0)5.0 (4.0-6.0)4.0 (3.0-5.0)0.017Body temperature on admission Patients, n (%)439 (100.0)139 (100.0)300 (100.0)— Temperature, median (IQR), °C41.0 (40.0-41.8)41.0 (40.0-41.5)41.0 (40.0-42.0)0.154Laboratory findings, median (IQR) PaO2/FiO2 ratio225.0 (135.5-306.5)226.0 (164.0-287.0)223.0 (128.0-316.0)0.825 White-cell count, 109/L11.7 (8.1-15.6)12.4 (8.6-16.8)11.3 (8.0-15.0)0.290 Lymphocyte count, 109/L1.7 (0.8-2.8)1.5 (0.7-2.3)1.9 (0.9-3.0)0.105 Platelet count, 109/L92.0 (56.0-138.0)101.0 (66.8-147.3)88.0 (54.0-130.0)0.039 Hemoglobin, g/L124.0 (109.0-139.0)122.0 (109.0-136.0)124.5 (109.0-140.0)0.311 Albumin, g/L35.3 (32.5-38.9)35.1 (32.0-38.5)35.4 (32.5-39.0)0.544GI symptoms2, n (%) Total patients85 (19.4)18 (12.9)67 (22.3)0.021 Diarrhea60 (13.7)13 (9.4)47 (15.7)0.073 Flatulence25 (5.7)7 (5.0)18 (6.0)0.685 Nausea/vomiting14 (3.2)7 (5.0)7 (2.3)0.134 Bloody stools8 (1.8)0 (0.0)8 (2.6)0.052Complications, n (%) Disturbance of water and electrolyte271 (61.7)79 (56.8)192 (64.0)0.151 Rhabdomyolysis81 (18.5)20 (11.6)61 (20.3)0.135 Myocardial damage216 (49.2)65 (46.8)151 (50.3)0.486 Disseminated intravascular coagulation172 (39.2)50 (36.0)122 (40.7)0.349 Acute respiratory distress syndrome226 (51.5)74 (53.2)152 (50.7)0.616 Acute kidney injury233 (53.1)66 (47.5)167 (55.7)0.110 Acute liver function impairment332 (75.6)74 (53.2)258 (86.0)< 0.001 Central nervous system damage206 (46.9)62 (44.6)144 (48.0)0.507Treatments Use of continuous renal-replacement therapy, n (%)34 (7.7)10 (8.1)20 (7.5)0.826 Length of ICU stay, median (IQR), d2.0 (1.0-3.0)2.0 (1.0-3.0)3.0 (2.0-5.0)< 0.001Clinical outcomes at data cutoff, n (%) Hospital discharge210 (47.8)68 (48.9)142 (47.3)0.757 Death123 (28.0)24 (17.3)99 (33.3)< 0.001 Still hospitalization78 (17.8)36 (25.9)42 (14.0)0.002 Transferred to another hospital28 (6.4)11 (7.9)17 (5.7)0.3701The denominators of patients who were included in the analysis are provided if they differed from the overall numbers in the group. Percentages may not total 100 because of rounding.2Nausea/vomiting, diarrhea, flatulence, or bloody stools are defined as gastrointestinal symptoms.IQR: Interquartile range; GCS: Glasgow coma scale; NRS: Nutrition risk screening; GI: Gastrointestinal; ICU: Intensive care unit.Table 4Characteristics of the heatstroke patients with endotracheal intubation, according to received full enteral nutrition after intensive care unit admission or not1 All intubated patients (n = 439) Full EN whether P value Yes (n = 173) No (n = 266) Characteristic Age, median (IQR), yr71.0 (63.0-80.0)72.0 (63.0-80.0)71.0 (63.0-79.0)0.801 Female sex, n (%)197 (44.9)81 (46.8)116 (43.6)0.509 GCS score at ICU admission, median (IQR)4.0 (3.0-5.0)6.0 (4.0-8.0)5.0 (3.0-7.0)0.002 NRS score at ICU admission, median (IQR)4.0 (3.0-5.0)4.5 (4.0-5.0)4.0 (3.0-5.0)0.193Body temperature on admission Patients, n (%)439 (100.0)173 (100.0)266 (100.0)— Temperature, median (IQR), °C41.0 (40.0-41.8)41.0 (40.0-41.4)41.0 (40.0-42.0)0.128Laboratory findings, median (IQR) PaO2/FiO2 ratio225.0 (135.5-306.5)246.0 (191.0-334.0)222.0 (134.0-315.0)0.226 White-cell count, 109/L11.7 (8.1-15.6)12.6 (9.4-16.7)11.4 (8.0-15.3)0.287 Lymphocyte count, 109/L1.7 (0.8-2.8)0.8 (0.5-1.6)2.0 (0.9-3.0)0.084 Platelet count, 109/L92.0 (56.0-138.0)71.5 (37.0-113.8)89.0 (54.0-132.5)0.011 Hemoglobin, g/L124.0 (109.0-139.0)119.0 (107.5-129.0)125.0 (110.0-140.0)0.002 Albumin, g/L35.3 (32.5-38.9)32.0 (29.0-35.2)35.5 (32.5-39.2)< 0.001GI symptoms2, n (%) Total patients85 (19.4)25 (14.5)60 (22.6)0.036 Diarrhea60 (13.7)17 (9.8)43 (16.2)0.059 Flatulence25 (5.7)8 (4.6)17 (6.4)0.435 Nausea/vomiting14 (3.2)7 (4.0)7 (2.6)0.409 Bloody stools8 (1.8)0 (0.0)8 (3.1)0.021Complications, n (%) Disturbance of water and electrolyte271 (61.7)105 (60.7)166 (62.4)0.718 Rhabdomyolysis81 (18.5)20 (11.6)55 (20.7)0.013 Myocardial damage216 (49.2)83 (48.0)133 (50.0)0.678 Disseminated intravascular coagulation172 (39.2)60 (34.7)112 (42.1)0.119 Acute respiratory distress syndrome226 (51.5)92 (53.2)134 (50.4)0.566 Acute kidney injury233 (53.1)79 (45.7)154 (57.9)0.012 Acute liver function impairment332 (75.6)94 (54.3)138 (51.9)0.615 Central nervous system damage206 (46.9)80 (46.2)126 (47.4)0.817Treatments Use of continuous renal-replacement therapy, n (%)34 (7.7)14 (8.1)20 (7.5)0.826 Length of ICU stay, median (IQR), d2.0 (1.0-3.0)2.0 (1.0-2.0)3.0 (1.0-4.0)< 0.001Clinical outcomes at data cutoff, n (%) Hospital discharge210 (47.8)83 (48.0)127 (47.7)0.962 Death123 (28.0)29 (16.8)94 (35.3)< 0.001 Still hospitalization78 (17.8)46 (26.6)82 (20.8)0.339 Transferred to another hospital28 (6.4)15 (8.7)13 (4.9)0.1131The denominators of patients who were included in the analysis are provided if they differed from the overall numbers in the group. Percentages may not total 100 because of rounding.2Nausea/vomiting, diarrhea, flatulence, or bloody stools are defined as gastrointestinal symptoms.IQR: Interquartile range; GCS: Glasgow coma scale; NRS: Nutrition risk screening; GI: Gastrointestinal; ICU: Intensive care unit.Subgroup analysisSince the definition of GI manifestations was composite, we subsequently explore whether there was difference in the characteristics of patients with different symptoms. We selected patients with a single symptom and divided them into 3 groups according to different GI manifestations (Table 5). There was a statistically significant difference in temperature on admission between patients with diarrhoea, flatulence, and nausea/vomiting (uP = 0.003). Notably, there were significant differences in complications between the three subgroups, except for complications of disturbance of water and electrolyte. Although mortality was not different between subgroups, the difference in the number of patients who were still hospitalized was statistically significant (vP = 0.025).Table 5Clinical characteristics of the study patients, according to types of gastrointestinal symptoms1 GI Symptoms2 P value Diarrhea (n = 88) Flatulence (n = 27) Nausea/vomiting (n = 15) Characteristic Age, median (IQR), yr65.0 (56.0-76.0)69.0 (56.0-79.0)68.0 (57.5-78.0)0.565 Female sex, n (%)35.0 (39.8)14.0 (51.8)6 (40.0)0.529 GCS score at ICU admission, median (IQR)5.0 (3.0-7.0)6.0 (3.0-9.5)4.0 (3.0-6.0)0.540 NRS score at ICU admission, median (IQR)4.0 (3.0-5.0)4.5 (4.0-5.0)4.0 (3.0-5.0)0.193Body temperature admission Patients, n (%)88.0 (100.0)27.0 (100.0)15.0 (100.0)— Temperature, median (IQR), °C41.0 (40.0-42.0)40.0 (39.8-41.0)40.1 (39.8-41.0)0.003Complaints and symptoms, n (%) Fever62.0 (70.5)20.0 (74.0)8.0 (53.3)0.343 Altered mental state or behavior35.0 (39.8)13.0 (48.1)4.0 (26.7)0.395 Dry skin or excessive sweating19.0 (21.6)2.0 (7.4)2.0 (13.3)0.215 Rubefaction7.0 (8.0)2.0 (7.4)1.0 (6.7)0.983 Fast pulse32.0 (36.4)6.0 (22.2)4.0 (26.7)0.344 Polypnea29.0 (33.0)6.0 (22.2)3.0 (20.0)0.397 Headache1.0 (1.1)3.0 (11.1)0.0 (0.0)0.024 Syncope63.0 (71.6)13.0 (48.1)8.0 (53.3)0.052 Other22.0 (25.0)4.0 (14.8)2.0 (13.3)0.378Laboratory findings, median (IQR) White-cell count, 109/L13.0 (8.2-16.3)13.0 (8.2-16.3)13.0 (8.2-16.3)0.210 Hemoglobin, g/L122.0 (110.3-136.0)125.0 (110.5-137.5)120.0 (107.3-130.0)0.589 Albumin, g/L35.8 (32.9-38.8)35.3 (32.0-39.5)36.7 (29.3-39.5)0.969Complications, n (%) Disturbance of water and electrolyte63 (71.6)20 (74.0)11 (73.3)0.085 Rhabdomyolysis24 (27.3)6 (22.2)4 (26.7)0.003 Myocardial damage49 (55.7)11.0 (40.7)9 (60.0)0.008 Disseminated intravascular coagulation40 (45.5)9 (33.3)6 (40.0)0.010 Acute respiratory distress syndrome46 (52.3)9 (33.3)6 (40.0)0.012 Acute kidney injury52 (59.1)15 (55.6)10 (66.7)0.024 Acute liver function impairment52 (59.1)15 (55.6)10 (66.7)0.024 Central nervous system damage48 (54.5)15 (55.6)9 (60.0)0.019Treatments Use of continuous renal-replacement therapy, n (%)7 (8.0)3 (11.1)2 (13.3)< 0.001 Length of ICU stay, median (IQR), d1.0 (1.0-2.0)1.0 (1.0-1.0)2.0 (1.0-3.0)0.015Clinical outcomes at data cutoff, n (%) Hospital discharge44.0 (50.0)11.0 (40.7)5.0 (33.3)0.400 Death16.0 (18.2)2.0 (7.4)4.0 (26.7)0.240 Still hospitalization19.0 (21.6)13.0 (48.1)5.0 (33.3)0.025 Transferred to another hospital9.0 (10.2)1.0 (3.7)1.0 (6.7)0.5471The denominators of patients who were included in the analysis are provided if they differed from the overall numbers in the group. Percentages may not total 100 because of rounding.2Nausea/vomiting, diarrhea, flatulence, or bloody stools are defined as gastrointestinal symptoms.IQR: Interquartile range; GCS: Glasgow coma scale; NRS: Nutrition risk screening; GI: Gastrointestinal; ICU: Intensive care unit.As we observed that the onset of GI symptoms was significantly different between patients, we further divided patients with GI symptoms into two categories: Those with GI symptoms on ICU admission and those with GI symptoms developed during ICU stay. The patient characteristics of both groups are shown in Table 6. The patients who had GI symptoms on admission were younger (vP = 0.050), had a higher BMI (22.7 vs 21.1, wP = 0.050), and had a lower nutrition risk screening (NRS-2002) score on admission (3.0 vs 4.0, xP = 0.009) than had those who developed symptoms later on. Patients who had less GI symptoms on admission had a lower number of comorbidities, including diabetes (1/68, 1.5% vs 9/64, 14.1%, yP = 0.009), but more complications, including haemorrhage of the digestive tract (23/68, 33.8% vs 12/64, 18.8%) and disseminated intravascular coagulation (38/68, 55.9% vs 24/64, 37.5%). Nevertheless, there is no difference in mortality and ICU length of stay.Table 6Heat stroke patient characteristics according to the time of onset of gastrointestinal symptoms GI symptoms1 P value On admission (n = 68) Developed in ICU stay (n = 64) Characteristic Age, median (IQR), yr67.0 (57.0-76.0)70.0 (64.0-80.0)0.050 Female sex, n (%)28 (41.2)32 (50.0)0.310 BMI, median (IQR), kg/m222.7 (20.2-24.8)21.1 (20.0-23.3)0.050 GCS score at ICU admission, median (IQR)5.0 (3.0-8.0)5.0 (3.0-7.0)0.814 NRS score at ICU admission, median (IQR)3.0 (3.0-4.5)4.0 (3.0-6.0)0.009Fever on admission Patients, n (%)68 (100.0)64 (100.0)— Temperature, median (IQR), °C41.0 (40.0-42.0)41.0 (40.0-41.3)0.265Complaints and symptoms on admission, n (%) Fever54 (79.4)39 (60.9)0.020 Altered mental state or behavior25 (36.8)30 (46.9)0.239 Dry skin or excessive sweating13 (19.1)7 0.190 Rubefaction5 (7.4)6 (9.4)0.674 Fast pulse27 (39.7)13 (20.3)0.015 Polypnea24 (35.3)14 (21.9)0.089 Headache1 (1.5)2 (3.1)0.524 Syncope49 (72.1)36 (56.2)0.058 Other20 (29.4)7 0.009Coexisting disorder, n (%) Diabetes1 (1.5)9 (14.1)0.009 Hypertension15 (22.1)21 (32.8)0.166 Chronic obstructive pulmonary disease11 (16.2)10 (15.6)0.931 Chronic cardiac insufficiency5 (7.4)11 (17.2)0.084 Hepatitis B infection1 (1.5)0 (0.0)0.330 Cancer1 (1.5)2 (3.1)0.524 Chronic renal disease0 (0.0)2 (3.1)0.142 Immunodeficiency1 (1.5)2 (3.1)0.524Laboratory findings PaO2/FiO2 ratio240.0 (141.8-316.0)217.0 (165.0-285.8)0.775 White-cell count, 109/L12.1 (7.8-16.2)11.8 (8.2-14.2)0.667 Lymphocyte count, 109/L2.1 (1.0-3.3)1.2 (0.6-2.0)0.023 Platelet count, 109/L90.0 (60.5-120.3)98.5 (67.8-150.5)0.256 Hemoglobin, g/L126.0 (112.0-135.5)118.5 (103.5-137.5)0.106 Albumin, g/L36.5 (33.3-40.0)35.0 (32.1-38.6)0.093Other findings, median (IQR) C-reactive protein, mg/L5.0 (0.5-10.0)5.0 (1.0-14.9)0.605 Procalcitonin, ng/mL1.4 (0.4-8.2)9.3 (2.2-29.3)0.055 Lactate dehydrogenase, U/L465.0 (314.3-810.3)382.5 (282.8-537.8)0.263 Aspartate aminotransferase, U/L123.9 (58.3-272.3)107.2 (44.0-268.3)0.152 Alanine aminotransferase, U/L46.5 (26.2-112.4)48.0 (27.1-89.5)0.247 Total bilirubin, μmol/L18.5 (12.8-25.9)15.6 (10.1-23.0)0.211 CK-Mb, U/L10.1 (2.9-33.1)8.7 (3.3-41.0)0.214 Creatinine, μmol/L131.1 (105.3-182.8)137.0 (97.5-171.0)0.628 D-dimer, mg/L3.9 (2.5-11.5)4.6 (1.9-12.7)0.524Minerals, median (IQR), mmol/L Sodium132.0 (129.0-137.0)133.9 (128.5-136.8)0.959 Potassium3.3 (3.0-3.9)3.5 (2.9-3.8)0.849 Lactate4.2 (3.1-5.5)3.6 (2.4-5.3)0.649Complication, n (%) Disturbance of water and electrolyte51 (75.0)44 (68.8)0.424 Rhabdomyolysis21 (30.9)13 (20.3)0.165 Myocardial damage40 (58.8)30 (46.9)0.122 Disseminated intravascular coagulation38 (55.9)24 (37.5)0.034 Acute respiratory distress syndrome39 (57.4)27 (42.2)0.082 Acute kidney injury45 (66.2)38 (59.4)0.419 Acute liver function impairment41 (60.3)40 (62.5)0.795 Central nervous system damage42 (61.8)30 (46.9)0.086Clinical outcomes at data cutoff, n (%) Hospital discharge35 (51.5)29 (45.3)0.479 Death17 (25.0)9 (14.1)0.114 Still hospitalization13 (19.1)20 (31.3)0.108 Transferred to another hospital3 (4.4)6 (9.4)0.2581Nausea/vomiting, diarrhea, flatulence, or bloody stools are defined as gastrointestinal symptoms.IQR: Interquartile range; CK-Mb: Creatine kinase; GI: Gastrointestinal; ICU: Intensive care unit; BMI: Body mass index.We divided patients who developed GI symptoms during their ICU stay into the early-onset (< 3 d of ICU stay) and late-onset groups (≥ 3 d of ICU stay) groups. As shown in Table 7, there was a significant statistical difference in EN support between the two groups. Fewer patients received EN support in the early-onset than in the late-onset group (29/41, 70.7 vs 22/23, 95.7%, zP < 0.001). The early-onset group received less EN calorie [752.0 kcal/d (IQR: 500.0–1007.5) vs 1292.0 kcal/d (IQR: 750.0-1560.0)], protein [20.0 g/d (IQR: 17.6–32.5) vs 28.0 g/d (IQR: 15.0–57.0)], and EN volume [600.0 mL/d (IQR: 147.5–1000.0) vs 900.0 mL/d (IQR: 461.2–1500.0)]. Moreover, patients in the early-onset group received EN support for a shorter time than did those in the late-onset group [3.0 d (IQR: 1.8–5.0) vs 7.0 d (IQR: 4.0–11.0)].Table 7Heat stroke patient characteristics according to the time of onset of gastrointestinal symptoms1 Early onset, n = 41 Late onset, n = 23 P value Characteristic2 Age, median (IQR), yr69.0 (62.3-79.0)64.0 (56.0-76.0)0.856 Female sex, n (%)23 (56.1)8 (34.8)0.102Fever on admission Patients, n (%)41 (100.0)23 (100.0)— Temperature, median (IQR), °C41.0 (40.0-42.0)40.7 (39.6-41.3)0.338Body temperature on admission, n (%) Fever23 (56.1)15 (65.2)0.475 Altered mental state or behavior19 (46.3)11 (47.8)0.909 Dry skin or excessive sweating4 (9.8)3 (13.0)0.686 Rubefaction4 (9.8)2 (8.7)0.889 Fast pulse11 (26.8)2 (8.7)0.084 Polypnea9 (22.0)5 (21.7)0.984 Headache2 (4.9)0 (0.0)0.282 Syncope21 (51.2)13 (56.5)0.683 Other3 (7.3)4 (17.4)0.215EN support EN, n (%)29 (70.7)22 (95.7)< 0.001 Average EN calorie, median (IQR), kcal/d752.0 (500.0-1007.5)1292.0 (750.0-1560.0)< 0.001 Average EN protein, median (IQR), g/d20.0 (17.6-32.5)28.0 (15.0-57.0)0.003 Average EN volume, median (IQR), ml/d600.0 (147.5-1000.0)900.0 (461.2-1500.0)< 0.001 Duration of EN, median (IQR), d3.0 (1.8-5.0)7.0 (4.0-11.0)0.011Laboratory findings, median (IQR) White-cell count, 109/L10.6 (7.8-13.5)13.5 (8.6-15.8)0.351 Hemoglobin, g/L117.0 (106.0-128.0)128.5 (105.3-141.5)0.187 Albumin, g/L35.4 (33.7-39.2)34.4 (30.8-36.8)0.239Complications, n (%) Disturbance of water and electrolyte26 (63.4)17 (73.9)0.391 Rhabdomyolysis6 (14.6)6 (26.1)0.261 Hemorrhage of digestive tract8 (19.5)3 (13.1)0.511 Myocardial damage17 (41.5)12 (52.2)0.409 Disseminated intravascular coagulation14 (34.1)9 (39.1)0.691 Acute respiratory distress syndrome17 (41.5)6 (26.1)0.855Clinical outcomes at data cutoff, n (%) Hospital discharge17 (41.5)11 (47.8)0.622 Death7 (17.1)2 (8.7)0.355 Still hospitalization12 (29.3)8 (34.8)0.648 Transferred to another hospital4 (9.8)2 (8.7)0.8891Nausea/vomiting, diarrhea, flatulence, or bloody stools are defined as gastrointestinal symptoms.2The denominators of patients who were included in the analysis are provided if they differed from the overall numbers in the group. Percentages may not total 100 because of rounding.IQR: Interquartile range; EN: Enteral nutrition.To further explore the relationship between the duration of GI symptoms and prognosis of heatstroke patients, we stratified patients into those who had GI symptoms for more than 4 d and those who did for had GI symptoms for less than 4 d, as shown in Table 8. Patients with GI symptoms for at least 4 d had lower albumin levels (37.0 g/L vs 34.4 g/L, P = 0.310) and more complications, including disseminated intravascular coagulation (27.8% vs 54.2%, P = 0.007) and acute respiratory distress syndrome (12.0% vs 54.2%, P < 0.001). They also showed higher recovery rates than did those who had symptoms for more than 4 d (56.3% vs 27.8%, P = 0.004).Table 8Heat stroke patient characteristics according to duration of gastrointestinal symptoms1 Last < 4 d, n = 96 Last ≥ 4 d, n = 36 P value Characteristic Age, median (IQR), yr70.0 (61.5-78.3)67.0 (59.0-76.0)0.659 Female sex, n (%)43 (44.8)17 (47.2)0.803 Body temperature on admission Patients, n (%)96 (100.0)36 (100.0)- Temperature, median (IQR), °C41.0 (40.0-41.5)41.0 (40.0-42.0)0.948Complaints and symptoms on admission, n (%) Fever69 (71.9)24 (66.7)0.559 Altered mental state or behavior39 (40.6)16 (44.4)0.692 Dry skin or excessive sweating16 (16.7)4 (11.1)0.428 Rubefaction9 (9.4)2 (5.6)0.889 Fast pulse32 (33.3)8 (22.2)0.767 Polypnea31 (32.3)7 (19.4)0.147 Headache2 (2.1)1 (2.8)0.811 Syncope67 (69.8)18 (50.0)0.034 Other19 (19.8)8 (22.2)0.758Laboratory findings, median (IQR) White-cell count, 109/L11.0 (7.5-14.2)7.3 (9.2-16.3)0.062 Hemoglobin, g/L122.0 (110.5-135.5)118.0 (106.0-132.5)0.941 Albumin, g/L37.0 (33.3-39.7)34.4 (31.1-36.4)0.031Complications, n (%) Disturbance of water and electrolyte69 (71.9)20 (55.6)0.075 Rhabdomyolysis28 (29.2)6 (16.7)0.144 Hemorrhage of digestive tract28 (29.2)7 (19.4)0.26 Myocardial damage50 (52.1)20 (55.6)0.722 Disseminated intravascular coagulation27 (27.8)20 (54.2)0.007 Acute respiratory distress syndrome12 (12.0)20 (54.2)< 0.001Clinical outcomes at data cutoff, n (%) Hospital discharge54 (56.3)10 (27.8)0.004 Death17 (17.7)9 (25.0)0.348 Still hospitalization21 (21.9)12 (33.3)0.176 Transferred to another hospital4 (4.2)5 (13.9)0.0481Nausea/vomiting, diarrhea, flatulence, or bloody stools are defined as gastrointestinal symptoms.IQR: Interquartile range.DISCUSSIONIn this retrospective, multi-center study, we reported the incidence of GI manifestations among critically ill adult patients with heatstroke admitted to ICUs in the Sichuan Province, China. Our data demonstrated that patients with GI symptoms had a significantly longer ICU stay compared with those without. As a manifestation of systemic organ damage in heatstroke, the appearance of GI symptoms affect patients’ EN therapy outcomes. Patients with older age and a lower GCS score on admission were more likely to experience GI symptoms. Our study provides valuable real-world evidence regarding the associations between heatstroke and GI symptoms with, to our knowledge, the highest number of patients from multiple centres to date.Conventionally, critically ill patients with have GI dysfunction; however, there is little evidence supporting this phenomenon among heatstroke patients. Due to the lack of standardization of the diagnostic and therapeutic approaches, in this study, we evaluated the GI tract according to its symptoms and found that 18.5% of patients with heatstroke suffered from said symptoms during their stay. Compared with other non-heat stroke critically ill patients, the incidence of GI symptoms in our cohort is relatively low[9,10]. This is partly due to the fact that our study only used symptoms to evaluate GI function, though other high-incidence studies generally included physical examination, including that for bowel sounds, for comprehensive evaluation. When comparing the same symptoms, such as vomiting, between patients with heatstroke and those in the ICU, we observed that the incidence of GI symptoms in heatstroke patients is still lower than that of patients in the general ICU. One reason behind this is that heatstroke patients do not have GI structural damage from the perspective of pathogenesis, but patients in the general ICU comprise those who underwent abdominal surgery, that is, those who already have GI structural disorders. Another reason is that our study only assessed GI symptoms without other indicators such as physical examination, which may have led to the underestimation of the incidence of GI dysfunction. Nevertheless, our research suggests that GI injury is an important high-incidence mani-festation of organ failure among heatstroke patients.Our study found that heatstroke patients with older age and lower GCS score were more likely to experience GI symptoms. Multiple clinical studies had described risk factors for GI dysfunction in critically ill patients, including older age, larger BMI, lower Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores, surgical laparotomy, and use of mechanical ventilation, analgesic sedation, and vasopressors[9-11]. Similar to other studies, our study also observed that older patients were more likely to experience GI symptoms after heatstroke. Of note, our study found that the degree of nervous system damage, as quantified by the GCS score, is also related to the occurrence of GI dysfunction. This may be because heat damages the enteric nervous system, as well as the central nervous system. Moreover, patients with lower GCS scores were more likely to receive mechanical ventilation and vasopressors, posing an impact on the intestinal blood supply and, consequently, possibly leading to GI failure. The causes of GI dysfunction caused by heatstroke warrant further research.We also observed that the presence of GI symptoms may affect EN support therapy. In our study, we found heatstroke patients who did not receive EN therapy within 48 h after ICU admission experienced more GI symptoms with more complications, longer ICU stay, and higher ICU mortality. The emergence of GI symptoms is the reason why EN cannot be started. Simultaneously, the failure to start EN support is also a reason for the deterioration of GI function. We also observed that a considerable proportion of patients with heat stroke still cannot implement total EN within 2 wk, suggesting that, for patients with heatstroke, further research to develop individualized nutrition support strategies is warranted.We also performed various subgroup analyses to discuss different GI symptoms and whether their timing and duration had an impact on patient prognosis. First, we found that patients with different GI symptoms have different clinical features. Different symptoms may indicate that the severity of heatstroke in these patients varies, and whether this reflects their prognosis to some extent requires further study. The onset of GI symptoms in patients also differed. Overall, the earlier the GI symptoms appeared, the severer the patient's condition was. At the same time, due to GI symptoms, such patients could not tolerate EN or could not meet EN standards, further impairing their GI function and forming a vicious circle. Better approaches for EN support in these patients are warranted. Finally, we discussed the duration of the patient's GI symptoms. This was the same as we previously realized: The longer the duration of GI symptoms, the worse their prognosis. These patients were unable to start EN therapy early on. In contrast, they were more likely to have GI microcirculation disorders and damage to the intestinal barrier.Currently, the cause of GI dysfunction caused by heatstroke is not particularly clear. Several reports have documented increased intestinal permeability during exercise with and without heat stress[4,12,13]. A murine model of classic heatstroke that induced a body core temperature as high as 42.7 °C showed considerable gut histological injury[14,15]. Studies have shown that one of the important mechanisms of heatstroke is the excessive opening of intestinal tight junctions, destruction of intestinal cell structure and function, increase in intestinal mucosal permeability, and introduction of endotoxin into the blood[16,17]. One of the most frequently mentioned mechanisms of how heatstroke causes GI symptoms is the leaky gut hypothesis. Our results also suggest that while heat can cause changes in the state of consciousness caused by central nervous system damage, it may also cause damage to the enteric nervous system, thereby causing GI dysfunction. Such inferences need further research to confirm in the future.The retrospective design of this study offers several benefits, including a high quality of data and a large number of patients. Our study provides a real-world representation of the current clinical practices for heatstroke in a mixed population of critically ill adult patients treated in ICUs in Sichuan Province, China. The patient sample size provides a robust representation of the target population, increasing the generalizability of our findings. Overall, our study provides important insights into the prevalence of GI symptoms among critically ill heatstroke patients and its relationship with risk factors and clinical outcomes. The findings of this study have important implications for the management and care of critically ill patients with heatstroke. Previous literature has demonstrated the vulnerability of the digestive tract to abnormal conditions, including hypoxia and elevated temperatures[4,12,13,18,19]. Studies have also indicated that most patients experience some form of GI symptoms during intense physical activity and elevated body temperature. Our study on GI symptoms following heatstroke incorporates risk factors and provides a comprehensive understanding of the subject, thereby supplementing previous research.Nevertheless, this study had some limitations. First, the use of GI symptoms to respond to GI dysfunction is one-sided. Another limitation is the exclusion of the most critically ill patients who had already passed away and those admitted to general wards. While this selection criterion was a necessary aspect of the research program, it is possible that the inclusion of these patients would not have greatly impacted the overall prognosis, as previously discussed. Our study was an observation of symptoms and did not address possible effects of treatment on GI function. Additionally, there is a high rate of missed diagnoses due to a lack of awareness of heatstroke in remote mountainous areas and the inadequate identification of heatstroke in a timely manner.CONCLUSIONThe incidence of GI symptoms among heatstroke patients admitted to the ICU was reportedly 18.5% in our study. Patients who are older and with a lower GCS score on admission have an increased likelihood of developing GI symptoms. Heatstroke patients with GI symptoms found it more difficult to tolerate EN therapy than did those without. Patients with GI symptoms were found to have a higher incidence of complications. The earlier the GI symptoms appeared and the longer the duration of GI symptoms, the more difficult it was for patients to tolerate EN, and the worse the predicted prognosis.ARTICLE HIGHLIGHTSResearch backgroundExtreme heat exposure is a growing health problem. The effects of heat on the gastrointestinal tract is unknown.Research motivationIt was intended to summarize the effects of heat on the gastrointestinal (GI) tract of intensive care unit (ICU) patients.Research objectivesThis study aimed to assess the incidence of GI symptoms associated with heatstroke and its impact on outcomes.Research methodsWe conducted a retrospective, multi-center, observational cohort study to analyze outcomes between patients.Research resultsThe timing and duration of gastrointestinal symptoms affects heatstroke patient's prognosis and enteral nutrition (EN) therapy. The status of EN therapy is related to heatstroke patients’ outcomes. Advanced age and low Glasgow coma scale (GCS) scores are risk factors for gastrointestinal symptoms in heatstroke patients.Research conclusionsThe GI manifestations of heatstroke are common and appear to impact clinically relevant hospitalization outcomes.Research perspectivesThis was a retrospective, multi-center, observational cohort study that involved patients admitted to 83 ICUs located in 16 cities in the Sichuan Province, China between June 1 and October 31, 2022. Results showed older heatstroke patients with a lower GCS score on admission were more likely to experience GI symptoms, which had statistical difference. Clinicians should pay attention to the time at which heatstroke patients started manifesting gastrointestinal symptoms, as well as the duration of said symptoms, to ensure that patients are timely treated with the proper EN therapy and have the best prognosis possible.ACKNOWLEDGEMENTSThe authors would like to acknowledge all investigators who participated in this trial. A full list of members who participated in this study from the Heatstroke Research Group in Southwest China: Wei Tang (Dazhou Central Hospital), Yi-Song Ren (Chengdu Pidu District Traditional Chinese Medicine Hospital), Qiong-Lan Dong (The Third People's Hospital of Mianyang), Mao-Juan Wang (Deyang People's Hospital), Liang-Hai Cao (Yibin Second People's Hospital), Xian-JunWang (The Second People's Hospital of Jiangyou City), Lang Tu (Shehong People's Hospital), Guang-Hong Ye (Anyue County Hospital of Traditional Chinese Medicine), Li Chen (Guang'an District People's Hospital, Guang'an City), An-Qin Li (The Fourth People's Hospital of Jiangyou City), Zhi-Fu Liu (Jingyan County People's Hospital), Xiao-Lin Han (Pengzhou Traditional Chinese Medicine Hospital), Yu Dai (Mianyang Anzhou District People's Hospital), Xiong Yang (Langzhong People's Hospital), Jun Chen (Jianyang People's Hospital), Xian-Peng Qiu (The Fourth People's Hospital of Zigong City), Sen-Zhong Cheng (Zhongjiang County People's Hospital), Bo Qi (Sichuan Jiangyou 903 Hospital), Pan Yang (Meishan Cancer Hospital), Xu-Ting Deng (Luxian People's Hospital), Qin-Ya Ding (Linshui County Traditional Chinese Medicine Hospital), Chun-Mei Huang (Nanjiang County Traditional Chinese Medicine Hospital), Fang-Pei Zhang (Hejiang County Hospital of Traditional Chinese Medicine), Hong-Yu Yang (Mianyang Fulin Hospital), Xiao-Cui Wang (Deyang Hospital of Integrated Traditional Chinese and Medicine), Jia-Jin Li (Mianyang People's Hospital), Xiao-Jiao Liu (Guanghan People's Hospital), Xue-Mei Ye (Meishan People's Hospital), Fang Wu (Ziyang Lezhi County People's Hospital), Tao Ye (Leshan Traditional Chinese Medicine Hospital), Zhuo Tang (Zizhong County Traditional Chinese Medicine Hospital), Liang He (Pyeongchang County Traditional Chinese Medicine Hospital), Jian Gong (Ziyang People's Hospital), Hong-Xu Chen (Leshan Traditional Chinese Medicine Hospital); Qiong-Hua Hu (Mianyang Central Hospital); Zhen Wang (Shifang Second Hospital); Rong Hu (Xingwen County People's Hospital); You-Wei Li (The Third People's Hospital of Anyue County); Lin Hou (Bazhong Enyang District People's Hospital); Zhuo Chen (The Third People's Hospital of Yibin); Hua-Qiang Shen (Bazhong Central Hospital); Jian Wang (Guang'an People's Hospital); Ping Fang (Neijiang Second People's Hospital); Yu Liu (Lezhi County Traditional Chinese Medicine Hospital in Sichuan Province); Yan Luo (Luzhou Traditional Chinese Medicine Hospital); Wei-Peng Xu (Fushun County People's Hospital); Chun-Lin Zhang (Yibin Traditional Chinese Medicine Hospital); Xiao-Qi Tang (Ziyang Yanjiang District People's Hospital); Yu-Liu (Lezhi County People's Hospital); Jing-Mei Yang (Qingshen County People's Hospital); Xin-Pin Yang (Pyeongchang County People's Hospital, Bazhong City); Liang Chen (Gaoxian People's Hospital); Guang-Wei Yang (Huaying People's Hospital); Chun-Hong Tang (Jintang County First People's Hospital); Shi-Hao Ren (Zitong County People's Hospital); Jie Zhao (Ziyang People's Hospital); Pin Li (Shehong Hospital of Traditional Chinese Medicine); Ya Qiang (Mianyang Traditional Chinese Medicine Hospital); Hong-Zhou Wang (Sichuan Science City Hospital); Sen-Zhong Cheng (Sichuan Mianyang 404 Hospital); Song-Lin Wu (Affiliated Hospital of Southwest Medical University); Lu Liu (Luzhou Xuyong County People's Hospital); Xue-Cha Li (Neijiang Dongxing District People's Hospital); Shi-Hu Zhang (Ya'an Yucheng District People's Hospital); He Lin (Zizhong County People's Hospital); Xiao-Dong Feng (Mianzhu People's Hospital); Jun Liang (Zitong County People's Hospital of Mianyang City).

PMC

Indian Journal of Plastic Surgery : Official Publication of the Association of Plastic Surgeons of India

PMC10049807

12-11-2022

10.1055/s-0042-1758452

Reconstruction of the Axillary Region after Excision of Hidradenitis Suppurativa: A Systematic Review

Amendola Francesco, Cottone Giuseppe, Alessandri-Bonetti Mario, Borelli Francesco, Catapano Simone, Carbonaro Riccardo, Riccardi Francesca, Vaienti Luca

Introduction Hidradenitis suppurativa (HS) is a chronic, debilitating, recurrent, auto-inflammatory disease of the pilosebaceous units of the skin. The axillary region is the most affected anatomical site and its reconstructive options include skin grafts, local random plasties, regional axial flaps, and regional perforator flaps. The main aim of this systematic review is to identify the best surgical technique for axillary reconstruction in the context of HS, in terms of efficacy and safety. Methods We adhered to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) throughout the whole review protocol build-up. The literature search was performed using MEDLINE, Embase, and Cochrane library databases, updated to March 2021. Quality was assessed for each study, through the National Institutes of Health Quality Assessment Tool. Results A total of 23 studies were included in the final analysis. We reviewed a total of 394 axillary reconstructions in 313 patients affected by HS Hurley Stage II or III. Skin grafts were associated with the highest overall complication rate (37%), and highest rate of reconstruction failure (22%). Between thoraco-dorsal artery perforator flap, posterior arm flap, and parascapular flap, the latter showed fewer total complications, recurrences, and failures. Conclusion Regional axial flaps should be considered as the best surgical approach in the management of advanced HS. The parascapular flap emerges as the most effective and safest option for axillary reconstruction. Local random flaps might be considered only for selected minor excisions, due to the higher risk of recurrence. The use of skin grafts for axillary reconstruction is discouraged.

Introduction Hidradenitis suppurativa (HS) is a chronic, debilitating, recurrent, inflammatory disease of the folliculo-pilo-sebaceous units of the skin, with an estimated prevalence of 1 to 4% in the general population. The axillary, the perineal, and the inframammary regions appear to be frequently involved. 1 2 The chronic and recurrent formation of abscesses and sinus tracts worsens the quality of life of affected patients, with both physical and psychological consequences. 3 4 Disease activity is classified following the Hurley score. 1 Medical therapy, including oral antibiotics or immunomodulating drugs, is usually the first-line treatment. Nevertheless, HS Hurley stage III usually presents with extensive cases refractory to the noninvasive therapies, requiring large excisions of the entire affected area, 5 with surgical margins extending beyond the clinical borders of disease activity. 6 While wide excisions offer a good treatment in the long term, the reconstruction of the excised area represents a challenge in the short term. HS most commonly presents in the axillary region 7 and several reconstructive options have been proposed in the literature, including skin grafts, local random flap, regional axial flaps, regional perforator flaps, and secondary intention healing. The ideal reconstructive option should be thin, large, and pliable, to recreate the concavity of the axillary region without impairing the shoulder motion. Skin grafts are commonly large and thin; however, they generally lack elasticity and retract over time. Regional axial flaps or perforator flaps are large and pliable, but usually fail to recreate the concavity, ending up in bulky reconstructions. Local random flaps maintain a similar texture to the axillary region; but they are not indicated in wide excisions. Currently, a gold standard technique for axillary reconstruction after HS excision has not been recognized yet. 3 The main aim of this work is to systematically review the literature of the past 40 years, to identify the best surgical technique for axillary reconstruction in terms of efficacy (lack of recurrence) and safety (lack of complications).Materials and Methods We adhered to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) throughout the whole review protocol build-up. 8 The review was not registered in any systematic review and meta-analysis registry. However, no study investigating axillary reconstruction in HS was identified in PROSPERO database. Research question and PICO: What is the best surgical technique for reconstruction of the axilla after excision of the area affected by HS in terms of efficacy and safety among all those described in literature?Population: Patients suffering from axillary hidradenitis, requiring immediate surgical reconstruction.Intervention and comparison: Skin grafts, local random flap (defined as pure-skin flap with random vascularization), regional axial fasciocutaneous flaps, and regional perforator flaps.Outcomes: Efficacy in terms of recurrence of hidradenitis, safety of the techniques expressed in terms of complications (recurrence, minor healing delay, infection, necrosis/failure). Failure was defined as a necrosis of the flap/graft requiring reoperation for achieving the coverage of the defect.Literature research: MEDLINE, Embase, and Cochrane library were searched. We searched MeSH terms “axilla” OR “hidradenitis” AND “reconstruction” OR “flap” OR “graft.”Inclusion criteria: Patient with axillary HS Hurley stage II/III, clear description of patient characteristics and outcomes, follow-up length clearly stated. Only English language studies were included.Exclusion criteria: Patients with HS involving areas other than axillary region; studies describing patients affected by hidradenitis in which data about axillary reconstruction were not extractable, studies not clearly reporting the grade or a histological or clinical diagnosis of HS; HS Hurley stage I requiring simple excision without reconstruction, follow-up length not clearly stated. Study selection and data extraction: analysis of the literature was performed by two coauthors independently and then matched. For every study, quality was assessed with National Institutes of Health (NIH) Quality Assessment Tool, 9 excluding from our research those with a score < 2. The score used for analytic studies was converted in a 1 to 9 scale, to be comparable with the case series scores. There were not disagreements among the interviewers regarding the quality of the studies included. Results Overall, 2447 articles were retrieved from the preliminary search, updated to March 2021. The flowchart of the study selection process is outlined in Fig. 1 . After removal of duplicates, 268 articles were screened by title and abstract. Only 57 articles were assessed for full-text eligibility, and among those, 22 studies were eventually included by criteria defined in the protocol ( Table 1 ). 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Of the 35 articles excluded after full text screening, the majority was rejected due to follow-up length not clearly stated or patient characteristics not well described. Mean quality score was 6.4, ranging from 2 to 9, according to NIH Quality Assessment Tool. Fig. 1 Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Flow Diagram. Adapted from Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group . P referred R eporting I tems for S ystematic Reviews and M eta- A nalyses: The PRISMA Statement. PLoS Med 6: e1000097. For more information, visit www.prisma-statement.org . Table 1Characteristics of the included studiesAuthors (year)DesignNIH assessment toolNumber of patientsNumber of axillaeTreatment Elboraey et al 12 CS868Propeller flap on thoracodorsal or intercostal system Sirvan et al 30 CS71417Posterior arm flap Marchesi et al 13 CS8121715 TDAP, 2 MSLD Elgohary et al 11 Cohort92028TDAP Nail-Barthelemy et al 10 CS91317TDAP Schmidt et al 27 CS62031Posterior arm flap Ching et al 28 CS545Inner arm perforator flap Wormald et al 26 Cohort91515TDAP1212Skin graft Alharbi et al 29 CS71012Inner arm perforator flap Nesmith et al 25 CS41115Parascapular Flap Hallock 24 CS523VY thoracodorsal perforator Flap Jandali et al 22 CS559TDAP Alharbi et al 31 CS71616Limberg random flap55Parascapular flap Gonzaga et al 23 CS648Skin graft Busnardo et al 21 Cohort81224TDAP Varkarakis et al 20 CS41521Limberg random flap Altmann et al 19 CS52025Limberg random flap Geh and Niranjan 18 CS647VY random flap Schwabegger et al 17 CS278LTAP Soldin et al 16 Cohort81616Skin graft3636Limberg random flap77Parascapular flap Elliot et al 14 CS81722Posterior arm flap Morgan et al 15 Cohort81010Skin graftAbbreviations: CS, case series; LTAP, lateral-thoracic artery perforator; MSLD, muscle sparing latissimus dorsi; NIH, National Institutes of Health; TDAP, thoraco-dorsal artery perforator. A total of 394 axillary reconstructions, in 313 patients affected by AH at advanced stages, have been reviewed. All the papers included in the study deal with patients at Hurley stage III, except for Elgohary et al who included patients at Hurley stage II or III. 11 The median age was 32 years with a median duration of disease of 72 months before intervention. The median follow-up was 16 months. The median axillary area affected measured 85 cm 2 . The areas were reported in each study multiplying the height per the width of the defect after the primary excision ( Table 2 ). Only seven of the included studies described patients' anamnestic information such as tobacco use. 10 12 13 20 23 30 31 However, according to the available data, 57% of the patients reported to be active smokers. Table 2Patients' characteristicsSGLRFRAFPFNumber of treated axillae46113107143Mean patient's age (years)32,434,937,635 Defect width range (cm 2 ) 88 15 –437,5 24 85 25 39,5 31 –487,9 26 58 10 –160,41 22 From diagnosis to surgery range (months) 36 24 –42 17 42 17 –84 19 42 17 –112,8 14 123,6 30 Mean follow-up (months)2112,12617Abbreviations: LRF, local random flap; PF, perforator flap; RAF, regional axial flap; SG, skin graft. Among the various surgical techniques, perforator flaps were used in 146 cases (37%), local random flaps in 105 cases (27%), regional axial flaps and skin grafts in 97 (24%) and 46 (12%) cases, respectively. Among perforator flaps the most used resulted to be the thoraco-dorsal artery perforator flap (TDAP) ( n = 108), while the most used regional axial flap was the posterior arm flap ( n = 70) followed by parascapular flap ( n = 27). Among the local random flaps, the Limberg flap ( n = 98) was the most described technique. The overall complication rate was 71 (17%). Skin grafts were associated with the highest complication rate ( n = 17, 37%), followed by local random flaps ( n = 22, 19%) and perforator flaps ( n = 20, 14%). The regional axial flaps demonstrated the lowest complication rate ( n = 12, 11%). Local random flaps demonstrated the highest rate of recurrence ( n = 9, 8%). Perforator flaps, regional axial flaps, and skin grafts demonstrated comparable rates of recurrence ( n = 3, 1, and 1, respectively). Skin grafts resulted in the highest rate of minor healing delays ( n = 7, 13%) and failure ( n = 10, 22%). Low incidence of infection was reported for all the reconstructive techniques included in the study. Details about specific and global complication rates are shown in Table 3 . Table 3Results of each type of reconstructionSGLRFRAFPFTotalNumber of treated axillae4610597146394Total complications17 (37%)22 (19.5%)12 (11%)20 (14%)71 (17.4%)Recurrence1 (2.2%)9 (8%)1 (1%)3 (2%)14 (3.4%)Minor healing delay6 (13%)7 (6.2%)9 (8.4%)12 (8%)34 (8.3%)Infection001 (1%)2 (1.4%)3 (0.7%)Failure10 (22%)6 (5.3%)2 (1.9%)5 (3.5%)23 (5.6%)Abbreviations: LRF, local random flap; PF, perforator flap; RAF, regional axial flap; SG, skin graft.No clear differences were noted between perforator flaps and regional axial flaps, in terms of total complications, specific complications, and recurrences. However, both regional axial flaps and perforator flaps showed reduced failure rate compared with skin grafts (3% and 2 vs. 22%), but no remarkable differences in terms of recurrences, infections, or minor healing delays.Among the most used singular techniques, the parascapular flap (7% of overall complications, none of recurrences and failures) proved to be associated with fewer complications than the TDAP (13% overall complications, 3% recurrences, 4% failure) and the posterior arm flap (15% overall complications, 1% recurrences, 2% failure).Discussion HS is a chronic inflammatory disease of the follicular pilo-sebaceous unit. 32 It usually presents with painful nodules, which may be complicated by abscesses, leading to sinus tract formation, scarring, and fibrosis. 16 32 Surgical management demonstrated to significantly improve the quality of life in patients affected by advanced HS. 33 However, no actual gold standard reconstructive technique has been indicated after local excision of the HS affecting the axillary area. Following our systematic review of the literature, a total of 409 axillae were reconstructed with different techniques, ranging from skin graft to perforator flaps. We identified several small case series and few comparative cohort studies, with no large high-quality datasets. Different surgical techniques demonstrated to be effective. Local random flaps showed the highest rate of disease recurrence, confirming the limited role for minor surgical excision in the treatment of axillary HS. Therefore, wide local excision of the entire hair bearing area should be considered as the most effective surgical approach. 16 34 Skin grafts were associated with the highest overall complication rate, with high rate of failure of the reconstruction. Perforator and regional axial flaps were characterized by the lowest complication rates, thus being the safest techniques for axillary reconstruction. Perforator flaps were the most used reconstructive technique for advanced axillary HS. Among perforator flaps, the TDAP flap was the most frequently performed and it demonstrated acceptable complication and recurrence rates. 11 The TDAP flap includes the advantages of a similar texture, color, and thinness to the axillary area. It also allows the maintenance of the axilla's diamond shape, with low rate of bumps and distortions. 11 21 Furthermore, it can be used for extensive resection without long-term retractions. 11 21 However, Elgohary et al described a 10.71% prevalence of scar widening and 10% of donor site morbidity (hypertrophic scar or seroma). 11 The TDAP flap also requires a meticulous preoperative planning, with the mapping of the perforators, and a consistent operative time. 11 13 26 Wormald et al compared the use of TDAP with the skin graft for axillary reconstruction in patients affected by HS, and showed a significantly improved quality of life in the TDAP group. 26 Similarly, Busnardo et al demonstrated a significant increase in arm abduction and mobility of the arm and shoulder at 6 months follow-up using the TDAP flap after excision of severe axillary HS. 21 Regional axial flaps demonstrated the lowest overall complication rate in our study. Among the 107 regional axial flaps used for axillary reconstruction, the posterior arm flap was the most common, with a 15% of overall complication, 1% of recurrences, and 2% of failures. The pedicle of the flap is defined as the cutaneous branch of the artery supplying the medial head of the triceps muscle, which arises from either the brachial or deep brachial artery. 27 30 35 This flap is considered to be safe and feasible for some authors, which observed a constant anatomy and a vigorous blood supply. 27 30 Although donor site morbidity is considered low and comparable to a posterior brachioplasty, the donor site scar is usually visible when wearing short-sleeved T-shirts, differently from other reconstructive techniques, and be troublesome for some patients. 27 Moreover the posterior arm flap tends to be bulky in the axillary reconstruction due to the fact that the arm tissue is relatively thicker than the axillary tissue. 14 30 The TDAP flap showed similar outcomes of posterior arm flap, but with more recurrences and failures. The parascapular flap was the second most used regional axial flap, and demonstrated better outcomes compared with the posterior arm flap, with similar rate of complications and no recurrences or failures registered. Local random flaps were the second most used technique, but they resulted to be significantly associated with disease recurrences than the other techniques included in our study, mainly due to the associated smaller excisions. Varkarakis et al reported satisfying reconstructions using the Limberg local plasty. However, 9.5% of patients had a delayed restoration of shoulder motion requiring physical therapy. 20 Skin grafts were associated with the highest complication rate, reporting numerous failures and minor healing delays. Several authors discourage the use of skin graft for axillary reconstruction. Other than poor graft takes long recovery, skin grafts are often cosmetically unsatisfactory and often develop retractions leading to joint contractures, thus impairing the upper limb motion. 16 26 29 The extension of the defect guides the reconstructive option. In fact, HS affects only the hair bearing area of the axilla, not extending beyond the posterior axillary fold. Therefore, a regional flap from the scapular region is almost always feasible. Even if the reconstructive ladder imposes the use of local random flaps as the first choice for small defect, in this context small excisions were shown to be burdened by a higher risk of relapse. Thus, a wide excision of the entire axillary hair-bearing region is advisable to reduce recurrences. According to our analysis, regional axial flaps should be considered as the first choice in axillary reconstruction after HS demolition at advanced stages, because they demonstrated to be effective in preventing postexcision disease recurrence and to be associated with the lowest complication rate. The parascapular flap emerged as the most effective and safest option for axillary reconstruction. The second option should be the posterior arm flap, safer and cosmetically more acceptable than the TDAP flap. Based on the findings of our study, we discourage the use of skin grafts for axillary reconstruction.The main limits of our work include the overall low quality of evidence of the studies included in the review due to absence of randomized controlled trials. The majority of patients' data were obtained from case reports and small case series and therefore a formal metanalysis could not be performed. Further weakness of the available dataset is the lack of information about patients' comorbidities, with only seven of the included studies reporting anamnestic information of the treated patients, as well as the lack of evidence in patient-reported quality of life after every single reconstructive technique. We are also conscious of the possible unreliable assessment of the Hurley stage by the different groups.ConclusionBased on the available literature on axillary HS, perforator and regional axial flaps show better outcomes and low rates of complications. However, regional axial flaps have the most consistent safety and efficacy profile. Among regional axial flaps, the parascapular flap appears to be the most reliable and safe procedure.Due to the low quality of the studies available in literature and the lack of patients reported outcomes, further investigations are warranted before a determined surgical approach could be considered as the gold standard treatment option.

PMC

International Wound Journal

37849027

PMC10828731

10-17-2023

10.1111/iwj.14434

Effectiveness of nursing intervention in the operating room to prevent pressure ulcer and wound infection in patients undergoing intertrochanteric fracture: A meta‐analysis

Qiu Fei‐Fei, Huang Si‐Mei

AbstractIn this study, a meta‐analysis was conducted to comprehensively assess the effectiveness of nursing intervention in the operating room to prevent pressure ulcers and wound infections in patients with intertrochanteric fractures. A computerised search of PubMed, Cochrane Library, Embase, China National Knowledge Infrastructure (CNKI), VIP Database of Chinese Technical Periodicals, and Wanfang databases was performed to identify randomised controlled studies (RCTs) on the effectiveness of nursing intervention in the operating room for patients undergoing intertrochanteric fractures from the time of construction of the respective databases to June 2023. Two researchers independently searched and screened the literature, extracted information and performed quality assessments of the included literature. The meta‐analysis was performed using RevMan 5.4 software. Eighteen studies were finally included, including 1517 patients, with 757 in the intervention group and 760 in the control group. The results showed that nursing intervention in the operating room significantly reduced the incidence of postoperative pressure ulcers in patients with intertrochanteric femoral fractures compared to the control group (1.69% vs. 6.01%, odds ratio [OR]: 0.32, 95% confidence interval [CI]: 0.18–0.57, p < 0.001) and reduced the incidence of surgical site wound infection (1.00% vs. 6.15%, OR: 0.23, 95% CI: 0.11–0.50, p < 0.001). Current evidence suggests that nursing intervention in the operating room is superior to routine care in reducing the incidence of pressure ulcers and wound infections in patients with intertrochanteric fractures and that such interventions should be promoted for clinical use.

1INTRODUCTIONIntertrochanteric fracture is a fracture that occurs from the base of the femoral neck to just above the lesser trochanter and is one of the most prevalent types of fracture in the elderly. 1 , 2 , 3 According to one study, it is estimated that hip fractures will increase globally from 1.66 million in 1990 to 4.26 million in 2050 as life expectancy increases and the number of older people continues to rise. 4 Intertrochanteric fractures account for approximately 50% of all hip fractures. 5 These fractures are mostly caused by direct violence or indirect violence such as collision, fall, and so forth. 6 The reason they usually occur in elderly patients may be because most of the elderly people suffer from osteoporosis and have a significant loss of motor ability and observation skills, which makes the hip area likely to fracture after a fall. 7 Typical symptoms are pain in the hip and inability to stand and walk normally. 1 As society ages and the number of elderly people increase, peri‐trochanteric, intertrochanteric, and subtrochanteric fractures are also on the rise, and most of these patients require surgical treatment. 8 During the surgical treatment of intertrochanteric fractures, postoperative wound infections and pressure ulcers not only affect the therapeutic effect but also bring great mental pressure and economic burden to the patients. 9 , 10 At present, the main measure to prevent postoperative infections in orthopaedic surgery is the application of antimicrobial drugs, but the irrational use of antimicrobial drugs often leads to the increase of bacterial resistance and the burden of liver metabolism. 11 Moreover, in the treatment of intertrochanteric fractures, the length of the operation is usually more than 2 h; moreover, after the operation, the patient needs to be in a passive position in bed, and most the patients are elderly, which makes them more susceptible to pressure ulcers. 12 Therefore, it is important to seek reasonable preventive measures.Nursing intervention in the operating room is a nursing method guided by modern nursing concepts that are patient‐centred and closely linked to all aspects of nursing work. The following actions may be employed: during the nursing period, timely and effective solutions to the concerns of patients and their families may be implemented to improve the patient's doctor–patient cooperation in the operating room; in the process of surgical treatment, to strengthen the management of the operating room environment, goods, and personnel, strict implementation of the aseptic operation, active cooperation with the surgeon and anaesthesiologist, reasonable adjustment of the position, timely delivery of instruments, inquiry of the patient's feelings during the operation, and close monitoring the vital signs may be performed; and finally, postoperative observation of anaesthesia awakening may also be performed. After sending the patients back to the ward, the ward is handed over to the ward nurses, who emphasise the postoperative precautions and make return visits to observe the wounds to reduce the occurrence of wound infections, improve the rate of incision healing, and at the same time, increase the satisfaction of nursing care. 13 , 14 Ouyang et al. showed that nursing intervention in the operating room could significantly reduce the incidence of postoperative wound infection and shorten the healing and recovery times after minimally invasive proximal femoral nail antirotation (PFNA) treatment of intertrochanteric fractures. 15 Li and Feng also showed that operating theatre nursing intervention can improve surgical indexes to a certain extent, reduce the incidence of postoperative wound infections and pressure ulcer complications in intertrochanteric fracture of the femur, as well as alleviate patients' pain, improve joint function and postoperative quality of life and increase patient satisfaction. 16 In recent years, there have been many clinical studies on the effects of nursing intervention in the operating room on pressure ulcer and wound infection in patients undergoing intertrochanteric fracture, but the conclusions of the studies are not yet consistent. Therefore, this study aimed to assess the effect of nursing intervention in the operating room on pressure ulcers and wound infections in intertrochanteric fractures by means of a meta‐analysis, with the secondary aim of providing medical evidence for clinical practice.2MATERIALS AND METHODS2.1Literature searchA computerised search of the PubMed, Cochrane Library, Embase, Knowledge, VIP and Wanfang databases was used to find randomised controlled trials (RCTs) on the effectiveness of nursing intervention in the operating room for pressure ulcers and wound infection in patients undergoing intertrochanteric fractures from the time each database was built until June 2023. A literature search was conducted using the following keywords: (intertrochanteric OR intertrochanteric fracture OR pertrochanteric OR trochanteric OR extracapsular hip fractures OR intramedullary fixation OR cephalomedullary nail) AND (operating room nursing OR nursing of operating room OR operating room nursing management OR nursing management of operating room). The search was restricted to Chinese and English languages. To prevent omissions, the references of the included literature were queried and the auxiliary group hand‐searched the relevant journals.2.2Inclusion and exclusion criteriaThe inclusion criteria were as follows: study participants: surgical patients diagnosed with intertrochanteric fracture by imaging; interventions: intervention group for nursing intervention in the operating room and control group for the study of traditional routine care methods; outcomes: wound infection and pressure sores and study design: RCTs. The exclusion criteria were as follows: studies that did not meet the above inclusion criteria; duplicate publications; conferences, abstracts, reviews, case reports or expert opinions and studies in which the full text was not available or for which data were incomplete and contacting the authors was unsuccessful.2.3Data extraction and quality assessmentBased on the inclusion and exclusion criteria, the titles and abstracts of the retrieved literature were initially screened separately by two researchers who read the full text of the initially included literature and extracted the information. Different opinions in the literature were discussed, and if not resolved, the decision was discussed with a third researcher. Two researchers independently extracted data from the full text using a uniform data sheet, including the first author, year of publication, sample size, age and sex. The included studies were independently evaluated according to the Cochrane Handbook quality assessment criteria. 17 The evaluation included random sequence generation, allocation concealment, blinding of investigators and participants, blinding of outcome assessors, completeness of outcome data, selective reporting and other sources of bias. Each item was evaluated as ‘low’, ‘unclear’ or ‘high’ risk of bias. A formal literature quality assessment form was developed, and dissenting literature was discussed and, if unresolved, discussed and decided upon by a third researcher.2.4Statistical analysisMeta‐analyses were performed using RevMan 5.4 software provided by the Cochrane Collaboration. Odds ratios (ORs) and 95% confidence intervals (CIs) were used as the effect sizes for dichotomous variables. The mean difference (MD) and 95% CI were used as the effect sizes for continuous variable information. Heterogeneity of the included literature was tested according to I 2, when p 50% indicated that there was a great deal of heterogeneity, and meta‐analysis was carried out using a random effects model; when p > 0.1 and I2 < 50% indicated that there was no significant heterogeneity, the meta‐analysis was carried out using a fixed effects model. To evaluate the stability of the combined OR and MD in this study, the included studies were excluded individually from the sensitivity analysis. When the number of literature was 10 and above, funnel plots were used to assess the presence of publication bias.3RESULTS3.1Study selection and quality assessmentA flowchart of the literature screening is shown in Figure 1. According to the proposed search strategy, 382 documents were retrieved and imported into Endnote X9 literature management software, 179 duplicate documents were excluded, 137 were excluded by reading the titles and abstracts of the documents, and 66 documents were initially screened. After carefully reading the full text, 18 documents were finally included, 13 , 16 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 including 1517 patients, with 757 in the intervention group and 760 in the control group. The risk of bias of the included RCTs is shown in Figure 2. The basic characteristics of the included studies are shown in Table 1.FIGURE 1Selection process.FIGURE 2Risk of bias summary of the included studies.TABLE 1Characteristics of the included studies.AuthorYearNumber of patientsAge (years)Gender (male/female)InterventionControlInterventionControlInterventionControlCai2020303072.5 ± 4.9 (63–82)73.5 ± 4.9 (63–84)20/1017/13Chen2023404071.25 ± 6.0871.39 ± 6.2523/1726/14Cui2019505071.58 ± 1.9571.63 ± 1.4228/2227/23Du2021373869.34 ± 1.08 (61–83)69.24 ± 1.12 (60–82)15/2214/24Fan2022353569.94 ± 3.87 (60–81)69.99 ± 3.91 (60–82)16/1921/14Li (a)2021434269.82 ± 11.6472.28 ± 12.0726/1722/20Li (b)2021252368.25 ± 3.45 (61–75)68. 43 ± 3. 16 (63–78)21/417/6Pan2023343470.5 ± 4.1 (63–7871.1 ± 4.7 (65–80)22/1223/11Ren2022404568.34 ± 4.5568.22 ± 3.5125/1531/14Shao2022242472.53 ± 7.47 (71–80)72.31 ± 7.69 (70–80)16/814/10Su2021464670.19 ± 3.2670.21 ± 3.5925/2124/22Wen2022454572.0 ± 4.0 (60–85)72.0 ± 4.0 (60–83)24/2126/19Yang2022353566.23 ± 5.32 (54–83)67.17 ± 6.04 (53–8212/2315/20Yu202310010070.32 ± 4.23 (60–8370.34 ± 4.25 (60–82)59/4157/43Zhang2021353567.41 ± 3.6567.54 ± 3.4817/1818/17Zhao2021565671.69 ± 4.98 (65–84)71.88 ± 4.76 (63–82)30/2631/25Zhao2022303072.5 ± 4.9 (63–82)73.5 ± 4.9 (63–84)17/1317/13Zhao2023525268.72 ± 1.11 (60–80)68.34 ± 1.24 (60–79)32/2030/223.2Pressure ulcerSeventeen of the included literatures 13 , 16 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 27 , 28 , 29 , 30 , 31 , 32 , 33 reported pressure ulcers, with 712 patients in the intervention group, 12 of whom developed pressure ulcers, and 715 patients in the control group, 43 of whom developed pressure ulcers. There was no statistical heterogeneity among the studies (I 2 = 0%, p = 1.00), and a fixed effects model was used. The results showed that the incidence of pressure ulcers in the nursing intervention in the operating room group was significantly lower than that in the control group, and the difference was statistically significant (1.69% vs. 6.01%, OR: 0.32, 95% CI: 0.18–0.57, p < 0.001), suggesting that application of the nursing intervention in the operating room was able to reduce the incidence of postoperative pressure ulcers in patients with intertrochanteric fractures (Figure 3).FIGURE 3Forest plot for pressure ulcer.3.3Wound infectionEleven of the included studies 16 , 18 , 20 , 22 , 23 , 26 , 27 , 28 , 30 , 31 , 32 reported wound infection, and a total of 500 patients were included in the intervention group, of which 5 patients developed wound infection. A total of 504 patients were included in the control group, of which 31 patients developed wound infection. There was no statistically significant heterogeneity among the studies (I 2 = 0%, p = 1.00), and a fixed effects model was used. The results showed that the incidence of wound infection was significantly lower in the nursing intervention in the operating room group than in the control group, and the difference was statistically significant (1.00% vs. 6.15%, OR: 0.23, 95% CI: 0.11–0.50, p < 0.001), suggesting that the application of the nursing intervention in the operating room was able to reduce the incidence of postoperative wound infection in patients with intertrochanteric femur fractures (Figure 4).FIGURE 4Forest plot for wound infection.3.4Publication biasThe results are shown in Figures 5 and 6; the studies were symmetrically distributed in the funnel plot, indicating that no obvious publication bias existed in the included literature.FIGURE 5Funnel plot for pressure ulcer.FIGURE 6Funnel plot for wound infection.4DISCUSSIONWith the accelerated aging of the population, intertrochanteric femoral fractures in the elderly are becoming a major social health problem worldwide. Intertrochanteric femoral fracture treatments are divided into conservative and surgical treatments. Because of the high complication rate of conservative treatment and the need for bed rest and limb traction, the mortality rate within 1 year after injury is relatively high. 34 , 35 Therefore, most scholars believe that surgical treatment is the first choice for patients with intertrochanteric femoral fractures. 36 Surgical treatment is primarily divided into intramedullary and extramedullary fixations. However, both extramedullary and intramedullary fixations must be performed in the operating theatre, and foreign bodies must be implanted. These foreign bodies are sterile and do not cause any discomfort. However, the long duration of femoral intertrochanteric fracture surgery, the large incision and the large area of exposed human muscle tissue lead to a high possibility of bacterial invasiveness, and the risk of postoperative incision infection is significantly increased. 37 , 38 At the same time, as there are more elderly people with intertrochanteric fractures, there are problems of age, body function and immune function decline, and the long period of bed rest after the operation makes pressure ulcers more likely to occur. 39 Pressure ulcer formation and wound infection, as common complications of intertrochanteric fractures, not only add physical and psychological pain to the patient but also increase the length and cost of hospitalisation and the possibility of life‐threatening implant infections. 40 , 41 , 42 Previous studies have shown that the incidence of wound infections and pressure ulcers in patients may be more than 10%, and the length of hospital stay is long under the traditional model of routine care. 43 , 44 Nursing intervention in the operating room can reduce the incidence of wound infections by 5%–8%, and patient satisfaction with care can exceed 90%. 45 The need to reduce this risk is even greater for a prolonged procedure such as intertrochanteric fracture of the femur with implantation of a foreign body. Nursing intervention in the operating room is proactive in all aspects, with rigorous plans to enhance aseptic practice by operating theatre nurses and surgeons and good preventive measures for pressure ulcers during surgery, leading to better prevention of postoperative complications. Eighteen studies were included in this analysis. The results of this study showed that nursing intervention in the operating room significantly reduced the incidence of pressure ulcers and wound infections in patients with intertrochanteric fractures. Ren and Wang analysed 95 patients with intertrochanteric fractures who underwent surgical treatment and found that nursing intervention in the operating room could reduce the rate of wound infections, alleviate the patients' postoperative pain and improve the patients' quality of life, which is consistent with the results of this study. 23 In a study of 90 elderly patients with PFNA of trochanteric fracture of the femur, Wen and Tan found that nursing intervention in the operating room could reduce postoperative complications of trochanteric fracture of the femur, improve surgical efficacy and alleviate patients' stress reaction, but there was no statistical significance in reducing the incidence of wound infection. 26 This finding contradicts the results of the present study. Yang 's study of 70 patients with trochanteric fractures who underwent surgical treatment also showed that nursing intervention in the operating room reduced the overall complication rate but did not reduce the incidence of wound infection. 27 This finding was contrary to the results of the present study. Cui et al.'s study of 100 elderly patients with internal fixation of trochanteric femoral fractures showed that nursing intervention in the operating room significantly reduced the incidence of pressure ulcers and infections, shortened the duration of the operation and improved the safety of the operation, a finding consistent with that of the present study. 19 This is the first comprehensive study to explore the effectiveness of nursing intervention in the operating room in preventing pressure ulcers and wound infections in patients with intertrochanteric fractures. However, several limitations should be addressed: the included literature is from the Chinese population and has a small sample size, which may have the possibility of selective bias; nursing interventions in the included studies were independent of each other, and many factors were difficult to control, which may have affected the external veracity of the results and the literature selectively reports the results, which may lead to a risk of selective bias in the final results.5CONCLUSIONIn summary, the available evidence suggests that nursing intervention in the operating room is more effective in reducing the incidence of pressure ulcers and wound infections in intertrochanteric fractures compared with conventional fractures. However, owing to the limitations of the quantity and quality of the included literature, more high‐quality studies are needed at a later stage to further validate our conclusions.FUNDING INFORMATIONThis study was supported by the 2022 Chinese Medicine Research Projects of the Guangdong Provincial Bureau of Traditional Chinese Medicine (no. 20221188).CONFLICT OF INTEREST STATEMENTThe authors declare that there is no conflict of interest.

PMC

Therapeutic Advances in Gastroenterology

PMC10338719

7-11-2023

10.1177/17562848221104953

Pilot study on a new endoscopic platform for colorectal endoscopic submucosal dissection

Maselli Roberta, Spadaccini Marco, Galtieri Piera Alessia, Badalamenti Matteo, Ferrara Elisa Chiara, Pellegatta Gaia, Capogreco Antonio, Carrara Silvia, Anderloni Andrea, Fugazza Alessandro, Hassan Cesare, Repici Alessandro

Background:The endoscopic submucosal dissection (ESD) is a technically demanding and time-consuming procedure, with an increased risk of adverse events compared to standard endoscopic resection techniques. The main difficulties are related to the instability of the operating field and to the loss of traction. We aimed to evaluate in a pilot trial a new endoscopic platform [tissue retractor system (TRS); ORISE, Boston scientific Co., Marlborough, MA, USA], designed to stabilize the intraluminal space, and to provide tissue retraction and counter traction.Method:We prospectively enrolled all consecutive patients who underwent an ESD for sigmoid/rectal lesions. The primary outcome was the rate of technical feasibility. Further technical aspects such as en-bloc and R0 resection rate, number of graspers used, circumferential incision time, TRS assemblage time, submucosal dissection time, and submucosal dissection speed were provided. Clinical outcomes (recurrence rate and adverse events) were recorded as well.Results:In all, 10 patients (M/F 4/6, age: 70.4 ± 11.0 years old) were enrolled. Eight out of 10 lesions were located in the rectum. Average lesion size was 31.2 ± 2.7 mm, and mean lesion area was 1628.88 ± 205.3 mm2. The two sigmoid lesions were removed through standard ESD, because the platform assemblage failed after several attempts. All rectal lesions were removed in an en-bloc fashion. R0 resection was achieved in 7/8 (87.5%) patients in an average procedure time of 60.5 ± 23.3 min. None of the patients developed neither intraprocedural nor postprocedural adverse events.Conclusion:TRS-assisted ESD is a feasible option when used in the rectum, with promising result in terms of efficacy and safety outcomes. Nevertheless, our pilot study underlines few technical limitations of the present platform that need to be overcome before the system could be widely and routinely used.

BackgroundColorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide.1–3 Although endoscopic mucosal resection (EMR) is the most common treatment approach for superficial colorectal lesions, when dealing with larger lesions, en-bloc EMR might be unfeasible and unsafe and a piecemeal resection may hinder histologic assessment and lead to an increased risk of local recurrence.4,5 Endoscopic submucosal dissection (ESD) may overcome these limitations, allowing dissection of larger lesions in one piece. Indeed, the European Society of Gastrointestinal Endoscopy suggests to consider ESD for the removal of colorectal lesions with a high risk of superficial submucosal invasion, in case they cannot be removed en-bloc by standard polypectomy or EMR. 6 It is also true that ESD is technically demanding and time-consuming, and an increased risk of adverse events has been reported.7,8The main difficulties are related to the instability of the operating field, due to the physiologic peristalsis, and to the loss of traction, due to the single operating channel. Recently, several new techniques and devices have been developed to facilitate ESD and to overcome the difficulties related to challenging situations.9–11Tissue retraction system (TRS; ORISE, Boston scientific Co., Marlborough, MA, USA) is a new endoscopic platform designed to stabilize the intraluminal space, to help visualizing the dissection plane, and to provide tissue retraction and counter traction. It consists of an expandable and dynamically controlled intraluminal chamber, mounted on a flexible overtube, and two associated specifically designed retractor graspers. The system is front-loaded over the endoscope and introduced into the colon, theoretically designed to reach at least the splenic flexure. When the target area is reached, the cage is deployed creating an expanded, optimally reconfigured and stable operating field around the target lesion. Then endoscopic removal of the lesion is performed using standard endoscopic instruments (injection needles, knives, snares, etc.) through the operating channel of the scope with assistance of one or two retractor graspers handled by a second operator. Each accessory within the TRS can be moved forward and backward, left or right, rotated 360°, and can be advanced out and pulled in, regardless of the TRS, allowing for the maximal tensile force needed to visualize the dissection plane.12–14The platform is not yet commercially available, and only two case reports of TRS-assisted ESDs have been published so far.14,15 No studies have been performed yet in humans to evaluate its feasibility and safety in speeding up colorectal ESDs.We performed a pilot study to evaluate the feasibility, the efficacy, and the safety in patients undergoing ESD of colorectal lesions with the assistance of the TRS.MethodsPatients and study designWe prospectively enrolled all consecutive patients ⩾18 years who underwent a sigmoid colon or rectal ESD in our institution, Humanitas Research Hospital. A detailed consultation and written informed consent were obtained from all patients prior to the procedure.Out of Good Clinical Practice, we enrolled only patients whose lesions could not have been optimally and/or radically removed with standard polypectomy or EMR. 6 All patients whose lesions expressed any risk for deep submucosal invasion such as distorted pit (Kudo’s type V) and/or capillary (Sano’s type III) patterns 16 were excluded from this trial. We did not enroll patients in poor general condition (American Society of Anesthesiologists score ⩾ 3), known for coagulation disorders, those who were pregnant and/or breastfeeding, and those who were not able to sign the informed consent.The study was conducted in accordance with the declaration of Helsinki and was registered on the ClinicalTrial.gov (NCT:03553199). The study protocol was approved by the local Institutional Review Board. The methods of our study were based on the STROBE recommendations. 17 Endoscopic procedureAll procedures were performed by expert operators (AR and RM), defined as endoscopists who had already performed at least 80 ESDs, 6 and were carried out under deep sedation administered by a dedicated anesthesiologist.A standard gastroscope (ELUXEO, EG-760R; FUJIFILM Co., Tokyo, Japan) with CO2 insufflation was used for all the procedures.After submucosal injection with saline tinged with methylene blue, we delimited the resection area by performing a circumferential mucosal incision keeping a 4–5 mm margin from the lesion using a T-type Hybridknife (ERBE, Marietta, GA, USA). Once the incision was performed, we pulled out the endoscope defining the end of the circumferential incision time (CIT) and starting time of TRS assemblage time (AT). The TRS was thus mounted and delivered: once the endoscope with the overtube was reinserted, we defined the ending AT (ORISE tissue retractor system components are illustrated in Figure 1).Figure 1.ORISE tissue retractor system. (a) Tissue retractor system cage architecture details. (b) Tissue retractor system devices. (B1) OIGs which are flexible conduits, guiding graspers. The OIG is available in three tip configurations; with 45°, 60°, and 90° tip bend angles. (B2) OTR overtube consisting of a handle and a flexible shaft with expandable distal end (Cage); (B3 and B4) graspers; and (B5) cage. (c) Tissue retractor system assembled over a gastroscope with two inserted retractor graspers.OIGs, ORISE Instrument Guides; OTR, ORISE tissue retractor.The ORISE TRS was then placed over the lesion. The lesion edge was then grasped with the dedicated grasper to provide traction, and submucosal dissection was performed (Figure 2).Figure 2a.TRS-aided ESD. (a) Rectal lesion. (b) TRS luminal distension and stabilization properties. (c) Lesion retracted cranially by the two graspers allowing easy injection/incision with the ESD knife.Figure 2b.TRS-aided ESD.EDS, endoscopic submucosal dissection; TRS, tissue retractor system. (a) ORISE TRS placed over a rectal lesion and (b-d) Lesion retracted cranially by one grasper allowing easy injection/incision with the ESD knife.After resection, the cage was closed and the specimen was retrieved. The specimen was finally placed on a polystyrene rigid support and fixed with pins, then saved in formalin solution, and sent to the pathologist. All procedural adverse events were recorded.Endoscopists’ perception and feelings through the different procedural steps (assemblage, resection assistance, specimen retrieval) were graded according to a Visual Analogue Scale.Follow-upInpatients clinical status was assessed the day after the procedure and daily until discharge. Outpatients received a phone call on day 1, day 7, and day 14 after the endoscopic procedure to investigate the possible postprocedural delayed AEs related to the procedure.Patients with complete endoscopic excision and no indication for surgery based on final histology were advised to undergo the first endoscopic surveillance at 6 months, 18 then at intervals of 12, 36, and 60 months. 6 If any recurrence was detected, this was resected and sent for histology; in the case of suspicious invasive neoplasia, biopsies were taken.Outcomes and definitionsComplete resection was defined according to current guidelines. 6 The resection was considered complete and defined as R0 when the neoplastic/dysplastic tissue was removed en bloc with free lateral and vertical margins. The endoscopic resection was considered incomplete in two cases: when the lateral or vertical margins were positive for neoplastic/dysplastic invasion (R1) and when the margins were not evaluable because of artificial burn effects (RX). Adverse events were defined according to ASGE lexicon as the occurrence of either intraprocedural or immediately postprocedural unintentional perforation, peritonitis, pneumoperitoneum, bleeding requiring hemostasis or transfusion, and procedure-related death. 19 The endoscopist’s (first operator) feelings about assemblage, resection, and specimen retrieval was graded from 1 to 10. According to the study by Suzuki et al., we defined submucosal dissection time (SDT) as the time (min) from the creation of the first traction with the TRS to the end of ESD. The area of the specimen was calculated by multiplying the halves of both height and length of the resected piece, then multiplied by 3.14. Submucosal dissection speed (SDS) was defined by dividing the specimen area by the SDT (min). 20 Finally, considering also both the CIT and the AT as previously defined, total ESD procedure time was as follows: Totalproceduretime=CIT+TRSAT+SDT Data collectionData were collected on a dedicated CRF spreadsheet using Windows Microsoft Excel (version 16, Microsoft Corp, Redmond; WA. USA). It included patient demographics, characteristics of the lesion (size, location, macroscopic appearance, pit, and vascular patterns), piecemeal or en-bloc removal, resection status (R0/R1), histological report, number of graspers used, CIT, TRS AT, SDT, SDS, and rate and type of adverse events related to the procedure and/or to the device.Statistical analysisQuantitative variables were expressed as means [standard deviation or median (range)]. Qualitative variables were described by frequencies with percentages. STATA (version 15) was used for the statistical analysis.ResultsBaselineWe enrolled 10 patients (M/F 4/6, age: 70.4 ± 11.0 years old). Most of the lesions (n = 7) were laterally spreading tumor (LST) granular mixed, two were non-granular LSTs, and the last one was an LST granular type. Eight out of 10 lesions were located in the rectum. Two lesions were located in the sigmoid colon. Average lesion size was 31.2 ± 2.7 mm, and mean lesion area was 1628.88 ± 205.3 mm2. Baseline characteristics are summarized in Table 1 and lesions characteristics are extensively reported in Supplemental Table 1.Table 1.Baseline characteristics.Baseline characteristicsValueAge, years old Mean (SD)70.4 (11.0)Gender (n) Male4 Female6LST type, n Granular1 Non-granular2 Granular mixed7Kudo pit pattern, n IIIL4 IIIs1 IV5Lesion area (mm2) Mean (SD)1628.88 (205.3)LST, laterally spreading tumors; SD, standard deviation.OutcomesProcedureThe two sigmoid lesions were removed through standard ESD, because the platform assemblage failed after several attempts due to difficulties in positioning and narrower lumen. On the other hand, an en-bloc resection was successfully completed in all the rectal cases (8/10, 80%), using the TRS assistance (Table 2).Table 2.Outcomes.OutcomesValuesEn bloc lesion removal achievement, n (%)8 R0 resection, n (%)7 (87.5)Timings Total time of the procedure, mean (SD) min(13.5) AT, mean (SD) min5.3 (4.8) SDT, mean (SD) min(19.7) SDS, mean (SD) mm2/min36.3 (33.9)Adverse events, n (%)0 AT, assembly time; SD, standard deviation; SDS, submucosal dissection speed; SDT, submucosal dissection time.The average total procedure time was 60.5 ± 23.3 min. The average time spent for assembling TRS was 6.5 ± 4.8 min. SDT resulted to be 45.1 ± 19.7 min, while SDS was 30.6 ± 23.9 min/mm2. In all, but two procedures, only one of the two graspers were used. Procedural characteristics and endoscopist’s feelings are extensively reported in Supplemental Table 2.A complete resection (R0) was achieved in 7 (87.5%) out of 8 patients. Only one lesion (pt. 2) resulted in a rectal cancer (adenocarcinoma), with deep submucosal involvement and positive deep margins. The patient was indeed referred for surgery. Other lesions were adenomatous lesions (six high-grade and one low-grade adenomas).None of the eight patients developed neither intraprocedural nor postprocedural adverse events.Follow-upAfter 6 and 12 months from the procedures, all the seven patients underwent a follow-up endoscopy: the scar was regular in all the cases, with no signs of residual adenomatous tissue.DiscussionESD is a well-established method for endoscopic removal of colorectal lesions in the East. Recently, it has also been gaining recognition in the Western world, where CRC prevalence is even more relevant. As a matter of fact, by allowing en-bloc excision regardless of the lesion size, ESD aims to increase the possibility of curative resection even for superficially invasive CRC. Nevertheless, ESD is a technically challenging, time-consuming procedure, which implies a significantly higher risk of adverse events if compared to EMR, necessitating ongoing innovations to overcome its complexity. Conventional ESD has a limited armamentarium of devices and maneuvers helping the endoscopist in improving the access to the submucosal space and the identification of the proper dissection plane (i.e. distal attachment, fluid injections, patient repositioning).According to our pilot study, TRS-assisted ESD is a feasible option when used in the rectum, with promising results in helping the operator in achieving adequate efficacy and safety outcomes as shown by the 100% of en-bloc resection coupled with the almost 90% of R0 resection on one side and the absence of procedural-related adverse events on the other.The theoretical advantage of the TRS platform over the number of accessories and techniques previously proposed for ESD assistance is the ability to provide both dynamic tissue retraction and instrument triangulation, which are essential for ideal visualization and access to dissection plane. In this regard, in our opinion, the TRS system was not taken the full advantage, as the endoscopist only used the two graspers twice in our series. However, it could be argued that as ‘pure endoscopists’, the authors are not used to the ‘surgical concept’ and this device could have been used better. Furthermore, the platform provided a stable operating space during the procedure and works as a convenient specimen-retrieval device after resection. These technical advantages aim at first to improve the safety and efficacy outcomes of colorectal endoscopic resections; however, they might also consistently affect resection speed, overcoming one of the key barriers preventing the definitive recognition of ESD potential over EMR. As a matter of fact, considering the number of colorectal lesions that Western endoscopists need to manage, the systematical adoption of ESD for large colorectal lesions would add an unaffordable burden on most of the western endoscopy services. Significantly reducing the procedural times would be the real game changer for ESD affirmation. In this regard, if comparing the SDS with TRS in our series with the SDS of western expert endoscopists in previously published series, we showed a faster dissection speed (30.6 ± 23.9 versus 16.7 mm2/min, respectively). 21 At the same time, this allowed to reach a dissection speed comparable to the one reported by eastern expert endoscopists (30.6 ± 23.9 versus 29.1 ± 3.2 mm2/min, respectively).20,22In our opinion, despite the several advantages of this new technology, few drawbacks may still prevent its large-scale use, and should be overcome. First, despite improving over the procedures due to the endoscopist learning curve, the AT should be further reduced by re-designing the deployment strategy of the working chamber. The inconvenient assemblage strategy prevented the use of the TRS platform for endoscopic resection proximally to the rectum, where the deployment complexity is increased. It could be argued that the rectum is where patients would benefit the most of an en-bloc resection given the higher risk of submucosal invasion. Nevertheless, overcoming the technical limitations of standard ESD even in other colonic tracts would have been even more relevant for a new resection-assisting platform.Moreover, even assuming the use of the TRS platform only for rectal resections, a second limitation is due to the design of the overtube, developed for reaching the splenic flexure.This forces the endoscopist to work far away from the patient when performing rectal ESD, losing part of the technical gain in terms of scope control. Considering that the TRS platform had been developed aiming to improve the procedural outcomes through an endoscopist-friendly assistance, this limitation may paradoxically may have a role in decreasing the willingness to use the device in daily practice for rectal lesions.The main drawbacks of the study are the lack of comparison with conventional ESD and the limited sample size, preventing conclusive statement on TRS. However, reassuring on feasibility and safety, this pilot experience permits to design future comparative studies. Moreover, its strict protocol, coupled with the prospective setting, permitted to lucidly analyze its pros and cons as discussed.ConclusionIn conclusion, the TRS platform provides a stable working space, dynamic tissue retraction, and instrument triangulation. In spite of few technical limitations which would need to be overcome, the time for technology to permit a decisive step toward a mini-invasive intraluminal surgery has finally come.Supplemental Materialsj-docx-1-tag-10.1177_17562848221104953 – Supplemental material for Pilot study on a new endoscopic platform for colorectal endoscopic submucosal dissectionClick here for additional data file.Supplemental material, sj-docx-1-tag-10.1177_17562848221104953 for Pilot study on a new endoscopic platform for colorectal endoscopic submucosal dissection by Roberta Maselli, Marco Spadaccini, Piera Alessia Galtieri, Matteo Badalamenti, Elisa Chiara Ferrara, Gaia Pellegatta, Antonio Capogreco, Silvia Carrara, Andrea Anderloni, Alessandro Fugazza, Cesare Hassan and Alessandro Repici in Therapeutic Advances in Gastroenterology

PMC

American Heart Hournal Plus: Cardiology Research and Practice

38510498

PMC10946039

12-01-2022

10.1016/j.ahjo.2022.100234

Comparison of CT acquired cardiac valvular calcification scores in hemodialysis and peritoneal dialysis patients undergoing open heart surgery

Kanaan Christopher N., Layoun Habib, Kondoleon Nicholas P., Fadel Remy, Mirzai Saeid, Schold Jesse, Arrigain Susana, Daou Remy, Mehdi Ali, Taliercio Jonathan J., Unai Shinya, Kapadia Samir, Harb Serge, Nakhoul Georges N.

Study objectiveData is scarce regarding which dialysis modality portends more severe cardiac valvular calcification (CVC). Our aim was to compare the degree of CVC in hemodialysis (HD) and peritoneal dialysis (PD) patient cohorts prior to open heart surgery (OHS) using a CT calcium score.Design, setting, and participantsDialysis patients who underwent OHS at our institution from 2009 to 2019 and who had pre-surgical cardiac CT were included in our study. We obtained duration of dialysis modality prior to their surgical date. There were two study cohorts to evaluate outcomes of interest: mitral and aortic calcification. CVC was assessed using the Agatston score. Logistic regression was performed to test for the association of PD and HD cumulative dialysis duration with presence of CVC.ResultsA total of 214 and 166 patients met inclusion for the mitral and aortic strata, respectively. Age, female sex, and BMI were associated with higher odds of presence of mitral calcification. Age and BMI were associated with higher odds of presence of aortic calcification, while female sex was associated with lower odds in the aortic strata. Cumulative years on PD and cumulative years on HD were not significantly associated with presence of CVC in either cohort.ConclusionPresence of mitral and aortic calcification for patients undergoing OHS was not significantly associated with cumulative length of PD or HD after adjusting for age, gender, and BMI suggesting that there may be more factors at play in the progression of CVC in end stage renal disease patients than what was previously established.

1IntroductionCardiovascular disease is the leading cause of mortality in both intermittent hemodialysis (HD) and peritoneal dialysis (PD) populations . These patients often suffer from downstream complications including vascular or tissue calcification after years of therapy, which has been noted to increase cardiovascular mortality up to 100-fold higher than in the general, age-matched population . The prognostic role of valvular calcification in chronic dialysis patient is well recognized and has even been equated by some authors to that of atherosclerotic vascular disease . Several factors have been identified as independent risk factors for cardiac valvular calcification (CVC). Those include age, inflammatory conditions, loss of calcification inhibitors, and dysregulated bone mineral metabolism , , in particular elevated phosphate levels . However, the data is lacking on which dialysis population portends more severe CVC. While the complications of hyperphosphatemia have been linked to increased CVC and mortality in the HD population, this association is less established among PD patients , , . PD patients have lower phosphate levels , and so theoretically, they could be expected to have less calcification. To this day, there is scarcity of data comparing the degree of CVC between dialysis modalities, as most research has been dedicated to comparing the level of coronary artery calcification (CAC). We aimed to compare the degree of aortic valve and mitral annulus calcification in HD and PD patients who underwent open heart surgery (OHS) using a computed CT score.2Materials and methodsWe used our institution's cardiothoracic surgery database and identified patients that had dialysis prior to heart surgery. We included records from October 2009 to October 2019. We included the first surgical record per patient for patients undergoing isolated coronary artery bypass graft (CABG), or CABG+valve surgery (repair or replacement), or valve-only surgery (repair or replacement). Only patients receiving dialysis for end stage renal disease (ESRD) were included in our study. Patients with congenital heart disease and those lacking information on the length of dialysis were excluded.We had two different study cohorts to evaluate each of the outcomes of interest: mitral and aortic calcification. To evaluate mitral calcification, we excluded any patients who previously underwent mitral valve surgery, such as those with a history of mitral valve repair or replacement. To evaluate aortic calcification, we excluded any patients who previously underwent aortic valve surgery, such as those with a history of aortic valve repair or replacement. Patients were included in both cohorts if they met criteria for both, and in one cohort if they met criteria for only one. Patients missing data to calculate a calcification score for their respective strata were excluded from the strata (Fig. 1). This study was approved by the Institutional Review Board of the Cleveland Clinic Foundation and was conducted in accordance with the Declaration of Helsinki (as revised in 2013).Fig. 1Flow chart for patient selection; CABG, coronary artery bypass graft; HD, hemodialysis; PD, peritoneal dialysis; AV, aortic valve; MV, mitral valve.Fig. 1We obtained cumulative length of PD and HD at the time of surgery from the United States Renal Data System (USRDS) prescription history and supplemented the data with chart review. We categorized patients based on their history of prior dialysis as: prior PD only, prior HD only, or both prior PD and HD.Our study outcomes were mitral and aortic calcification scores. We obtained calcification scores prior to the surgical interventions through chart review. The Agatston score was used to quantify calcification on mitral and aortic valves. The score was automatically calculated by the software on a manually defined volume of interest. CT reading and calcium scoring were done using TeraRecon Aquarius iNtuition (TeraRecon Headquarters, Durham, NC). In addition to evaluating continuous calcification scores, we evaluated calcification present (above 0) vs. absent. The calcification scores were calculated using cardiac CT in end-systole and imaging review was done by an expert cardiac imaging CT reader from our institution.For each study strata, we compared patient characteristics by prior PD only, prior HD only, and prior PD and HD using Chi-square and Kruskal-Wallis tests for categorical and continuous variables, respectively. We summarized and compared calcification scores across the different dialysis modalities using Kruskal-Wallis tests. We categorized calcification scores into presence of any calcification (>0) vs. no calcification, and used logistic regression analysis to evaluate the association between cumulative years on each dialysis modality and presence of calcification while adjusting for age, sex, BMI, and serum calcium. We had phosphorus values on a subset of the study patients and as a sensitivity analysis, we evaluated a similar model adjusting for the calcium phosphorus product instead of solely the serum calcium.3ResultsA total of 296 patients met inclusion criteria for at least one of the strata in our study. Of those, 214 met inclusion for the mitral strata, and 166 met criteria for the aortic strata.Of the 214 patients included in the mitral strata, 16 had only prior PD, 166 had only prior HD, and 32 had prior PD and HD. The median age of patients was 65.5 and 68 % were male. Table 1 shows patient characteristics for each group. The median number of years on PD was 2.1 and 1.7 respectively for those in the PD only group vs. PD and HD group. The median number of years on HD was 2.8 and 1.8 respectively for those in the HD only group vs. PD and HD group.Table 1Descriptive statistics by prior PD/HD at surgery (Mitral strata).Table 1FactorOverall(N = 214)Prior PD only(N = 16)Prior HD only(N = 166)Prior PD and HD(N = 32)P-valueAge65.5 [57.0,73.0]63.5 [48.0,75.0]67.0 [58.0,74.0]63.0 [51.5,68.5]0.063bGender0.80c Female69 (32.2)4 (25.0)54 (32.5)11 (34.4) Male145 (67.8)12 (75.0)112 (67.5)21 (65.6)Race0.35d American Indian2 (0.93)0 (0.0)2 (1.2)0 (0.0) Black65 (30.4)1 (6.3)53 (31.9)11 (34.4) Hawaiian1 (0.47)0 (0.0)1 (0.60)0 (0.0) Multiracial9 (4.2)0 (0.0)9 (5.4)0 (0.0) Unknown1 (0.47)0 (0.0)1 (0.60)0 (0.0) White136 (63.6)15 (93.8)100 (60.2)21 (65.6)BMI27.4 [24.4,31.2]29.0 [26.2,31.2]26.9 [23.7,31.2]28.1 [26.1,31.2]0.18bCalcium mg/dl9.0 [8.5,9.5]8.8 [8.2,9.1]9.1 [8.5,9.5]8.9 [8.5,9.8]0.19bPTH pg/ml232.0 [123.0,345.0]234.0 [130.0,431.0]230.0 [120.0,335.0]322.0 [124.0,380.0]0.80bPhosphorus mg/dl4.0 [3.0,5.0]5.5 [4.7,6.7]3.8 [2.9,4.7]4.6 [4.2,5.6]0) was 43.8 %, 44.0 %, and 53.1 % respectively for those in the PD only group, HD only group, and combined PD and HD group (P = 0.63).In the logistic regression model, age, female sex, and BMI were associated with higher odds of presence of mitral calcification (Table 3). Cumulative years on PD and cumulative years on HD were not significantly associated with presence of mitral calcification. The odds ratio per 1 year of PD was 1.23 (95 % CI: 0.95, 1.58) and 1.05 per 1 year of HD (95 % CI: 0.97, 1.13) shown in Fig. 2. There were 147 of 214 patients in the mitral strata who had phosphorus values and were included in the sensitivity analysis. The model from the sensitivity analysis that adjusted for calcium‑phosphorus product produced similar results, but sex was not significantly associated with presence of mitral calcification.Fig. 2Adjusted odds ratios for presence of calcification in mitral and aortic strata. PD, peritoneal dialysis; HD, hemodialysis.Fig. 2Of the 166 patients included in the aortic strata, 20 had only prior PD, 115 had only prior HD, and 31 had prior PD and HD. The median age of patients was 62 and 56 % were male. Table 2 shows patient characteristics for each group. The median number of years on PD was 2.1 and 1.1 respectively for those in the PD only group vs. PD and HD group. The median number of years on HD was 2.6 and 1.7 respectively for those in the HD only group vs. PD and HD group.Table 2Descriptive statistics by prior PD/HD at surgery (Aortic strata).Table 2FactorOverall(N = 166)Prior PD only(N = 20)Prior HD only(N = 115)Prior PD and HD(N = 31)P-valueAge62.0 [51.0,70.0]65.5 [56.0,73.5]62.0 [53.0,71.0]57.0 [48.0,65.0]0.019bGender0.48c Female73 (44.0)10 (50.0)47 (40.9)16 (51.6) Male93 (56.0)10 (50.0)68 (59.1)15 (48.4)Race0.77d Asian1 (0.60)0 (0.0)1 (0.87)0 (0.0) Black60 (36.1)6 (30.0)44 (38.3)10 (32.3) Multiracial5 (3.0)0 (0.0)5 (4.3)0 (0.0) White100 (60.2)14 (70.0)65 (56.5)21 (67.7)BMI27.6 [23.8,30.6]28.1 [24.0,29.5]27.7 [24.1,33.1]27.0 [22.4,29.9]0.60bCalcium mg/dl8.9 [8.4,9.4]8.9 [8.3,9.4]9.0 [8.4,9.4]8.8 [8.5,9.5]0.99bPTH pg/ml234.0 [126.0,377.2]332.5 [173.0,514.0]230.0 [120.0,308.0]349.6 [124.0,1955.0]0.30bPhosphorus mg/dl3.9 [3.1,5.2]5.3 [4.7,8.1]3.6 [2.8,4.5]4.8 [3.8,6.5]0) was 60.0 %, 45.2 %, 51.6 % respectively for those in the PD only group, HD only group, and combined PD and HD group (P = 0.43).In the logistic regression model, age and BMI were associated with higher odds of presence of aortic calcification, while female sex was associated with lower odds (Table 4). Cumulative years on PD and cumulative years on HD were not significantly associated with presence of aortic calcification. The odds ratio per 1 year of PD was 1.27 (95 % CI: 0.99, 1.64) and 1.02 per 1 year of HD (95 % CI: 0.93, 1.13). There were 134 of 166 patients in the aortic strata who had phosphorus values and were included in the sensitivity analysis (Fig. 2). The model from the sensitivity analysis that adjusted for calcium phosphorus product produced similar parameter estimates but no variables were significantly associated with presence of aortic calcification.4DiscussionOur study investigated whether valvular calcification is worse in one dialysis modality over the other. The results of our study show that the degree of CVC in the aortic and mitral valves did not differ between dialysis modalities based on cumulative dialysis years on either PD or HD, nor did it differ if patients had years on both modalities as opposed to just one modality.In the general population, the prevalence of CVC is up to five times higher in dialysis patients than in those not on dialysis, ranging between 32 and 47 % in PD patients and 19–84 % in HD patients , , , . PD patients are expected to maintain more optimal bone mineral disease profiles. This is due in part to fewer hemodynamic changes and less hyperdynamic circulation, but also because many of these patients retain residual renal function (RRF), and thus the ability to clear small solutes, maintain fluid balance, and control phosphorus levels . To this point, some studies have illustrated an inverse relationship between the maintenance of renal function and decreased vascular/valvular calcification , . Given this information, it would be expected that PD patients, with their increased residual renal function compared to HD counterparts, might therefore manifest significantly less CVC over years on this modality. However, our findings did not support this hypothesis. When adjusting for covariates in our logistic regression model, we did not see a difference in calcium levels or calcium phosphate product in either the mitral or aortic strata. Rather, our descriptive data surprisingly showed a trend toward higher phosphorus and calcium phosphate product in patients who only received PD as opposed to HD or a combination of HD and PD. Various reasons can account for this unexpected trend. First, PD patients have been shown to have a higher degree of overall phosphorus exposure. For instance, in a study by Evenepoel et al. , a validated mathematical model was used to calculate time-averaged-concentration of phosphorus between HD and PD patients rather than relying on a single phosphorus measurement, and noted that PD patients had a higher degree of overall phosphorus exposure. This was attributed to inferior phosphorus clearance in the PD population, and challenged commonly held convictions . Second, the introduction of convective therapies and use of HD membranes with higher efficiency and permeability could optimize phosphate removal compared to the lower efficiency therapy of PD . Third, the loss of RRF has been linked to increased inflammation and calcium phosphate product, contributing to a higher burden of calcification ; however, we did not have data regarding the RRF of our patient sample.Data on the direct comparison of CVC in PD and HD populations is scarce. To our knowledge, only one study directly compared CVC in the two adult populations, reporting a lower prevalence of CVC in PD as opposed to HD patients, which was hypothesized as being due to the presence of RRF contributing to tighter phosphate control and removal of uremic toxins in PD patients , , , . Despite the lack of data on direct CVC comparisons, our study results are in line with much of what has been published when comparing the dialysis populations in CAC. Jansz et al. reported that PD patients did not develop less CAC compared to HD patients, despite the fact that HD patients had a 6-month longer median time on dialysis than their PD counterparts. Kim et al showed similar findings, with no difference in CAC score between PD and HD patients. In contrast, Srivaths et al reported a higher incidence of CAC in patients on HD compared to PD, a finding that was attributed to better control of mineral imbalance; however, this study was carried out on a pediatric population.Our study showed that age was associated with higher odds of presence of mitral and aortic calcification. This is not surprising as it is well established that the prevalence of CVC increases with age, after years of lipid accumulation, chronic inflammation and endothelial dysfunction , . While our study showed an association between female sex and increased mitral calcification, several studies have shown a higher prevalence of CVC among males . Some have hypothesized that the sexes differ by way of pathogenesis and extracellular matrix remodeling. Others attribute the difference to hormonal variability such that there is a clear role for androgens in the promotion of calcific nodule and reactive oxygen species formation . In an in-depth review on whether chronic kidney disease modifies vascular calcification risk, heterogeneous conclusions were found such that some large scale studies yielded neutral results with a few noting higher vascular calcification risk in females, similar to our study . Interestingly, our results for the prevalence of mitral and aortic calcification are concordant of those published in patients without ESRD. Repeated studies have demonstrated that men have a greater degree of aortic valve calcification as well as faster rates of calcification progression along the valve leaflets , . On the other hand, women have shown an enhanced predisposition toward mitral annular calcification compared to men, which was corroborated by our study , . While the molecular mechanisms have not been distinctly identified, such findings suggest a gender-related pathophysiologic process responsible for calcification at certain sites. Clearly, there are many factors unaccounted for that modulate the deposition of calcium between sexes. Whether dialysis modality has an impact on the accumulation of valvular calcification is unknown and cannot be determined by the results of our study, though there may be insights to be gained from future studies.Our results require cautious interpretation as the size of our cohorts was small, especially that of the PD population. The number of years on PD was also limited and could have influenced our outcomes. PD patients had higher rates of smoking and diabetes, though not significant, which could have played a role in the progression of valvular calcification. These results also could have been explained by a potentially lower degree of compliance exhibited by peritoneal dialysis patients in the face of more demanding daily regimens . The degree of calcification was analyzed as presence vs. absence because there were not enough patients to stratify their scores into mild, moderate, and severe. Therefore, we opted to focus on the prevalence of CVC. It is also important to note that these patients may have had differing lengths of pre-existing chronic kidney disease, which may have in part contributed to the degree of CVC beyond that of solely the period the patients were on dialysis. Seeing as the patients presented to our institution at different points in their disease process, the length of baseline CKD could not be quantified prior to dialysis commencement. Generalizations to a wider cardiac population (e.g. congenital heart disease, those lacking information of dialysis length) should be made with caution. Last, it is important to note that patients who undergo OHS typically tend to have lower degrees of valvular calcification.Our study is unique in that there are few studies that directly compared cardiac valvular calcification, rather than coronary artery calcification, in HD and PD populations using CT scoring. Further, both groups were comparable in their baseline characteristics and risk factors, as this study only included dialysis patients who had progressed to the point of requiring cardiothoracic intervention.5ConclusionIt appears that the severity of valvular calcification may not be worse in the hemodialysis patient population, and that there may be more factors at play in the progression of valvular calcification in end stage renal disease patients than what was previously thought. Future studies with larger populations should be designed to investigate whether a specific dialysis modality predicts a higher burden of valvular calcification, and whether this leads to worse outcomes after open heart surgery.Abbreviations CVCcardiac valvular calcificationHDhemodialysisPDperitoneal dialysisOHSopen heart surgeryCACcoronary artery calcificationCABGcoronary artery bypass graftESRDend stage renal disease Sources of supportNone.CRediT authorship contribution statementConceptualization: C Kanaan, H Layoun, S Harb, G Nakhoul; Data Curation: C Kanaan, H Layoun, N Kondoleon, R Fadel, S Mirzai; Formal Analysis: S Arrigain, J Schold, H Layoun; Roles/Writing: C Kanaan, H Layoun, S Arrigain, S Harb, G Nakhoul; Manuscript Review: All authors.Declaration of competing interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

PMC

Arthroplasty Today

PMC10517266

9-19-2023

10.1016/j.artd.2023.101186

Safe Zones and Trajectory of Femoral Pin Placement in Robotic Total Knee Arthroplasty

Carvajal Alba Jaime, Constantinescu David S., Lopez Juan D., Lepkowsky Eric R., Hernandez Victor H., Jose Jean

BackgroundRobotic-assisted total knee arthroplasty may result in array pin-related complications. Lack of knowledge on ideal pin placement results in varied insertion sites and trajectory, with unknown risks to surrounding neurovascular structures.MethodsThis study included 10 lower-extremity magnetic resonance images. Images were subdivided into 6 zones of study. Zones consisted of a correlating axial image with femoral pin placement replicated by drawing a line angled 45° from the anterior to posterior reference in the anteromedial to posterolateral femoral quadrants. The distances from the pin paths to the neurovascular structures were measured.ResultsZone 2C demonstrated femoral pin trajectory an average of 14 mm from the femoral artery/vein. In Zone 2B, proximity increased to an average of 30 mm to the femoral artery and 29 mm to the femoral vein. At Zone 1A, the popliteal artery and vein were on average 22 mm from the femoral pin, while the common peroneal nerve was an average of 21 mm. Placing pins in Zone 1A poses a high risk of injury to the genicular arteries. Women demonstrated greater proximity to neurovascular structures than men in 66% of the sites (P < .05).ConclusionsThis classification system for safe zones and trajectory of femoral pin placement in robotic-assisted total knee arthroplasty demonstrates that proximally, the profunda femoris and femoral artery/vein are at risk of injury, while distally, the genicular arteries, common peroneal nerve, and popliteal artery/vein are at risk. Caution should be exercised if femoral pins are inserted with an angle less than 45°, especially in women.

IntroductionThe emerging popularity of technology-assisted total knee arthroplasty (TKA), particularly robotic-assisted total knee arthroplasty (RATKA), has introduced a unique set of complications when compared to conventional instrumentation . The need for femoral and tibial arrays creates the potential for pin-related complications [, , , ]. While increasing awareness focuses on the risk of pin-site fracture or infection, scarce literature exists on the risk of iatrogenic injury to the surrounding soft tissues due to pin placement. As such, the risk to surrounding neurovascular structures has yet to be determined.Limited knowledge of ideal pin placement leads to varied insertion and trajectory, largely based on surgeon preference. Extensive research has been conducted on complications associated with external fixation pin sites, primarily focusing on the incidence of pin-site irritation and infection, and placing significant emphasis on preventive measures [6,7]. Among the reported complications, one notable concern is the occurrence of bone loss after pin removal, leading to the formation of a stress riser and subsequently increasing the risk of fractures . However, there is a lack of studies investigating the potential damage to neurovascular structures, with limited findings restricted to case reports [8,9]. Placement of tibial array pins can be safely performed using knowledge of external fixation of the lower extremity . Insertion of the femoral array pin is typically performed from an anteromedial to posterolateral direction, which differs from conventional femoral external fixator application . Avoidance of a direct anterior to posterior pin placement should be exercised as it has been demonstrated to be associated with increased periprosthetic fracture . Available literature demonstrates that a high risk to neurovascular structures exists when utilizing manufacturer recommended technique for femoral array pin placement . Therefore, the primary aim of our study was to measure the proximity of surrounding neurovascular structures to the projected femoral pin placement throughout the femur. Our secondary aim included analyzing the difference between sexes. We hypothesize that an increased risk to vascular structures is present proximally in the femur and to women compared to men.Material and methodsThe study included 10 lower-extremity magnetic resonance imaging (MRI) images from the hip through the knee, comprising 5 age-matched pairs of men and women. The femurs were divided into 3 equal zones by drawing a line from the greater trochanter to the knee joint on the coronal MRI in order to standardize measurements across varied heights. These were labeled as Zone 1 being the most distal zone, Zone 2 being the middle zone, and Zone 3 being the most proximal zone. Zones 1 and 2 were further subdivided into 3 sections to create 6 total zones of study (Fig. 1), labeled A, B, and C from distal to proximal within their respective zones. Zone 3 was excluded as this was deemed to be too proximal for feasible pin placement during surgery.Figure 1Coronal MRI images of a right femur divided into zones of projected femoral pin insertion sites. MRI, magnetic resonance imaging.For each zone of study on a coronal MRI of the femur, the correlating axial image was produced using scout mode technique (Fig. 2a). Whiteside’s line and a line perpendicular to it were drawn on each of the corresponding axial images to divide the femur into 4 quadrants in this plane. Consistent with manufacturer technique, femoral pin placement was replicated by drawing a line angled 45° from the anterior to posterior reference in the anteromedial quadrant (Fig. 2b). This line was extended beyond bicortical limits in order to most accurately depict the risk that femoral pin placement poses. The distances to identified neurovascular structures were then measured and recorded (Fig. 3).Figure 2Coronal MRI of right femur with corresponding scout mode axial MRI with neurovascular structures measured from projected femoral pin at 45° (a) and correlating intraoperative image of femoral array in insertion (b). MRI, magnetic resonance imaging.Figure 3Representative axial MRI of femur at the various zones of study with nearby neurovascular structures identified and distance measured. MRI, magnetic resonance imaging.The distances from the projected femoral pin paths to the surrounding neurovascular structures were measured using Philips IntelliSpaceRadiology 4.7 and recorded on Microsoft Excel (Microsoft Excel for Mac 2022; version 16.66.1). This software was additionally used to compute mean values and standard deviations for men, women, and combined for each of the 6 zones. In order to compare men and women, a paired 2-tailed T-test function was performed on Excel. A statistically significant result was determined by setting the P-value at 0.05.Study exemption was approved by the institutional review board.ResultsOf the 10 MRIs reviewed (5 men, 5 women), the mean age was 72.2 (men: 72.2, women: 72.2, P > .05). The laterality consisted of 6 right femurs and 4 left femurs. The popliteal artery and vein, as well as tibial nerve and common peroneal nerve, were within proximity in Zone 1A. Proximally to the bifurcation, in Zones 1C-2C, the femoral artery as well as sciatic nerve were in proximity to the proposed femoral pin path. Most proximally, at Zone 2C, the profunda femoris becomes an additionally present vascular structure. These results, with specific measurements, are summarized in Table 1.Table 1Distance to neurovascular structure of a femoral array pin according to zones along the femur.ZoneDistance to neurovascular structure ± standard deviation (mm)Femoral arteryProfunda femorisFemoral veinPopliteal arteryPopliteal veinSciatic nerveTibial nerveCommon peroneal nerve1A---22 (±4.4)22 (±5.3)-31 (±9.8)21 (±9.2)1B29 (±5.6)---22 (±4.1)--31 (±10)1C38 (±6.7)---30 (±6.2)35 (±10)--2A40 (±8.0)-38 (±8.8)--35 (±11)--2B30 (±9.0)-29 (±6.8)--36 (±8.1)--2C14 (±7.4)23 (±7.3)14 (±7.6)--32 (±8.3)--The closest neurovascular structure to the proposed femoral pin path differed according to zone along the femur. In 4 out of 6 zones, women had a statistically significant shorter distance to neurovascular structures than men (Table 2).Table 2Distance to neurovascular structure of a femoral array pin according to zones along femur, grouped by sex.ZoneNeurovascular structure in closest proximityDistance to neurovascular structure ± standard deviation (mm)P valueMenWomen1ACommon peroneal nerve26 (±7.6)14 (±7.0).03a1BPopliteal vein25 (±4.2)20 (±2.6).071CPopliteal vein35 (±3.2)26 (±5.1).01a2ASciatic nerve42 (±8.7)28 (±7.4).03a2BFemoral vein34 (±3.2)25 (±7.1).04a2CFemoral artery16 (±9.4)11 (±3.7).24aP < .05 denotes statistical significance.DiscussionCurrently, there is no established standard for the placement of femoral pins in RATKA with regards to the safety of neurovascular structures. Neurovascular injury in TKA has been studied extensively in both primary and revision settings [, , ]. When performing RATKA, the femoral array pins must be placed more proximally, so the concerns of pin placement exist throughout the length of the femur. The most important finding of this study is that femoral pin placement from the anteromedial to posterolateral quadrant at a 45° angle poses significant risk to the neurovascular structures within 1-2 cm throughout the proximal and distal femur. Specifically, Zone 2C demonstrated femoral pin trajectory an average of 14 mm from the femoral artery and vein, and Zone 1 A demonstrated the popliteal artery and vein were on average 22 mm from the femoral pin, while the common peroneal nerve was an average of 21 mm. This risk was additionally increased in women. Furthermore, placing pins at the most distal zone (1A) poses a high risk of injury to the genicular arteries. In a cadaveric study, Barner et al. mapped out the genicular arteries of 46 knees and found that the superior medial geniculate and superior lateral geniculate were 57.3 mm ± 8.1 mm and 55.2 ± 6.2 mm from the joint line, respectively. In the 10 studied MRIs, the most distal zone is consistently just below this level (Fig. 4). Given these findings, we recommend avoiding pin placement this distally in the femur.Figure 4Coronal MRI of right femur with corresponding scout mode axial MRI at most distal zone (1A) with geniculate arteries labeled (white arrows) and projected femoral pin at 45°. MRI, magnetic resonance imaging.While study on pin site placement has been sparse, there is previous literature that does report on the potential dangers to neurovascular structures. In their cadaveric study, Marchant et al. described pin placement in the distal most quadrant of the femur with a trajectory of 30 degrees as passing within 5 mm of the sciatic and peroneal nerve in 100% of cases. In comparison, our measurements performed at a 45° angle increased the average distances to 21 mm. This highlights the posterior location of neurovascular structures and that surgeons should be cautious to not err with an acute angle trajectory less than 45° from the anterior to posterior plane. Our finding that women had a greater risk of proximity to neurovascular structures is also consistent with the established literature .Our study did have several limitations. First, the limited sample size of 10 (5 men and 5 women) MRIs potentially reduces the accuracy and generalizability for the results of the mean distance values from neuromuscular structures. The limitation arose largely due to the narrow age group of focus. Future studies can expand on the number of samples analyzed. Next, significant differences in femur size and morphology exist. To accurately determine pin placement throughout the femur, we proposed division into zones instead of distance from the joint line, as is frequently described in manufacturer technique guides. This accounts for differences in heights and lengths of femurs, although this would require intraoperative measurement and assessment by the surgeon for each individual patient. As age would be unlikely to create anatomic variation in the patient population that would be undergoing TKA, we chose the most common age group to receive TKA and compared findings between men and women . Although variations in anatomy have been described with the knees in varying degrees of flexion , our study was performed with all samples supine in the MRI scanner, which may not replicate intraoperative conditions. Image analysis showed that correcting for femoral version did not significantly impact the projected femoral pin insertion trajectory and thus no impact on distance from pin to neurovascular structures; however, considerations should be taken for pin placement with altered limb alignment.ConclusionsOur proposed classification system for safe zones and trajectory of femoral pin placement in RATKA demonstrates that proximally, the profunda femoris and femoral artery/vein are at risk of injury, while distally, the genicular arteries, the common peroneal nerve, and popliteal artery/vein are at risk. Caution should be exercised particularly if femoral pins are inserted with an angle less than 45°, especially in women.Conflicts of interestVictor Hugo Hernandez is a board member of AALOS, MOS, and AAHKS International Committee and received Research support from OMEGA and OREF. Jaime Carvajal Alba is a part of speakers bureau/paid presentations and a paid consultant for Smith and Nephew Consultant. All other authors declare no potential conflicts of interest.For full disclosure statements refer to

PMC

Journal of Physical Therapy Science

PMC10149295

5-01-2023

10.1589/jpts.35.312

The sitting active and prone passive lag test: a validity study in a symptomatic knee population

Deepak Sebastian, Priti George, , Zishu Tsang, Chetan Patel

[Purpose] This study aimed to determine the diagnostic utility of the sitting active and prone passive lag test in identifying terminal extension lag in unilaterally symptomatic knees. The lack of full extension at the knee leads to greater force of quadriceps activation, overloading of the weight bearing joints, abnormal gait mechanics, resulting in pain and dysfunction. [Participants and Methods] Participants were randomly assigned and evaluated by two blinded examiners, to determine the presence of extension lag at the knee. The reproducibility of test results between examiners was determined, for reliability. In addition, the ability of the test to identify the presence of extension lag in symptomatic knees and absence of extension lag in asymptomatic knees was assessed, for validity. [Results] The results revealed the test to possess an ‘almost perfect’ inter-rater reliability, high sensitivity, and moderate specificity. [Conclusion] The sitting active and prone passive lag test may be incorporated as a reliable and valid test to determine the presence of terminal knee extension lag in a unilaterally symptomatic knee population.

INTRODUCTIONThe knee is a mobile joint and the available mobility or range of motion (ROM) determines its ability to engage in activities of daily living. ROM in the knee is a composite parameter comprising extension and flexion. The total knee ROM ranges from 0° extension to 140° flexion. Since limitations of knee ROM are observed during dysfunctional states its measurement and management has been an integral part of physical therapy practice1). Knee flexion has procured more attention than its counterpart extension, owing to its functional capabilities2). More recently, the need for normal knee extension has been gaining importance3). The clinical and functional consequences of its absence has made rehabilitation professionals target its restoration during management.Rehabilitation professionals have devised methods for evaluating range of motion at the knee joint1). Both knee flexion and extension have garnered equal attention, as the lack of terminal knee extension and its consequences have been adequately described4,5,6,7,8). Clinically termed extension lag or flexion deformity, the causes described ranged from joint degeneration, trauma, surgery and arthrogenic muscle inhibition (AMI), to nerve compression and neurological weakness9,10,11,12,13,14,15,16). Clinicians have recognized that a lack of terminal knee extension resulted in overuse, increased energy expenditure, gait abnormalities, pain and dysfunction of the involved and adjacent joints, and poor functional outcomes4, 17,18,19,20,21,22). The need for a reliable and valid clinical test for its accurate assessment and subsequently management was hence imperative.Despite knee extension lag being a common presentation in rehabilitation settings, the description of validated testing methods to assess its presence is remote. One study recommended assessing knee extension lag in the supine position with the ankle supported on a roll and the knee left unsupported. A lack of terminal knee extension with reference to 0 degrees of extension as being normal was visually assessed. Additionally, open kinetic chain knee extension was performed in the supine position to visually observe for a lack in the terminal ranges of knee extension5). Limitations of this description was the exclusion of the influence of the hamstrings and gastrocnemius. Additionally, the reported results were descriptive and a methodology to determine the diagnostic utility of the test was lacking. A more recently described sitting active and prone passive lag test (SAPLT) appeared to address these limitations23). The SAPLT appeared novel as it addressed the above concerns and additionally used the contralateral asymptomatic knee as a reference. Previous studies used 0 degrees of extension at the knee as a reference standard for normal knee extension range of motion. A normal knee, however, may not present with 0 degrees of extension consistently as a few degrees of hyperextension is a common finding in asymptomatic knees54). Achieving 0 degrees of knee extension would still create a functional asymmetry if the contralateral knee presented with hyperextension. Additionally, the SAPLT was the only test that appeared to test both active and passive restraints for knee extension, concurrently.While the results revealed the operant definition and reproducibility of the SAPLT to be favorable, its validity, however, was not established. In other words, there was no description as to whether the SAPLT consistently identified the presence of knee extension lag on the symptomatic side (true positive) and its absence on the asymptomatic side (true negative). The consistent ability of the test to do this would establish its usefulness or validity in identifying extension lag at the knee, and hence the purpose of this study.Hands-on clinical tests have continued to prevail in clinical practice alerting the clinician of issues that may not be identified by imaging or lab analysis. They are especially valuable in assessing musculoskeletal movement disorders and are termed impairment-based tests. They are primarily used to identify movement dysfunction24) and are required to be reliable or reproducible between examiners25). Reproducible clinical tests, however, would still be lacking in validity if they do not identify what they are intended to identify. The variables of interest seen in the evaluation of clinical test validity are sensitivity (Sn), specificity (Sp), predictive values (PPV/NPV) and likelihood ratios (LR).PARTICIPANTS AND METHODSThis study was a methodological design to develop test validity estimating parameters of relevance such as test reliability, sensitivity, specificity, predictive values, likelihood ratios, and receiver operating characteristic (ROC) curves of an individual impairment based diagnostic test. While the larger purpose of this study was to establish the validity and subsequently the diagnostic utility of the SAPLT, its ease of administration across different experience levels was also brought to light. Hence, two orthopaedic board certified and orthopaedic manual therapy fellowship trained physical therapists, with 20+ years of experience in orthopaedic physical therapy, along with one general physical therapist with 3 years of experience in orthopedic physical therapy participated in the testing process. They initially conducted pilot training on unilaterally symptomatic knees to familiarize the methodology of performing the SAPLT and understand its operant definition. An active lag was determined by the inability of the erectly seated participant to actively extend the involved knee, with the ankle in maximum dorsiflexion, to the same level as the normal knee held in a full knee extension and ankle dorsiflexion. This was determined by the low position of the toes on the involved side (Fig. 1Fig. 1. Test position for ‘active lag’ showing positive active lag on the right.). A passive lag was determined by placing the participant prone with the knees just past the edge of the table. With both legs fully extended and resting, the high position of the heel compared to the heel on the normal side determined the presence of a passive lag (Fig. 2Fig. 2. Test position for ‘passive lag’ showing positive passive lag on the right.). The differentiation between an active and a passive lag was considered mandatory with the assumption that the most appropriate intervention could then be instituted. An active lag may indicate the need to address muscle strength, while a passive lag may indicate the need to address tightness of the passive restraints. Once the ‘operant definition’ for consistency of performance was clearly understood by all participating testers, the study proceeded.Upon the approval of the Institutional Review Board of Henry Ford Health System, Detroit, Michigan (approval number 14921), 27 participants with unilaterally symptomatic knees (n=27) were randomly assigned and independently examined by 2 blinded musculoskeletal physical therapists at a time, to determine the presence of an active or a passive, extension lag at the knee, or both. The researchers examined both the symptomatic and asymptomatic knee of the 27 assigned patients thereby examining 54 knees in total. The average age of the individuals tested was 51. The inclusion criteria were individuals presenting with unilateral knee pain, stiffness, and weakness, with a normal and asymptomatic contralateral knee. The normal and asymptomatic knee was used as a reference side for normal knee extension range, however blinded to the testers. Individuals presenting with acute injuries or acute orthopaedic and rheumatological conditions requiring immediate medical attention were excluded from testing. Individuals who were not willing to sign the informed consent were also excluded from testing. The diagnoses that the tested individuals presented with were knee osteoarthritis, patellofemoral pain, meniscus and ligament pathology, stabilized trauma and synovitis, and post-operative conditions, primarily knee joint replacements, ligament repairs and meniscectomies.The schedules of the individuals tested were uploaded by the administrative department into the computer scheduling system. This was done on a recurring referral basis. As random recurrent referrals from different referral sources of the health system appeared on the schedule, the ‘assigner’ oversaw identifying those patients who met the inclusion criteria and prepared them for testing. The preparation and testing of individuals who met the criteria were done in the same order as the recurring referral scheduling with no alterations for convenience. The ‘assigner’ was the physical therapist who positioned the participant in the test positions for the 2 blinded testers. The responsibility of the assigner was to position the participants in the sitting and prone test positions and summon the raters for testing. A total of 3 raters participated in the study however, at a given time, only two raters tested. The knees were draped, or the participant was allowed to have their exercise pants with shoes on, to blind the testers from signs of obvious swelling, redness or a surgical scar that may indicate the symptomatic side. Since test validity measures true positives (a positive test on the symptomatic side), the knees were concealed to not bias the tester whose reporting might then favor the symptomatic side. For this reason, the testers were not permitted into the testing area when the positions of the participants were changed. The test was performed once, and both clinicians observed the results simultaneously mainly by the positions of the heels, and toes. They independently recorded their finding for both parts of the test on the recording sheet. Upon completion of the testing, the assigner marked the symptomatic side on the recording sheet for future reference and statistical analysis. The clinicians were considered to be in agreement when they agreed individually on the active and the passive components of the test, identified as positive or negative. The test was considered valid if a positive test was identified consistently on the symptomatic side and a negative test was identified consistently on the asymptomatic side.Data analyses were performed on IBM statistical package for social sciences (SPSS) version 28.0.0.0 , Graph Pad kappa calculator, MedCalc statistical software and by utilizing mathematical formulas to calculate values obtained from the contingency table. The data collected consisted of True positives (TP), False positives (FP), False negatives (FN) and True negatives (TN) and were represented and calculated on a 2 × 2 contingency table26) (Table 1Table 1. Contingency tableTestPresentAbsentTotal+VETPFPTP+FP−VEFNTNFN+TNTotalTP+FNFP+TNTotalTP: true positives; FP: false positives; FN; false negatives, TN: true negatives.). The parameters of relevance were Sensitivity, Specificity, Predictive Values and Likelihood Ratios (Fig. 3Fig. 3. Nomogram for likelihood ratio.). Sensitivity (Sn) is the percentage of participants with a knee dysfunction who test positive on the dysfunctional side and is calculated as Sn=TP/(TP+FN). A test that is highly sensitive is considered good at ruling out disease or an impairment and are good for screening purposes. Specificity (Sp) is the percentage of participants without a knee dysfunction who test negative on the non-dysfunctional side and is calculated as Sp=TN/(FP+TN). A test that is highly specific is considered good at ruling in disease or an impairment and are good confirmatory tests. Likelihood Ratio (LR) represents the probability of a patient with a disease or dysfunction having a positive or negative test result in comparison to the probability of a patient without the disease having a positive or negative test result. Positive LR (+ve LR) suggests the number of times more likely a positive test result would be observed in the symptomatic knee versus the asymptomatic knee. It suggests how well disease is ruled in. Positive likelihood ratio is calculated as +ve LR=sensitivity/1-specificity. Negative LR (−ve LR) suggests the number of times more likely a negative test result would be observed in the symptomatic knee versus asymptomatic knee. It suggests how well disease is ruled out. Negative likelihood ratio is calculated as −LR=1-sensitivity/specificity. Positive predictive value (PPV) is the percentage of positive test results that are true positives and is calculated as PPV=TP/(TP+FP). Negative predictive value (NPV) is the percentage of negative test results that are true negatives and is calculated as NPV=TN/(FN+TN).The reliability of a clinical test is the extent to which results are consistent between different and blinded examiners across separate occasions of testing. The weighted kappa is used to measure association as the scale of measurement was ordinal. Hence, inter-rater reliability was determined using the kappa statistic27).Receiver operating characteristic (ROC) curves are a graphical depiction of a test’s overall diagnostic performance28). The Y axis represents Sensitivity, and the X axis represents 1-Specificity. The closer the curve fills out the top left corner, the better the test performance is quantified by the area under the curve (AUC). An AUC of 0.5 states that the test performs no better than chance (not useful). An AUC of 0.9 suggests a better-performing test.RESULTSFor the SAPLT, the inter-rater reliability was k=0.926 (SE of kappa=0.072, 95% confidence interval) indicating almost perfect agreement (Table 2Table 2. Reliability resultsABTotalA26127B12627Total272754Number of observed agreements: 26 (96.30% of the observations).Number of agreements expected by chance: 13.5 (49.93% of the observations).Kappa=0.926, SE of kappa=0.072.95% confidence interval: from 0.784 to 1.000.). Twenty five of the 54 knees tested resulted in a positive test elicited on the symptomatic side and 24 of the 54 knees tested resulted in a negative test on the asymptomatic side. Five of the 54 knees tested resulted as a false positive test (Table 3Table 3. Validity tableTestPresentAbsentTotal+VETPFPTP+FP=30255−VEFNTNFN+TN=24024TotalTP+FN=25FP+TN=2954TP: true positives; FP: false positives; FN; false negatives, TN: true negatives.). Calculation for test validity was performed utilizing the formulas to calculate Sn, Sp, positive and negative LR and PPV respectively (Table 4Table 4. Validity resultsStatisticValue95% CISensitivity (%)100.0086.28 to 100.00Specificity (%)82.7664.23 to 94.15Positive likelihood ratio5.80/5.52.61 to 12.87Negative likelihood ratio0.00Positive predictive value (%) 83.3369.25 to 91.73Negative predictive value (%) 100.00Overall accuracy (%)90.7479.70 to 96.92CI: confidence interval.).Test validity calculation revealed Sn=1.0, Sp=0.82, +ve LR=5.0, −ve LR=0.0, PPV of 0.83, NPV of 1.0 and an overall accuracy of 90.74% (Fig. 4Fig. 4. Receiver operating characteristic (ROC) analysis.). A highly sensitive test means that there are few false positive results, and thus fewer cases ‘with’ the disease are missed. A highly specific test means that there are few false negative results, thus fewer cases ‘without’ the disease are missed. A test with a sensitivity and specificity of around 0.9 (90%) is generally considered to have clinically meaningful and acceptable diagnostic performance. The results suggest the SAPLT to be a sensitive test, however lacking specificity. A positive LR of 10 or greater results in a large and significant increase in the probability of a disease, and a negative LR of 1 or lesser suggests less likely the disease or outcome29). The results of this study indicated a high negative likelihood ratio suggesting that if the SAPLT is negative, the less likely the individual will ‘have’ the impairment or disease. The greater the positive predictive value, the more specific the test and less likely an individual with a positive test will ‘not have’ the disease or impairment30). The results of this study revealed a greater negative predictive value indicating the SAPLT to be a more sensitive test and that an individual with a negative test is less likely to ‘have’ the impairment. ROC analysis revealed the curve filling out the top left corner with an AU of 0.964, suggesting a better-performing test (Fig. 4).DISCUSSIONThe results of this study suggest the SAPLT to be a reliable and valid test to evaluate the presence of extension lag at the knee. The results revealed the test to possess an ‘almost perfect’ inter-rater reliability, high sensitivity, and moderate specificity. While diagnostic tests assess the presence of a disease or disorder, the SAPLT was used to evaluate impairment and not disease. The SAPLT may hence be used as a predictor of pain, imminent dysfunction, or functional challenges such as gait disorders31). In diagnosis literature the term ‘gold standard’ or ‘reference standard’ is often encountered32). It is used to describe a diagnostic test that is regarded as definitive for a particular disease, and thereby becomes the ultimate measure for comparison. In methodological designs where the diagnostic utility of a particular test is studied, the blinded examiner applies the test on a symptomatic and asymptomatic population where the symptomatic participants are evaluated based on a gold standard. An impairment-based test, however, may often lack a gold standard as the impairment is visually or clinically obvious. The deep neck flexor endurance test (DNF) that is most preferred by clinicians to assess the strength of the deep flexors of the neck33) is an example as there is no gold standard described to assess weakness of the deep neck flexors. The SAPLT may also be considered an impairment-based test that may operate without a reference standard as there is no gold standard described to assess terminal extension lag at the knee. Although the results of this study suggest the SAPLT to be reliable and valid, the lack of a reference standard may mean that it is inadequate to test individuals with bilaterally symptomatic knees.The single largest advantage of an impairment-based test is that while it correlates current or predicts imminent dysfunction it also suggests the most appropriate and directly relevant treatment intervention. As an example, while weakness of the deep neck flexors has been described to cause neck dysfunction33), a positive DNF test also suggests the need for deep neck flexor strengthening. Similarly, the SAPLT firstly correlates current or predicts imminent knee dysfunction. In addition, it suggests appropriate management of flexibility or strength training, or both. If the passive component appears positive, the appropriate intervention may be to prioritize those structures passively. As a speculation the appropriate intervention may be to first lengthen the posterior capsule, hamstrings, and gastrocnemius prior to strengthening the quadriceps and the gastrocnemius for an effective plantarflexion knee extension force couple. This speculation however requires validation.An active knee extension lag is described as the inability to actively extend the knee into full range of extension, in the absence of a passive restraint. The quadriceps has been described as the primary mechanism that performs this activity. The quadriceps may be directly challenged by injury, surgery, or neurological compromise of the femoral nerve9,10,11,12,13,14,15,16). The femoral nerve can be compressed along its course due to penetrating, compressive, and iatrogenic injuries, mostly over the anterior aspect of the hip and inguinal area. Mechanical compression may range from prolonged postures of hip flexion as in sitting on a low chair35) or gynecologic procedures in the lithotomy position36). While a direct injury to the extensor mechanism disrupts the ability of the knee to extend fully, another mechanism that occurs following trauma, surgery, progressive wear and tear or immobilization is a process known as arthrogenic muscle inhibition (AMI). While AMI is ubiquitous across knee joint pathologies, its severity may vary according to the degree of joint damage, time since injury, and knee joint angle4). AMI is caused by a change in the discharge of articular sensory receptors, and challenges to the spinal reflex pathways. Additionally, a poor cortical representation can also contribute to inhibitory states of the quadriceps12, 34).Passive restraints to knee extension are relatively rigid and sometimes termed as flexion deformity. The description of a passive restraint is the inability to extend the knee fully both passively and actively with the continued ability to flex the knee. The posterior capsule of the knee is described as the primary passive restraint to terminal knee extension with the hamstrings, popliteus, and gastrocnemius as adaptive contributors13, 15, 37). While all causes described for an active lag may cause a passive lag, the assumed position of comfort of knee flexion in a painful knee38) and the avoidance of terminal knee extension in individuals who present with ACL deficient knees are other causes39, 40).The presence of an extension lag at the knee might indicate a compromise of the normal functioning of the lower kinetic chain during functional activities. The implication is higher when this compromise is persistent for an extended period. In closed kinetic chain, the quadriceps and the plantarflexion knee extension couple have been described to work eccentrically during the early stance phase of gait and to assist in knee extension during terminal stance, respectively4, 41). This collectively is described to sufficiently absorb shock, reducing impulsive loading at the knee. Quadriceps weakness has also been associated with an increased rate of loading at the knee joint, contributing to persistent knee pain, patellofemoral cartilage loss and tibiofemoral joint space narrowing42,43,44).The motions at the knee, flexion, and extension, are accompanied by rotation45, 46). Extension at the knee is accompanied by tibial external rotation (11.40° ± 3.0°), also termed ‘screw home’45, 46) and flexion is accompanied by tibial internal rotation (11.55° ± 3.20°), with musculature responsible for the rotations produced47). The clinical measurement of knee range of motion of flexion and extension has been standardized1), but a clinical method to measure or demonstrate the presence of tibial external and internal rotation is currently unavailable. While the literature supports tibial internal and external rotation to be occurring with knee flexion and extension respectively45), the assumption that lack of terminal knee extension has rendered the tibia to be in a position of relative internal rotation may be justified. In other words, the presence of a positive SAPLT may indicate the presence of tibial internal rotation (lacking screw home), however, not specified in degrees. This clinical finding, as described in introductory section, has strong clinical and functional implications warranting resolution, to prevent undue stress of the vulnerable structures. Uncontrolled or persistent internal rotation of the tibia, especially in closed kinetic chain has been described to unduly stress the tibiofemoral articular cartilage48), the menisci49), the supporting ligaments principally the anterior cruciate ligament (ACL)50) and the patellofemoral joint51, 52).The SAPLT has demonstrated excellent reproducibility between examiners and has shown to be reliable and sensitive in identifying the presence of an extension lag in unilaterally symptomatic knees. Additionally, the test has demonstrated the ability to differentiate between the presence of an active versus a passive lag or identify the presence of both, enabling the clinician to institute the most appropriate management. There is ample evidence to suggest that the resolution of a persistent knee extension lag can decrease undue stresses over multiple structures in and around the knee, decreasing the risk for dysfunction. This warrants the prompt identification of the presence of a lack of knee extension and the SAPLT may serve as an easily administered reliable and valid method. The test however poses limitations as tightness of the iliopsoas in causing a lag is ignored53). Additionally, the maintenance of an erect and normal lordosis and maximum ankle dorsiflexion may be difficult to perform or missed during the testing process rendering results to be inaccurate23). While a reference standard may be appropriate for testing diagnostic utility, establishing one for an impairment-based test like the SAPLT might be a challenging task. While the gold standard for measuring ROM is electro goniometry, many ‘normal’ knees may hyperextend past zero degrees of extension, questioning ‘normal ‘even if accurate ROM is established. Clinically however, the reference standard limitation suggests that the SAPLT might be best suited for identifying the presence of a knee extension lag in unilaterally symptomatic knees. The SAPLT appears to differentiate an active versus a passive restraint for knee extension, however, does not denote the exact structure. Additional testing past the SAPLT might be required to identify the exact structure to offer a direction toward specific management. The SAPLT is a test applied in an open kinetic chain (OKC) position and is translated to activities and functional consequences in a closed kinetic chain (CKC). This assumes that an impairment observed in OKC can directly translate to an activity in CKC, a claim not fully justified. These limitations may be suggestions for future research.Funding and Conflict of interestNo funding was obtained by the researchers for this study and the authors of this study do not report any conflict of interest.

PMC

Diabetes, Metabolic Syndrome and Obesity

PMC10404410

8-02-2023

10.2147/DMSO.S416280

Survival of Patients with Hepatitis B-Related Hepatocellular Carcinoma with Concomitant Metabolic Associated Fatty Liver Disease

You Yajing, Yang Tao, Wei Shuhang, Liu Zongxin, Liu Chenxi, Shen Zijian, Yang Yinuo, Feng Yuemin, Yao Ping, Zhu Qiang

PurposeMetabolic associated fatty liver disease is a novel concept defined as fatty liver associated with metabolic disorders. We investigated the effect of metabolic associated fatty liver disease on hepatocellular carcinoma patient mortality.Patients and MethodsA total of 624 patients with hepatocellular carcinoma between 2012 and 2020 were enrolled in this retrospective study. Hepatic steatosis was diagnosed using computed tomography or magnetic resonance imaging. Metabolic associated fatty liver disease was defined based on the proposed criteria in 2020. Propensity score matching was performed for patients with metabolic associated fatty liver disease and those without the condition. A Cox proportional hazards regression model was used to evaluate the association between metabolic associated fatty liver disease and hepatocellular carcinoma patient outcomes.ResultsPatients with hepatocellular carcinoma and metabolic associated fatty liver disease tended to achieve better outcomes than did those without metabolic associated fatty liver disease after matching (p<0.001). Metabolic associated fatty liver disease was significantly associated with better prognosis in patients with concurrent hepatitis B infection (p<0.001). Moreover, high levels of hepatitis B viral DNA in serum samples was associated with a significantly increased risk of death in patients without non-metabolic associated fatty liver disease (p=0.045). Additionally, the association between metabolic associated fatty liver disease and survival in hepatitis B virus-related hepatocellular carcinoma was similar in all subgroups based on metabolic traits.ConclusionMetabolic associated fatty liver disease increases the survival rate of patients with hepatocellular carcinoma and hepatitis B virus infection. The potential interaction of steatosis and virus replication should be considered for future research and clinical treatment strategies.

Plain Language SummaryThe current study explores the association between metabolic associated fatty liver disease (MAFLD) and chronic hepatitis B-related hepatocellular carcinoma (HBV-HCC) in terms of the prognosis. It shows that HBV-related HCC patients with MAFLD have a better outcome than those without MAFLD. Risk factors for HCC, such as positive HBeAg (Hepatitis B e-antigen), high load of HBV DNA, and metabolic traits, do not have a significant effect on the MAFLD group. A novel finding was that patients with MAFLD achieved better survival among HCC patients with hepatitis B infection, which may be due to the interaction between steatosis and virus replication. Drawing on discoveries in the interaction between MAFLD and HBV-HCC, we propose that further patient stratification is necessary for MAFLD group, considering the heterogeneous nature of MAFLD and the complex pathogenesis of HBV.IntroductionMetabolic associated fatty liver disease (MAFLD) is a novel concept proposed by an international consensus in 2020, defined as hepatic steatosis with diabetes, obesity, or at least two metabolic abnormalities.1 Considering the increasing incidence of metabolic disorders, MAFLD has been associated with the development of various cancers.2,3 Hepatocellular carcinoma (HCC), the predominant primary malignancy of the liver, is one of the leading causes of mortality in patients with cancer.4,5 However, as MAFLD criteria have recently been developed based on consensus, there are limited data regarding the characteristics of patients with MAFLD-HCC, especially in Asians.The higher incidence of HCC in Asia than in other regions of the world is still related to predominance of chronic hepatitis B (CHB).6,7 Unfortunately, the previous term NAFLD was defined as the exclusion of other chronic liver diseases, making it harder to focus on metabolic disorders in CHB patients.8 Thus, the new MAFLD definition makes its coexistence with CHB possible. Accompanied by the rapid increase in the prevalence of metabolic disorders, the coexistence of metabolic syndrome and CHB is commonly observed.9–11 In hepatitis B virus (HBV) carriers, metabolic factors such as obesity and diabetes are well-established risk factors for HCC; however, hepatitis steatosis is found to be associated with a lower incidence of HCC.12 Thus, owing to the combined effects of these metabolic factors, MAFLD represents a complex multisystem disorder, and the impact of MAFLD on the clinical outcomes of CHB-HCC patients remains unknown.As such, we performed a retrospective analysis to investigate the effect of MAFLD on the long-term survival of patients with HCC. We also explore the association between MAFLD and CHB-related HCC in terms of the prognosis.Materials and MethodsPatientsThe study included a total of 848 patients with HCC who were admitted to Shandong Provincial Hospital Affiliated with Shandong First Medical University, Shandong, China, between June 2012 and June 2020. Patients underwent computed tomography (CT) or magnetic resonance imaging (MRI) before initial treatment. Patients were identified according to the following inclusion criteria: Age ≥18 years; Initial diagnosis of HCC confirmed by histopathology and radiographic evaluation; Available imaging data for the assessment of hepatic steatosis; and No other malignant neoplasms within the last 5 years. The exclusion criteria were as follows: Patients with HCC who underwent transcatheter arterial chemoembolization or other conservative treatments; Patients with hepatic steatosis with lacking body mass index (BMI) information; or Patients without any MAFLD inclusion criteria. A total of 624 patients were enrolled, including 199 with MAFLD and 425 without MAFLD (Figure 1). This study was approved by the institutional review board of Shandong Provincial hospital, and the requirement for informed patient consent was waived due to the retrospective nature of the study. Figure 1Patient flow diagram.Abbreviations: CT, computed tomography; HCC, hepatocellular carcinoma; MAFLD, metabolic dysfunction-associated fatty liver disease; MRI, magnetic resonance imaging; PSM, propensity score matching.Diagnosis of Liver DiseaseMAFLD cases were defined as radiologically diagnosed hepatic steatosis in combination with at least one of the following characteristics: Overweight (BMI ≥ 23 kg/m2 for Asians); Type 2 diabetes mellitus; and Evidence of metabolic dysregulation. Metabolic dysregulation was defined by the presence of two or more of the following metabolic abnormalities: Blood pressure ≥130/85 mmHg or specific drug treatment; 2) Plasma triglycerides ≥1.70 mmol/L or drug treatment; 3) Plasma HDL-C 0)45 (22.6)94 (22.1)0.89041 (22.0)45 (24.2)0.623AFP (>1000 ng/mL)37 (19.1)81 (19.6)0.88637 (20.3)42 (23.0)0.543Histopathology0.4260.310 Well17 (9.9)28 (7.0)17 (10.3)10 (5.7) Moderate120 (70.2)297 (74.4)115 (69.7)128 (73.1) Poor34 (19.9)74 (18.6)33 (20.0)37 (21.1)Tumor number (>1)32 (16.2)69 (16.2)0.98132 (17.2)29 (15.6)0.674Tumor size (cm)+3.8 (2.6–6.3)4.0 (2.7–6.9)0.1843.9 (2.6–6.4)4.5 (2.7–7.0)0.066Microvascular invasion42 (21.2)94 (22.2)0.78842 (22.6)52 (28.0)0.233Metastases9 (4.5)36 (8.5)0.0779 (4.8)16 (8.6)0.213Satellite nodules21 (10.6)50 (11.8)0.66521 (11.3)22 (11.8)0.871Child B and C18 (9.1)28 (6.6)0.27317 (9.1)18 (9.7)0.859MELD+7.0 (7.0–8.0)8.0 (7.0–9.0)0.0407.0 (7.0–8.0)8.0 (7.0–9.0)0.089BCLC0.8750.772 014 (7.2)23 (5.4)10 (5.4)12 (6.5) A38 (19.6)77 (18.2)37 (19.9)32 (17.2) B89 (45.9)197 (46.6)87 (46.8)80 (43.0) C50 (25.8)119 (28.1)49 (26.3)59 (31.7) D3 (1.5)7 (1.7)3 (1.6)3 (1.6)CNLC0.9830.762 I130 (67.0)278 (65.9)122 (65.6)113 (60.8) II13 (6.7)27 (6.4)13 (7.0)13 (7.0) III48 (24.7)110 (26.0)48 (25.8)57 (30.6) IV3 (1.6)7 (1.6)3 (1.6)3 (1.6)Initial therapy0.0080.587 Liver resection112 (56.3)281 (66.1)111 (59.7)103 (55.4) Laparoscopic resection61 (30.7)120 (28.2)56 (30.1)69 (37.1) Others26(13.0)24(5.7)19 (10.2)14 (7.5)HBeAg positive43 (21.6)95 (22.6)0.78940 (21.5)35 (18.8)0.518HBV DNA (log10 IU/mL)+2.8 (1.8–4.9)3.0 (1.6–4.8)0.7772.8 (1.9–4.9)2.3 (1.4–3.9)0.078Notes: +Data are medians, with interquartile ranges in parentheses. p value of < 0.05 was considered statistically significant and shown in bold values. Except where indicated, Data are numbers of patients, with percentages in parentheses.Abbreviations: AFP, alpha fetoprotein; ALBI, albumin-bilirubin grade; BCLC, Barcelona Clinic Liver Cancer staging system; BMI, Body mass index; Child B and C, Child-Pugh class B and C; CNLC, China liver cancer staging system; ECOG PS, Eastern Cooperative Oncology Group performance status; HBV, hepatitis B virus; HCV, hepatitis C virus; HBeAg, hepatitis B e antigen; HCC, hepatocellular carcinoma; MELD, model for end-stage liver disease; MAFLD, metabolic dysfunction-associated fatty liver disease; PSM, propensity score matching; SMD, standardized mean difference.Outcomes in HCC PatientsIn the total cohort, the median follow-up was 87 months (95% CI 80.2–93.8), and the median OS between patients with and without MAFLD was not significantly different (p = 0.48) (Figure 2A). After PSM, the median OS were 87 and 40 months in the MAFLD and non-MAFLD groups, respectively (p < 0.001) (Figure 2B). For patients with CHB, the median OS was significantly lower in the non-MAFLD (31 months) than in the MAFLD group (87 months) (p < 0.001) (Figure 2C). For patients without HBV infection, the median OS was not significantly different between the MAFLD and non-MAFLD groups (p = 0.56) (Figure 2D). Figure 2(A) OS before matching. (B) OS after matching. (C) OS in patients with hepatitis B after matching. (D) OS in patients without hepatitis B after matching.Abbreviations: HCC, hepatocellular carcinoma; MAFLD, metabolic dysfunction-associated fatty liver disease; OS, overall survival.Outcomes in MAFLD and Non-MAFLD PatientsFor CHB-HCC patients in the PSM cohort, the median OS of the MAFLD group was not significantly different between the hepatitis B e-antigen (HBeAg)-positive and HBeAg-negative groups (p = 0.19) (Figure 3A). A similar result was found among non-MAFLD individuals (p = 0.40) (Figure 3B). Moreover, the outcome of patients with MAFLD was not influenced by the serum HBV DNA level (p = 0.93), which was shown to be an important risk factor for non-MAFLD participants (p = 0.045) (Figure 3C and D). Figure 3(A) Patients with MAFLD stratified by HBeAg status. (B) Patients with MAFLD stratified by HBV DNA levels. (C) Patients with non-MAFLD stratified by HBeAg status. (D) Patients with non-MAFLD stratified by HBV DNA levels.Abbreviations: CHB, chronic hepatitis B; HCC, hepatocellular carcinoma; MAFLD, metabolic dysfunction-associated fatty liver disease; HBeAg, hepatitis B e-antigen; HBV, hepatitis B virus.Risk Factors for Outcomes of Patients with Hepatitis BFor patients with CHB after matching, the univariate analysis in Table 2 showed that Eastern Cooperative Oncology Group (ECOG), alpha fetoprotein (AFP), liver function reserve (Child–Pugh), and tumor characteristics (histopathology, tumor size, microvascular invasion, metastasis, and satellite nodules) were significant factors for poor OS, as expected. MAFLD (p 0)2.01 (1.35–2.99)0.0012.13 (1.40–3.24)0.000Alcohol1.32 (0.88–1.97)0.186AFP (>1000 ng/mL)1.77 (1.17–2.68)0.0070.81 (0.50–1.32)0.398Child B and C2.60 (1.54–4.38)0.0001.58 (0.87–2.88)0.133Histological grade0.0410.193 Well(ref)(ref) Moderate2.43 (0.76–7.76)0.1331.91 (0.46–7.94)0.372 Poor3.77 (1.13–12.61)0.0312.76 (0.63–12.00)0.177Tumor number (>1)1.42 (0.90–2.26)0.136Tumor size1.15 (1.09–1.21)0.0001.06 (1.00–1.13)0.048Microvascular invasion3.95 (2.67–5.83)0.0003.01 (1.96–4.64)0.000Metastases2.33 (1.27–4.25)0.0060.96 (0.48–1.93)0.911Satellite nodules2.05 (1.26–3.35)0.0041.19 (0.69–2.03)0.538Note: p value of < 0.05 was considered statistically significant and shown in bold values.Abbreviations: AFP, alpha fetoprotein; BMI, Body mass index; Child B and C, Child-Pugh class B and C; CI, confidence interval; ECOG PS, Eastern Cooperative Oncology Group performance status; HR, hazard ratio; HBsAg, Hepatitis B surface antigen; MAFLD, metabolic dysfunction-associated fatty liver disease.Subgroup AnalysesThe study was stratified according to the types of metabolic traits (BMI, type 2 diabetes mellitus, and metabolic abnormalities proposed in the MAFLD criteria), and the outcome of patients with CHB-HCC was observed. As shown in Figure 4, clinical outcomes were significantly better in the MAFLD group, whereas the interaction effects between survival and metabolic traits were not statistically significant. Figure 4 Subgroup analyses by two groups of non-MAFLD and MAFLD after matching.Abbreviations: HR, hazard ratio; MAFLD, metabolic associated fatty liver disease.DiscussionOur analyses showed that among patients with CHB-HCC, those with MAFLD experienced better long-term outcomes than did those without MAFLD. In addition, multivariate analysis indicated that MAFLD was an independent favorable prognostic factor for patients with CHB-HCC; moreover, the subgroup analyses defined by metabolic characteristics further confirmed these findings. Whilst the association between MAFLD and HCC has been explored in a few studies,19,20 the potential effect of MAFLD in patients with HCC with coexisting other etiologies remains largely unknown. The new definition of MAFLD has been endorsed and supported by the Chinese Society of Hepatology (CSH), and a large proportion of HCC patients in China has CHB as an etiology. With the increasing prevalence of MAFLD, it is relevant to explore the underlying interactions between MAFLD and CHB in HCC patients.21 To our knowledge, this is the first study wherein liver steatosis has been evaluated by quantitative imaging methods for the diagnosis of MAFLD to investigate the potential synergism between MAFLD and CHB in HCC progression.Unlike the previous definition of NAFLD, exclusion of other etiologies, such as alcohol and viral infections, is not a prerequisite for the diagnosis of MAFLD.22 The epidemiological features of the HCC population in our study indicate that the incidence of MAFLD is increasing with concomitant HBV infection (Supplementary Figure 1). Furthermore, while the subclassification of MAFLD has not been completed,1 our evidence highlights the importance of this new criterion in individuals with HBV-related HCC.Substantial epidemiological evidence indicates that being overweight is associated with an increased risk of HCC.23 Moreover, a recent meta-analysis demonstrated that overweight or obese is associated with higher all-cause mortality than is normal body weight.24 Conversely, we found a significantly lower risk of death in patients with HCC with a higher BMI (≥ 23 kg/m2). Controversial data have also been generated from NAFLD studies. Patients with lean NAFLD (BMI < 23 kg/m2) are at a higher risk of developing severe liver disease and worse outcomes, despite having a better histological and metabolic profile.19,25 However, our stratified analyses determined that both lean and overweight patients with MAFLD achieved a significant improvement in the clinical outcome of HCC. The main reason could be the heterogeneity of MAFLD influenced by multiple factors including age, sex, surveillance, alcohol intake, viral infection, and metabolic status.26–31 The clinical course of HCC is based on the balance of these diverse inputs. To reduce selection bias and the effect of baseline differences, strict PSM and multivariate analyses were performed. The results showed that the protective effect of MAFLD on HCC patient survival is independent of tumor characteristics and obesity. Nevertheless, the expected prognostic advantage of MAFLD might be counterbalanced by other causes in the total cohort, such as different treatment options, more aggressive tumor phenotypes, and worse health status of patients.20Hepatic steatosis, defined as the abnormal accumulation of triglycerides within hepatocytes, is correlated with the progression of inflammation, fibrosis, and carcinogenesis.32,33 Although liver biopsy is a reference standard, non-invasive quantification of liver fat is now possible due to advances in imaging modalities conducted to evaluate the fat content in MAFLD participants.34–36 However, our results suggest that hepatic steatosis may not be an important factor for MAFLD-HCC patient outcomes (Supplementary Figure 2A and B). One potential reason is that 84.9% of the patients were classified as having mild steatosis, while the proportion of those with moderate to severe fatty liver was lower. Another possibility is that viral infections and cirrhosis status might confer a protective effect on steatosis, which remains controversial in clinical practice.37–39HBV infection remains a global public health problem and plays a pivotal role in hepatocarcinogenesis.40 According to the updated Clinical Practice Guideline,41 HBeAg is essential for immune tolerance in patients with CHB, and positive serum HBeAg is associated with poor outcomes in HCC.42 However, in the MAFLD and non-MAFLD groups, no differences in survival were detected between HBeAg-positive and HBeAg-negative patients in our study. A previous study has verified that an increasing risk of HCC seen in HBeAg seropositive cases might be due to higher HBV DNA viral loads rather than the effect of HBeAg status.43 Interestingly, higher HBV DNA levels were significantly correlated with unfavorable outcomes in non-MAFLD participants, whereas no differences were detected in MAFLD patients. One prospective study found that hepatic steatosis was associated with a three-fold increase in hepatitis B surface antigen (HBsAg) seroclearance rate in HBV carriers.44 Moreover, the presence of hepatic steatosis was associated with a lower HBV DNA load in HCC patients,39 which might account for our results. Also, recent studies showed that steatosis in CHB patients was significant associated with lower risk of HCC incidence.39,45 Contrastingly, previous studies have shown that MAFLD is associated with the risk of liver fibrosis and inflammatory activity in CHB patients.46 Thus, additional well-designed studies are needed to confirm these data, which will be important to clarify the effect of MAFLD on liver disease progression in patients with CHB.This study has several limitations. First, although we performed PSM and multivariate analysis to balance the variables and enhance intergroup comparisons, there was still unidentified bias due to the retrospective nature of the study. Second, data on 3 metabolic parameters (waist circumference, insulin resistance, and C-reactive protein level) used to confirm the diagnosis of MAFLD were not available; thus, a few patients with lean MAFLDs were excluded. Third, because of incomplete information on antiviral treatments, treated patients may be biased to have more favorable outcomes than those of untreated patients. Additionally, the study by Goh et al showed that statin use is associated with a reduced risk of HCC development in patients with CHB;47 however, fewer patients in this study had dyslipidemia and required statins.ConclusionIn conclusion, our study shows an increasing prevalence of MAFLD-HCC and indicates that MAFLD is associated with lower mortality in CHB-HCC patients, independent of metabolic risk factors. In addition, a novel finding of our study was that patients with MAFLD achieved better survival among HCC patients with hepatitis B infection. This may be due to the interaction between steatosis and virus replication. Further work is needed for patient stratification, considering the heterogeneous nature of MAFLD and the complex pathogenesis of HBV.

PMC

BMJ Open

37620268

PMC10450086

8-24-2023

10.1136/bmjopen-2023-071927

Examination of gaze behaviour in social anxiety disorder using a virtual reality eye-tracking paradigm: protocol for a case

Zeka Fatime, Clemmensen Lars, Arnfred Benjamin Thorup, Nordentoft Merete, Glenthøj Louise Birkedal

IntroductionSocial anxiety disorder (SAD) has an early onset, a high lifetime prevalence, and may be a risk factor for developing other mental disorders. Gaze behaviour is considered an aberrant feature of SAD. Eye-tracking, a novel technology device, enables recording eye movements in real time, making it a direct and objective measure of gaze behaviour. Virtual reality (VR) is a promising tool for assessment and diagnostic purposes. Developing an objective screening tool based on examination of gaze behaviour in SAD may potentially aid early detection. The objective of this current study is, therefore to examine gaze behaviour in SAD utilising VR.Methods and analysisA case–control study design is employed in which a clinical sample of 29 individuals with SAD will be compared with a matched healthy control group of 29 individuals. In the VR-based eye-tracking paradigm, participants will be presented to stimuli consisting of high-res 360° 3D stereoscopic videos of three social-evaluative tasks designed to elicit social anxiety. The study will investigate between-group gaze behaviour differences during stimuli presentation.Ethics and disseminationThe study has been approved by the National Committee on Health Research Ethics for the Capital Region of Denmark (H-22041443). The study has been preregistered on OSF registries: participants will be provided with written and oral information. Informed consent is required for all the participants. Participation is voluntarily, and the participants can at any time terminate their participation without any consequences. Study results; positive, negative or inconclusive will be published in relevant scientific journals.

STRENGTHS AND LIMITATIONS OF THIS STUDYThe present study aims to examine gaze behaviour in three novel social evaluative environments using 360° videos in virtual reality (VR) on a clinical sample of social anxiety disorder (SAD).Using 360° videos as stimuli may enhance the sense of presence because of the enhanced realism of the videos making it a paradigm with high ecological validity in VR compared with VR-constructed environments.The chosen experimental tasks are novel and not previously used in research studies limiting the comparability with other studies.The current study only comprises a clinical population of SAD participants which precludes investigating whether the gaze patterns are specific to SAD.IntroductionSocial anxiety disorder (SAD) is characterised by a persistent and excessive fear or anxiety of being subject to scrutiny, criticism, rejection or humiliation in social and/or performance situations.1 Lifetime prevalence is estimated at 12.1%2 with higher prevalence among girls and women.3 4 SAD has an early age of onset (mean 14.5 years) compared with other psychiatric disorders5 as well as a high degree of comorbidity with other psychiatric disorders that are typically preceded by an SAD,6–8 suggesting that SAD may be a predisposition and a risk factor for development of other psychiatric disorders.9 In Denmark, anxiety disorders have an estimated lifetime prevalence between 13% and 29% and an incidence rate of approximately 17 000 new cases yearly.10 In addition, anxiety disorders are associated with an increased mortality,11 and economic burden of approximately 10 billion Danish Krone (DKK)10 signifying the importance of early detection and early treatment of the disorder.Early detection may prevent a chronic course of the disorder, the development of other psychiatric disorders, further burdens to the individual, impaired functioning and ultimately decrease the significant societal burden associated with the disorder.6 7 12 Early detection of the disorder may be aided by developing an objective screening tool for SAD that comprises behavioural markers for the disorder specifically by examining gaze behaviour, which is considered to be aberrant in SAD.1 13 Advances within virtual reality (VR) and eye-tracking technology are considered promising to improve early identification of SAD. This notion is corroborated by a growing body of research employing eye-tracking technologies to study attentional bias and gaze behaviour.14 15 Eye-tracking is argued to enable a direct measure of visual attention as it allows recording overt eye movements directly and continuously in real time.16Gaze behaviour in SADWhereas in many species, direct gaze is perceived as threatening, evoking an aversive response, the opposite is thought to happen in humans, where eye contact is believed to modulate communication and social interaction processes.13 17 18 Direct gaze perception is believed to play a pivotal role both in the development of the social cognition and in the social functioning of the individual.18 Direct gaze perception is associated with enhanced self-awareness, increased memory for face identity in adults, increased positive appraisals of others and with activate prosocial behaviours.18 Moreover, it is suggested that direct gaze causes affective reactions to the perceiver of the direct gaze,19 indicating it has a high social significance. In SAD, gaze behaviour is thought to be aberrant with SAD individuals showing inadequate eye contact in social situations characterised by fear and avoidance of direct eye contact.1 13 Theoretical models of SAD suggest that in social or performance-based situations, individuals with SAD tend to engage in avoidant behaviour by avoiding any potential real or perceived confrontation or negative evaluation from others. In situations where a total withdrawal from the situation is impossible, the avoidance may be subtle such as avoiding salient social stimuli (eg, avoidance of looking at faces, avoidance of gaze exchange, avoidance of eye contact). This avoidant behaviour is considered a safety behaviour; a maladaptive strategy that perpetuates the anxiety and, thus, serves as a maintenance factor of the disorder.20 21 The visual avoidance of faces has indeed been confirmed in many studies22; however, most studies have used paradigms (typically free viewing paradigms consisting of viewing photographs) that are considered to lack ecological validity.23 Additionally, many studies have used samples from the community as opposed to clinical samples of SAD. However, gaze behaviour findings from community may not generalise to the clinical populations of SAD.22 23Moreover, SAD individuals may have a selective attention to threat.21 In the so-called vigilance-avoidance hypothesis, it is theorised that SAD individuals may tend to have a selective attention to threat by initially paying attention to any external indicators of negative social evaluation followed by avoidance of such external indicators, which in turn perpetuates anxiety symptoms.24 25 The empirical evidence in this area is mixed with some studies demonstrating a maintenance of attention to threatening stimuli, while others show avoidance.14 The mixed results are argued to be due to methodological diversity in the experimental tasks used to study the selective attention to threat.23 Some authors have, therefore, studied hyperscanning, another aspect that is considered to indicate hypervigilance. A hyperscanning strategy defined as a vigilant strategy characterised by an excessive scanning of the environment typically for threat detection26 27 has indeed been confirmed in some studies making it a promising aspect of SAD to be further investigated.26–29Thus, a considerable amount of research on gaze behaviour has used paradigms that may lack ecological validity.23 30 This has led to the necessity of designing experiments as social interaction and/or performance-based tasks that resemble real-life situations. In these so-called ‘social evaluative tasks’, the participant is required to perform a real-life task with a risk of negative social evaluation (eg, performing a speech in front of a prerecorded audience)23 thus enhancing the ecological validity of the paradigm.Assessing gaze behaviour in SAD using VRVirtual reality (VR) has shown good potential in psychotherapeutic intervention but has been used to a lesser degree for assessment or diagnostic purposes.31 32 Empirical findings using VR in SAD have demonstrated that social fear can be successfully induced in VR environments,33–35 indicating its usefulness for conducting ecologically valid experiments. A sense of presence, that is, the experience of being present in a mediated environment and not in a physical environment,36 has been argued to be paramount for experiments conducted in VR. However, some studies have demonstrated that gaze behaviour in VR and in prerecorded videos may be experienced differently than in real life by having lesser physiological reactions in the VR and prerecorded videos compared to face-to-face interactions, suggesting that findings from studies conducted in VR may not necessarily apply to the real life.37 38 To date, a small number of studies have used VR to examine gaze behaviour in SAD.39–44 These studies report that compared with healthy control groups, individuals with SAD or individuals with social anxiety symptoms show an avoidant gaze behaviour in social and performance situations. Studies utilising immersive 360° video environments in VR are still scarce. 360° video environments in VR may enhance the sense of presence due to their immersive and realistic presentation45 and it can be hypothesised that it may be superior to animated environments in VR in terms of ecological validity although a study comparing the two has not yet been conducted. Holmberg et al have demonstrated that anxiety in SAD can be successfully triggered by 360° videos of real-life situations in VR.46 In addition, Rubin et al conducted the first study to assess gaze behaviour in participants with social anxiety symptoms using 360° stimuli (a public speaking task) in VR. The study showed that compared with a healthy control group, individuals with social anxiety symptoms showed a pattern of avoidance of social threat,47 thus demonstrating good potential of utilising 360° stimuli as a highly ecological environment for conducting research on gaze behaviour in SAD as well as using it for attention guidance training in SAD.48 We aim to extend these preliminary findings by including three novel naturalistic social evaluative tasks using 360° stimuli in VR in a clinical sample of SAD. The three tasks capture both the social and performance aspect of SAD and we believe that the tasks are relatable and recognisable to the participants since the tasks resemble everyday situations in VR.ObjectivesThe current study aims to identify eye gaze patterns in SAD using VR across three different social and performance real-life situations and, thus, extend the existing literature on the utility of VR for assessment purposes in SAD.In line with previous findings on gaze avoidance,44 49 50 we hypothesise that compared with a healthy control group (HCG), SAD participants will exhibit fewer number of fixations and less fixation duration (dwell time) on social areas (faces and body regions) of the participants presented in the three VR tasks.In line with previous findings,26–28 we hypothesise that SAD participants will show a hyperscanning strategy indicated by increased total length of scan path compared with the HCG across the three VR tasks.In line with the vigilance-avoidance model,24 we also hypothesise that SAD individuals will exhibit shorter time to first fixations on social stimuli compared with HCG across the three VR tasks.Additionally, we will examine the interaction of the tasks on gaze behaviour, hypothesising that both SAD participants and HCG participants will show greater avoidance and hyperscanning in task C compared with tasks A and B, given that task C contains an objective threat for negative evaluation.MethodsStudy designThe proposed study is a case–control study including a clinical sample of 29 individuals with SAD and a matched healthy control group of 29 individuals. A VR-based eye-tracking paradigm will be employed in which gaze behaviour as indicated by eye movements will be examined using the integrated eye tracker in the VR headset. Stimuli will be presented as 360° 3D videos in VR.SettingThe experimental study will be carried out by VIRTU research group at the Copenhagen Research Centre for Mental Health CORE, at Mental Health Center Copenhagen, Copenhagen University Hospital, Denmark. The inclusion period will start in January 2023 and is expected to last approximately 9 months.ParticipantsThe study will include a total of 58 participants comprising 29 individuals with an SAD diagnosis (ICD: F 40.1) referred to psychiatric clinics in the Capital Region of Denmark and 29 HCG individuals that will be recruited from the community, using ads at relevant institutions or via the research recruitment service www.forsoegsperson.dk.Participants from the HCG will be matched to the SAD sample (1:1) on age (±2 years) and gender (see table 1). For the social anxiety levels among the HCG, participants with a score above 30 on Liebowitz Social Anxiety Scale (LSAS) will be excluded from the study as this cut-off will exclude participants with subclinical levels of SAD.51Table 1Overview of inclusion and exclusion criteria for the SAD participants and healthy control group (HCG) participants Inclusion criteria for the SAD group Inclusion criteria for the HCG group Fulfilling diagnostic criteria for social anxiety disorder ICD: F40.1Age 18–75Sufficient knowledge of the Danish languageInformed consent Age 18–75Sufficient knowledge of the Danish languageInformed consent Exclusion criteria for the SAD group Exclusion criteria for the HCG group Psychotic disorders, autism spectrum disorders and personality disordersA diagnosis of alcohol or drug dependence (ICD: F10-19, 20–26)Significantly impaired vision hindering engagement in VR experiencesEpilepsy Any psychiatric diagnoses including alcohol and drug dependenceSignificantly impaired vision hindering engagement in VR experiencesEpilepsy HCG, healthy control group; SAD, social anxiety disorder; VR, virtual reality.Patient and public involvementA panel of patients have been involved in developing the stimuli material. The stimuli material was originally developed to be used for exposure purposes in the So-REAL study: a randomised control trial, evaluating a VR-based intervention for SAD. The videos have been developed by a team consisting of clinicians, a panel of patients with SAD in collaboration with the VR production company KHORA-VR. The videos consist of various real-life social and performance environments, typically feared by patients with SAD. Actors were paid to deliver the content in the different videos. The process of the development of the videos consisted of regular meetings between clinicians, patients and the VR production company. The experience of the patients (level of anxiety provoked by the videos, the validity of the videos) seeing the videos were considered for further development of the videos. In the final stage, the videos were tested by two clinicians in a group therapy format. This led to further feedback from the clinicians and patients on the videos and their use for exposure purposes in group therapy. The process of development lasted approximately 16 months producing 12 real-life situations for SAD.52The experimental tasksAll participants will be presented to high-resolution 360° stereoscopic videos in high-end VR head mounted display (HMD).The three experimental tasks are:Task A is a job interview situation that we conceptualise as a performance task. In the video, a male and a female interviewer ask a variety of job-related questions. After each question, a ‘listening loop’ of 30 s follows allowing the participant to answer while the two interviewers appear to listen. The participant will be instructed to act as if this was a real job interview and to respond to the questions. The job interview includes questions such as: ‘Can you tell us something about yourself’, what is your motivation for applying for this job’, ‘how can you contribute to this job?’. The attitude of both interviewers is welcoming and warm (see figure 1).Figure 1Demonstration of the VR job interview situation. VR, virtual reality.Task B is a social interaction situation comprising small talk/discussion in a canteen while eating lunch in a work setting. The participant is joined by four colleagues: two male and two female colleagues. The participant is instructed to act as if this was a real interaction. No direct questions are made to the participant and as such the participant is not required to verbally engage in the conversation but will be free to do so. The atmosphere is welcoming and warm. The colleagues look sporadically at the participant and smile at times (see figure 2).Figure 2Demonstration of the VR social interaction situation. VR, virtual reality.Task C is conceptualised as a performance task, where there is an objective threat for being negatively evaluated. The participant is instructed to verbally engage in the video. The participant is asked to do a presentation together with a colleague in front of a group of people. The task starts with the colleague introducing himself to the audience and asks the participant to present him/herself to the audience. The colleague must, however, leave the room after a phone call he receives, leaving the participant alone in the room. In addition, the computer in which the participant is to use for a power point presentation stops working and as such the participant must carry out the presentation without the support of power point. The atmosphere gets a bit tense, and the group of people appear to be impatient. However, the task ends before the participant must do a presentation (see figure 3).Figure 3Demonstration of the VR presentation situation. VR, virtual reality.ApparatusTo collect gaze data, eye movements will be tracked using eye-tracking integrated in the HTC VIVE Pro Eye HMD. Eye movements are tracked at 120 Hz (binocular). The integrated eye tracker has a trackable field of 110°, an accuracy of 0.5°−1.1° and a calibration of 5-point. A powerful gaming computer will serve as an interface between the eye-tracking software and the HMD.Data preparationThe Imotions software will be used to collect, calculate and analyse gaze-based data.53 54 Analysis will be carried out on predefined areas of interest (AOI) as well as on raw gaze-based data. In the current study, AOI will be predefined and categorised as social stimuli and non-social stimuli. The social stimuli comprising of face regions and body regions will be predefined and categorised separately while the non-social stimuli (the background) will be predefined and categorised as the rest of stimuli expect faces and body regions (see figure 4). An I-VT (Velocity-Threshold Identification) filter will be used to analyse the raw eye-tracking data and to classify fixations and saccades by comparing the speed of the eye’s movements to a velocity threshold. The velocity threshold will be of 100°/s and a minimum fixation of 100 ms. Thus, the I-VT filter classifies a fixation if the eye moves slower than this threshold, whereas if the eye moves faster than this threshold, the I-VT filter classifies it as a saccade. The total fixation time will be calculated as the sum of all fixations within an AOI.Figure 4Demonstration of a predefined and categorised area of interest (AOI).ProcedureEligible participants from the SAD and HCG will be invited to the VR lab, where they will undergo a clinical assessment conducted by experienced psychologists followed by completion of psychometric questionnaires (see online supplemental file 1). Prior to beginning the experiment, participants will be given verbal instructions about the nature of the experimental tasks. Each experimental task lasts approximately 2 to 2.5 min. After each experimental task, a pause of 10 s follows. The order of the tasks presented to the participants is randomised by the Imotion software. As stated earlier, participants will be instructed to answer the questions of the interviewers in task A, whereas in task B, the verbal communication is not mandatory. In task C, the participants are as well instructed to verbally engage with the people in the task. Before beginning the experiment, participants will also be calibrated to the HMD. To minimise distractions from the surroundings, the researcher will leave the room, when the VR tasks are being performed.10.1136/bmjopen-2023-071927.supp1Supplementary data After the completion of the experimental tasks, the participants are asked to fill in a presence scale questionnaire, using the Multimodal Presence Scale (MPS), which measures the level of the experience of presence in VR.36 Subjective distress will be measured before the experimental tasks and after completion of the experimental tasks using Subjective Units of Distress Scale (SUDS).55 At the end of the experiment, participants will be thoroughly debriefed and thanked for the participation.Assessment batteryParticipants from both groups will undergo an assessment consisting of the listed measurements.Mini International Neuropsychiatric Interview (MINI), V.7.0 for Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). Psychometric analyses of the MINI have demonstrated acceptable test–retest and inter-rater reliability.56 57 MINI will be used to screen for diagnosis and rule out psychiatric diagnoses in the HCG.Personal and Social Performance Scale (PSP) will be used to measure the social functioning of the participant.58LSAS will be used to measure symptom severity of SAD. LSAS assesses 24 situations typically feared by individuals with SAD, rated on anxiety and avoidance, divided into subscales of performance anxiety and social situations. It has acceptable psychometric properties.59 The LSAS will be used as a diagnosis supplement to MINI.The experience of presence in VR is measured using MPS developed and validated by Makransky et al.36 This scale consists of 15 items measuring aspects of the physical presence, social presence and self-presence using a 5-point Likert Scale (1=completely disagree, 2=disagree, 3=neither disagree nor agree, 4=agree, 5=strongly disagree).36Fear of Negative Evaluation using the Brief Version of the Fear of Negative Evaluation Scale (FNES).60SUDS55 will be used to rate the state anxiety pre and post the experimental tasks.Outcome measuresThe outcome measures are eye movement data derived from the eye-tracking and consist of the following: fixation-based parameters: total fixation duration (dwell time) in milliseconds, total number of fixations and mean fixation time measured on AOI. These parameters indicate high and low levels of gaze avoidance. Raw data parameters: scan path length, mean distance between fixations, time to first fixation and length of first fixation on social stimuli are indices that indicate hypervigilance and hyperscanning. Exploratory outcomes are association between eye movement data and LSAS, PSP, Presence in VR, and FNES.Sample size calculationOur primary hypothesis concerns differences in the number of fixations in SAD subjects compared with HCG. In a previous study of49 49 the HCG had a mean score of 56.7 (SD=5.1) on the number of fixations. We set the minimal clinically important difference to a true difference in the experimental and control means to 0.75 SD (=3.8). Corresponding to an expected mean score of 52.9 in patients with SAD. We calculated effect sizes revealing that 29 subjects are required in each group to detect the expected difference in a t-test with 80% power at the 0.05 significance level using a two-sided hypothesis.Statistical methodsData will be analysed using Stastical Package for the Social Sciences (SPSS). The analysis of data will be conducted using a mixed design Analysis of Variance(ANOVA) combining between-subject’s and within-subject’s analysis. Initially, a one-way ANOVA will be conducted to assess group (SAD vs HCG) differences on psychometric measures (LSAS, FNES, PSP, MPS and SUDS). Means, SD and F-scores will be reported.To examine gaze behaviour, three ANOVAs will be conducted with group (SAD vs HCG) as the between-subject factor and AOI (face vs body) as the within-subjects factor for each task (tasks A, B and C).To examine whether there is a difference in gaze behaviour between task A, B and C, a two-way Multivariate analysis of variance (MANOVA) will be conducted with task as a within-subject factor and group (SAD group vs HC group) as a between-subject factor. Order of task presentation will be included as a between-subject control variable. Eye-tracking metrics (the number of fixations and the total fixation duration and the mean duration of fixations on social areas, total scan path length and mean distance between fixations) are the dependent variables.Furthermore, exploratory analyses using a repeated measure ANOVA will be conducted on SUDS score with group (SAD vs HCG) as a between-subject factor and time (pre vs postexperimental task) as a within-subject factor.Exploratory analyses using Pearson correlational analyses will also be carried out on the SAD group assessing eye movement data and psychometric measures (LSAS, FNES, PSP, MPS).All statistical tests of significance will be two-tailed, with significance level set at p<0.05. Missing data will be handled by multiple imputations.Ethics and disseminationThe study will be conducted in accordance with the Helsinki Declaration. The study has been approved by the National Committee on Health Research Ethics for the Capital Region of Denmark (H-22041443). All participants will receive participant information 48 hours prior to the conduct of the study. Any adverse events will be monitored and recorded throughout the study period and reported to the Committee on Health Research Ethics of the Capital Region Denmark. VR may cause cyber sickness to some people, which corresponds to motion sickness. Given that VR is in general well tolerated, we do not expect any adverse events to happen.61 Participation is voluntary, and the participants can at any time terminate their participation without any consequences. Study results; positive, negative or inconclusive will be published in relevant scientific journals.DiscussionThe proposed study aims to examine gaze behaviour differences in a clinical sample of SAD compared with an HCG using a VR eye-tracking paradigm. Determining behavioural markers may have important clinical implications, not only for enhancing the understanding of the aetiology of SAD but also by attempting to develop an objective screening tool that may aid the assessment of SAD. Thus, the results from this study may provide the foundation for conducting subsequent studies evaluating VR-based eye-tracking as an objective, automated screening tool for SAD. However, aberrant gaze behaviour has also been found in other mental disorders such as autism.62 As the current study only comprises a clinical population of SAD participants, it precludes investigating whether the gaze patterns are specific to SAD. This may be explored further in future studies.Whereas studies using VR as a tool in psychotherapeutic intervention is growing,31 the use of VR for assessment or diagnostic purposes is still scarce. Conducting experimental research with VR-based paradigms may add further evidence on the usefulness of VR as an assessment tool capturing aspects of psychopathology. The potential of establishing a specific gaze pattern in SAD using VR may also have treatment-related implications by informing targets for interventions that may involve exposure of gaze avoidance and/or selective attention to threat.Supplementary MaterialReviewer commentsAuthor's manuscript

PMC

Journal of the Endocrine Society

PMC10554018

10-05-2023

10.1210/jendso/bvad114.2016

SAT545 Erythropoietic Thyroid Nodule in the Setting of Myelofibrosis

Vasquez Libia M, Bruder Jan M, Koops Maureen, Trejo Jonathan

Abstract Disclosure: L.M. Vasquez: None. J.M. Bruder: None. M. Koops: None. J. Trejo: None. Background: Extramedullary hematopoiesis is frequently seen in non-osseous locations but not endocrine organs, this is a case of hematopoesis in a thyroid nodule. Clinical Case: A 79-year-old man with myelofibrosis diagnosed at age 66, CAD post CABG, atrial fibrillation on anticoagulation and HFrEF was referred to the endocrinology clinic due to enlarging left lobe thyroid nodule. He was diagnosed with a goiter at age 72 but denied compressive symptoms. He had no history of radiation to his neck or family history of thyroid cancer. On physical exam, the left thyroid lobe was visibly enlarged with slight right tracheal deviation without lymphadenopathy. TSH level was normal. Thyroid ultrasound demonstrated a 6.4 x 4.0 x 4.8 cm, complex cystic-solid, taller than wide nodule in the left lobe and a sub-centimeter cystic nodule in the right lobe. FNA showed hematopoietic elements, including maturing cells in three lineages of hematopoiesis. On repeat ultrasound 5 years later. The left nodule increased in size to 7.3 x 5.4 x 4.4 cm and highly suspicious (TR4 and TR3) by Ti-RADS criteria. Repeat FNA of the nodule showed many red blood cells with few hematopoietic cells, flow cytometry did not show any abnormal populations. No thyroid follicular cells were reported. Myelofibrosis was diagnosed by bone marrow biopsy many years prior. He had maintained a stable CBC without palpable spleen or hepatomegaly. Given this history without thyroid dysfunction or reported symptoms, surgery was not pursued. External beam radiation was a treatment option should the nodule cause symptoms. He continued to have mild anemia not requiring blood transfusions. He passed away 2 years later in hospice care after hospitalization for severe heart failure decompensation. Conclusion: Extramedullary hematopoiesis involving the thyroid is rare but has been reported. This type of extramedullary hematopoiesis. can be treated with surgery or low-dose radiation because of the radiosensitivity of hematopoietic tissue . This was a potential treatment modality if compressive symptoms arose, but they did not. This case illustrates a situation in which surgical intervention on highly suspicious nodules would not have been the optimal treatment choice. Reference: 1. Jan I., Weng M., Wang C., et al. Extramedullary hematopoiesis involving the thyroid: A rare cytologic finding in otherwise healthy patients and review of literature. Journal of the Formosan Medical Association. 117, 1108-1114.2. Taher A, Vichinsky E, Musallam K et al., authors; Weatherall D, editor. Guidelines for the Management of Non-Transfusion Dependent Thalassaemia (NTDT) [Internet]. Nicosia (Cyprus): Thalassaemia International Federation; 2013. Chapter 11, EXTRAMEDULLARY HEMATOPOIESIS. Available from: Presentation Date: Saturday, June 17, 2023

PMC

HemaSphere

PMC10430980

8-08-2023

10.1097/01.HS9.0000970296.80801.95

P848: EXPRESSION PROFILE OF BCL-2 FAMILY PROTEINS IN MULTIPLE MYELOMA.

Sánchez Cristina De Ramón, Rojas Elizabeta A, Misiewicz-Krzeminska Irena, Cardona-Benavides Ignacio J, Cuadrado Myriam, Isidro Isabel, Jose Calasanz Maria, García-Sanz Ramón, Rosiñol Dachs Laura, Martinez-Lopez Joaquín, Baldé Joan, Lahuerta Juan J, San Miguel Jesus F, Mateos Maria-Victoria, Corchete Luis A, Gutierrez Norma Carmen Gutierrez

Abstract Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational ResearchBackground: Evasion of apoptotic mechanisms due to the deregulation of Bcl-2 family proteins may play an important role in the resistance of multiple myeloma (MM) to different therapeutic schemes. Therapies targeting these proteins, such as venetoclax, which inhibits the Bcl-2 protein, have recently been developed. To date, the expression of Bcl2 family proteins in MM patients have mostly been analyzed at RNA level, even though transcript levels by themselves may not be appropriate to predict protein levels. Although, some studies analyze the expression of apoptotic proteins in MM by immunohistochemistry techniques or Western blot, the low protein concentration obtained after CD138 isolation have largely limited the quantification of proteins so far.Aims: - To investigate the expression of the Bcl-2 family proteins in newly diagnosed MM (NDMM) patients, and its association with clinical features and cytogenetic abnormalities.- To analyze the prognostic impact of Bcl-2 family proteins in NDMM patients who underwent autologous stem cell transplantation.- To explore the Bcl-2/Bcl-xl, Bcl-2/Mcl-1 and Bcl-2+Bim+Bax/Bcl-xl protein ratios, which have been associated with response to venetoclax when analyzed at RNA level, depending on the genetic alterations.Methods: Bone marrow samples from 120 NDMM patients enrolled in the Spanish Myeloma Group clinical trial GEM2012MENOS65 were included in this study. The expression of the antiapoptotic (Bcl-2, Bcl-xl, Mcl-1) and proapoptotic (Bax, Bak, Bim, Puma, and Bad) proteins were analyzed by the capillary electrophoresis nanoimmunoassay methodology (ProteinSimple, WES™ system).Results: All the Bcl-2 family proteins analyzed were expressed in more than 80% of MM patients, except Bax which was found in less than 30%. Bak, Bim and Mcl-1 were the proteins with the highest level of expression.Patients with low expression levels of Bim protein had a significant higher incidence of plasmacytomas (p = 0.019). We found that Bak protein expression was significantly higher in patients with t(14;16) (p = 0.028), while Bcl-xl and Puma expression was lower in patients with t(11;14) and 1p deletion (p = 0.014, p = 0.046), respectively. High levels of Bad and Puma proteins were associated with longer OS (p = 0.017 and p = 0.001). Conversely, high levels of Bcl-2 had a negative impact on PFS (p = 0.018).When we analyzed the values of three ratios that have demonstrated to be predictors of the response to venetoclax, we found that patients with t(11;14) had higher levels of Bcl-2/Bcl-xl and Bcl-2+Bim+Bax/Bcl-xl ratios than patients without t(11;14) [14/18 (79%) vs. 41/91 (45%), and 14/18 (79%) vs. 42/91 (46%), respectively, p = 0.01], as has been published at RNA level. Strikingly, the high Bcl-2+Bim+Bax/Bcl-xl protein expression ratio was also enriched in patients harbouring TP53 mutations compared to patients without these mutations [7/8 (87%) vs. 40/82 (49%), p = 0.036].Summary/Conclusion: - The expression of anti and proapoptotic proteins was well-balanced in MM.- High levels of the proapoptotic proteins Bad and Puma were associated with favorable prognosis, while the overexpression of the antiapoptotic protein Bcl-2 had a negative impact on survival.- High Bcl-2/Bcl-xl and Bcl-2+Bim+Bax/Bcl-xl protein ratios were significantly enriched in patients with t(11;14). Patients harboring TP53 mutations were also significantly associated with high Bcl-2+Bim+Bax/Bcl-xl protein ratio.FUNDING: This study has been funded by ISCIII (PI19/00674) (co-funded by FEDER), Castilla y Leon (GRS 2058/A/19, GRS 2331/A/21) and AECC (PROYE20047GUTI). CDR, EAR and IJCB were supported by AECC (CLJUN18010DERA), “Consejería Educación Castilla y León” and ISCIII (FI20/00226), respectively.Keywords: Protein Expression, BCL2, Apoptosis, Multiple myeloma

PMC

Journal of Clinical and Translational Science

PMC10129444

10.1017/cts.2023.229

148 A Feasibility Study for the Implementation of a Hospital-based Violence Intervention Program in the Rural South

Lovelady Nakita, Curran Geoffrey, Richardson Joseph B., Wilson Michael, McBain Sacha, Urban Kelly, Washington Taylor, Rohm Laura, Zaller Nickolas

OBJECTIVES/GOALS: As hospitals across the nation respond to the need to address community violence, there is a dearth of Hospital-based Violence Intervention Programs (HVIPs) in the South despite having disproportionate rates. This research aims to identify key factors and strategies for implementation of an HVIP among rural patient populations in a southern state. METHODS/STUDY POPULATION: Semi-structured interviews will be conducted with medical providers, social service organizations, and patients transferred from four high-risk rural areas in Arkansas. Data will be analyzed using Framework Analysis, a rapid analysis approach involving framework development, code application, impactful statement identification, and content analysis. Evidence- Based Quality Improvement (EBQI), a group consensus making process, will be conducted to identify key implementation strategies and factors to adapt based interview findings. Priority areas for adaptation will be identified via systematic rating. The EBQI team, including researchers and key rural stakeholders will engage in a series of discussion, vote on final strategies, and develop a guide for future HVIP implementation and pilot testing. RESULTS/ANTICIPATED RESULTS: Findings from this study will result in a prioritized list of barriers and facilitators across sample groups. Factors will be rated by level of importance. Cluster maps will display the relationships among factors. Go and no-go zones will be identified based on importance and feasibility. Implementation strategies will be mapped to barriers and facilitators. DISCUSSION/SIGNIFICANCE: The findings will result in a culturally and geographically relevant HVIP model and package of implementation strategies to test in future hybrid trials (feasibility pilot & multi-site RCT); and shape the future of violence prevention efforts in healthcare settings across the rural South.

PMC

Journal of Pain Research

38444879

PMC10913792

3-01-2024

10.2147/JPR.S466139

Knowledge Mapping Analysis of Research on Pregnancy-Related Pelvic Girdle Pain (PPGP) from 2002 to 2022 Using Bibliometrics [Corrigendum]

Xu L, Li Y, He Y, et al. J Pain Res. 2024;17:643–666.The authors have advised Figure 1 on page 645 is incorrect. Due to an error at the time of resubmission Figure 1 and Figure 2 were inadvertently duplicated. The correct Figure 1 is shown below. Figure 1The flow chart of the screening process.The authors apologize for the error.

PMC

Lancet Regional Health - Americas

PMC10820257

1-08-2024

10.1016/j.lana.2023.100664

Double-edged sword of wastewater surveillance

Street Renée, Johnson Rabia, Guerfali Fatma Z.

The article by Oghuan and colleagues1 emphasizes significant advances in wastewater-based epidemiology (WBE) to support public health interventions. Propelled forward by the COVID-19 pandemic, WBE continues to grow rapidly. However, the ethical, legal, and social aspects of WBE have not kept up with the same momentum. Characterized by the analysis of an anonymous pooled community sample, WBE studies frequently circumvent the scrutiny of bioethics’ committees. The findings of Oghuan and colleagues1 underscore the importance of wastewater surveillance and the potential to identify specific locations of non-reported mpox infections within urban settings. The study goes further to suggest sampling upstream of communities, even at the individual building level. The recent global mpox outbreak predominantly affected, but was not limited to, gay, bisexual, and other men who have sex with men, primarily spreading from person-to-person through sexual networks.2 Currently, 65 countries have discriminatory laws against consensual same–sex relations, and in 12 of these countries, the death penalty is either imposed or remains a possibility.3 The spatial resolution of WBE, ranging from large sewersheds to single building level surveillance, needs to take into consideration legal and ethical implications along with social consequences, ensuring alignment of good clinical practice. By actively implementing such measures, WBE will prioritize the rights, safety, and well-being of individuals over the interests of science and society.4 As WBE continues to flourish as a public health tool, it is imperative to navigate emerging ethical and legal issues and safeguard against potential risks.ContributorsConceptualization: RS, FZG; Writing—original draft: RS, RJ, FZG; Writing—review and editing: RS, RJ, FZG.Declaration of interestsThe authors declare no competing interest.

PMC

Journal of Chemical Information and Modeling

37805934

PMC10598796

10-08-2023

10.1021/acs.jcim.3c01153

AmberTools

Case David A., Aktulga Hasan Metin, Belfon Kellon, Cerutti David S., Cisneros G. Andrés, Cruzeiro Vinícius Wilian D., Forouzesh Negin, Giese Timothy J., Götz Andreas W., Gohlke Holger, Izadi Saeed, Kasavajhala Koushik, Kaymak Mehmet C., King Edward, Kurtzman Tom, Lee Tai-Sung, Li Pengfei, Liu Jian, Luchko Tyler, Luo Ray, Manathunga Madushanka, Machado Matias R., Nguyen Hai Minh, O’Hearn Kurt A., Onufriev Alexey V., Pan Feng, Pantano Sergio, Qi Ruxi, Rahnamoun Ali, Risheh Ali, Schott-Verdugo Stephan, Shajan Akhil, Swails Jason, Wang Junmei, Wei Haixin, Wu Xiongwu, Wu Yongxian, Zhang Shi, Zhao Shiji, Zhu Qiang, Cheatham Thomas E., Roe Daniel R., Roitberg Adrian, Simmerling Carlos, York Darrin M., Nagan Maria C., Merz Kenneth M.

AmberTools is a free and open-source collection of programs used to set up, run, and analyze molecular simulations. The newer features contained within AmberTools23 are briefly described in this Application note.

IntroductionThe present status of the Amber (Assisted Model Building and Energy Refinement) suite of programs has been the product of decades of effort from a broad range of research groups, starting with the group of the late Peter Kollman in the early 1980s.1 Amber contains tools for energy minimization (EM), molecular dynamics (MD) simulations, free energy (FE) calculations, potential of mean force (PMF) capabilities, and all the needed tools to set up the modeling effort. The software stack has been reviewed in the past,2−4 and the manual contains detailed descriptions of all the algorithms in Amber as well as a full list of contributors to Amber over the years (see Besides the actual code, Amber is used to describe a series of highly regarded force fields5 for proteins,6−12 carbohydrates,13,14 nucleic acids,7,8,15 and lipids.16 The present Application Note will only describe the latest additions to the open-source AmberTools23 and as such is not meant to give a thorough exposition of all the methods and capabilities of AmberTools and Amber.Overview of Amber and AmberToolsAmber and AmberTools form a collection of programs that are designed to work together to facilitate system setup, MD simulations, and trajectory analysis for biomolecules. It is useful to note that the Amber force fields mentioned above can be used in a variety of molecular dynamics codes outside of AmberTools and Amber. The Amber code is updated in even-numbered years, and it uniquely includes the base MD code known as pmemd, which offers parallel and graphics processing unit (GPU)-accelerated versions of the MD codes along with some free-energy-based methods not implemented in AmberTools. Analogous MD function is available in sander in AmberTools. AmberTools is distributed under an open-source license, primarily the GNU General Public License, with some portions covered by other compatible open-source licenses. The Amber force fields are in the public domain and are distributed with AmberTools. The pmemd code is distributed as source code but has a separate license that contains restrictions on use and redistribution; there is no license fee for noncommerical use of pmemd. Full details on licensing and distribution can be found at WorkflowThe basic workflow for AmberTools is shown in the accompanying (see Figure 1), and it describes preparation, simulation, and analysis steps. Preparation starts at the top, since all MD simulations require some sort of starting three-dimensional (3D) structure, which for biomolecules is usually in the form of a PDB-format file; AmberTools has some model-building capabilities (e.g., PACKMOL-Memgen, see below), but other codes are generally used if experimental structures are not available. The prepareforleap step, which is recent and still under development, carries out tasks to map components in the input file to Amber nomenclature (especially useful for carbohydrates), add hydrogens, identify cross-links, assign histidine protonation states, and similar tasks. Next is the LEaP program, which is a workhorse program that connects the nascent structure to Amber’s built-in force fields for proteins, nucleic acids, carbohydrates, lipids, and common solvents and to bespoke force fields for other components like ligands and cofactors that can be created by programs like antechamber and mdgx (for general organic molecules) and pyMSMT (for metal ions). The LEaP code creates two files: an “inpcrd” file that has complete three-dimensional coordinates and a “prmtop” file that contains all other information needed for force field-based analyses of the system. The latter file can be examined and edited via parmed, which can also export similar files in the GROMACS or CHARMM format.Figure 1Common workflow in AmberTools. Flow went from top to bottom. Black boxes are for preparation, gray indicates an optional preparation step specific for membrane systems, blue for simulation, and red for analysis.The simulation phase is primarily the province of sander or pmemd. The “mdin” file contains a large number of parameters that control the type and length of the simulation to be carried out, the integration method, the use of a QM/MM (quantum mechanics/molecular mechanics) model, specification of enhanced-sampling and thermodynamic integration methods, and the like. Restraints on the system, often from NMR or X-ray data but more recently from cryogenic electron microscopy (cryoEM) and other sorts of integrative modeling, can also be input at this point.Snapshots of conformations are generally stored at regular intervals during a simulation and then serve as input for an analysis phase. The cpptraj program is the workhorse code here, providing geometric and energetic analyses, clustering algorithms, and many other routines. Three other codes, MMPBSA.py, FE_Toolkit, and FEW (Free Energy Workflow)17 are devoted to estimating free energy changes. More complete descriptions of all of this, including a full list of programs, encompassing nearly 1000 pages of text, are in the Amber23 Reference Manual.AmberTools23 UpdatesWe have a number of significant new features for AmberTools23 which include automated building of membrane-protein–lipid-bilayer systems, enhancements to the polarizable Gaussian multipole method, extensions to the Poisson-Boltzmann surface area (PBSA) method, enhanced free energy capabilities, enhanced QM and QM/MM capabilities, and a significant upgrade of the Amber Web site and tutorials. Each of these additions is summarized below.1Polarizable Gaussian Multipole Model in the SANDER ProgramThe polarizable Gaussian Multipole (pGM) model is a next-generation induced-dipole polarizable model aiming to balance accuracy and efficiency for molecular simulations of biomolecular systems.18−22 We recently developed a new framework for efficient computation of analytical atomic gradient for the pGM model.18 The pGM virial for constant pressure molecular dynamics simulations was also implemented in previous releases of Amber.19 The accuracy and robustness of the pGM model have also been validated on various molecular properties.20−22 In the AmberTools23 release, we further optimized the induced-dipole iteration algorithm. Specifically, we introduced maximum relative error as the convergence criterion to ensure energy conservation in molecular dynamics simulations. We also designed and implemented multiorder extrapolation (MOE) and local preconditioning conjugate gradient (LPCG) schemes to accelerate the induced-dipole iteration.23 Given the new developments, MD simulations with the pGM model are able to achieve a similar level of energy conservation as those with the point charge additive models, within 2–3 induction iterations.2New Features in the PBSA ProgramMM/PB(GB)SA24 is an end-point method for calculating the free energies of molecules in implicit solvent, i.e., Poisson–Boltzmann (PB) and generalized Born (GB). Solvation interactions, especially solvent-mediated dielectric screening and Debye–Hückel screening, are essential determinants of the structure and function of biomolecules. Several efficient finite-difference numerical solvers, both linear25−27 and nonlinear,28 are implemented in pbsa for various applications of the Poisson–Boltzmann method. The GPU support of those solvers is also implemented in pbsa.cuda.29−31 In the 2023 release, improvements to the pbsa program include the integration of the Machine-Learned Solvent Excluded Surface (MLSES) model,32 which provides a highly efficient and differentiable molecular surface for continuum solvation modeling of biomolecules. Various options for the MLSES model have been implemented, allowing users to optimize performance on both central processing unit (CPU) and GPU platforms using Fortran, the CUDA kernel, and LibTorch. This flexibility enables users to choose the best-suited hardware and software environments for their needs. Additionally, an MBAR/PBSA strategy has been developed combining the PBSA continuum solvent model with the Multistate Bennet Acceptance Ratio (MBAR) approach. This coupling allows for more accurate modeling of electronic polarization, leading to improved accuracy in absolute binding free energy simulations of highly charged ligands.33To date, the GB model in AmberTools could be specified with the following “igb” values: 1,34 2,35 5,36 7, and 8.37 In 2017, an accurate yet efficient grid-based surface GB model was introduced38 which is currently available in AmberTools as a stand-alone application named GBNSR6 ($AMBERHOME/bin/gbnsr6).39 GBNSR6 calculates the solvation free energy of an input structure on a single snapshot. In AmberTools23, GBNSR6 has been integrated into MMPBSA.py such that it runs over multiple snapshots extracted from the trajectories of protein, ligand, and complex structures. To run this model, “igb = 66” is now available in MMPBSA.py. All input parameters of the stand-alone GBNSR6 program can be modified through the MMPBSA.py input file.3PyRESP and PyRESP_GENAccurate modeling of electrostatic and polarization effects is crucial in molecular simulations. Many polarizable force fields have been developed to account for these important effects. Among these models, the polarizable Gaussian Multipole (pGM) model has emerged as a self-consistent approach in handling both short-range and long-range interactions.18−23 We have recently developed the PyRESP program41 for electrostatic parametrizations for point charge additive models and induced-dipole models, including the pGM model. By performing least-squares fittings to electrostatic potentials surrounding molecules, the PyRESP program extends functionalities of the ancestor RESP program that only perform parametrizations for point charge additive models.42 However, the process of generating input files for PyRESP is tedious and error-prone. In the AmberTools23 release, we implemented a flexible and user-friendly program, PyRESP_GEN,82 to minimize the user’s efforts to set up a PyRESP run. In addition, we also optimized the restraint weights for the pGM models with and without permanent dipoles. For the pGM-perm model, the optimal strategy for electrostatic potential fitting is also proposed.43D-RISMThe 3D reference interaction site model (3D-RISM) of molecular solvation43 is an implicit solvent model that calculates equilibrium density distributions and thermodynamics of explicit solvent models. The implementation in AmberTools permits MD, energy minimization, and trajectory analysis through sander, while rism3d.snglpnt provides standalone trajectory analysis.44 Recently, periodic boundaries were introduced, allowing application to crystal structure refinement and other periodic systems.45 In addition, computational scaling for open boundaries was improved via treecode summation for electrostatic interactions, providing a 2–4 times speedup for typical proteins and enabling application to large biomolecular complexes with more than 1 million atoms.465LibTorch Interface to AmberWe introduced a LibTorch interface to the 2023 release of AmberTools, which is a cutting-edge C++ runtime library developed by the PyTorch team.47 This library enables flexible tensor computations and dynamic deep neural network modeling. Amber now provides two options for enabling the LibTorch library: a built-in mode and a user-installed mode. With the LibTorch integration, the pbsa program supports both CPU and GPU environments, making it highly versatile. Additionally, user instructions and tutorials have been provided for configuring the LibTorch library, making it more accessible to researchers and developers working in Amber and AmberTools.6Free EnergyFree energy methods have been a mainstay of Amber for decades.48,49 Besides our existing free energy technology base this latest release of AmberTools includes a collection of new software tools for the robust analysis of free energy simulations (FE-ToolKit)50,51 as well as workflow tools for production free energy simulation setup and analysis (ProFESSA)52 using the GPU-accelerated Amber free energy engine with enhanced sampling features. This software is part of the Amber Drug Discovery Boost package.536.1FE-ToolKitThe FE-ToolKit contains two main utilities: edgembar for analysis of alchemical free energy simulations (e.g., such as those used for prediction of ligand-protein absolute and relative binding free energies in drug discovery54) and ndfes for analysis of multidimensional free energy profiles (e.g., such as those used for prediction of minimum free energy pathways in studies of enzyme mechanisms55,56).6.2EdgembarThe edgembar program performs analysis of alchemical free energy simulations using the multistate Bennett acceptance ratio (MBAR) method,57 the Bennett acceptance ratio (BAR) method,58 exponential averaging,59 thermodynamic integration,60 or combinations of these approaches. Alchemical free energy simulations often calculate a network of relative free energy differences between two environments. For example, ligand binding applications in drug discovery use a network of alchemical transformations between ligands, termed a “thermodynamic graph”, where each ligand represents a “node” in the graph and each “edge” represents an alchemical transformation between ligands bound to their target relative to that in aqueous solution. Given the alchemical simulation outputs from the independent trials in both environments, edgembar will perform a “network-wide” free energy analysis,51 including the imposition of cycle closure and, optionally, experimental constraints. The analysis produces a comprehensive report of the results, including uncertainties and warnings. The report identifies potential problems with simulations that may require further attention. The issues include: a lack of convergence, the analysis of too few statistically independent samples, poor phase space overlap between adjacent alchemical states,61 and poor reweighting entropy.626.3NdfesThe ndfes program evaluates multidimensional free energy surfaces from umbrella sampling.50 The analysis can be performed with the variational free energy profile (vFEP) method,63,64 MBAR,57 the weighted thermodynamic perturbation method (wTP),65 and the generalized weighted thermodynamic perturbation method (gwTP).66 The wTP and gwTP methods estimate the free energy surface of an expensive target-level of theory from the sampling performed with inexpensive reference potentials.66 The estimation of ab initio QM/MM free energy surfaces in condensed-phase environments has become more practical in the latest version of AmberTools with the combined introduction of the GPU-accelerated QUICK software67 and ndfes analysis program.6.4ProFESSAThe ProFESSA workflow52 uses the GPU-accelerated AMBER free energy engine. The workflow establishes a flexible, end-to-end pipeline for performing alchemical free energy simulations that brings to bear technologies including new smoothstep softcore potentials and optimized alchemical transformation pathways,68 the alchemical enhanced sampling (ACES) method,69 and a network-wide free energy analysis51 with optional imposition of cycle closure and experimental constraints implemented in FE-ToolKit.7Quantum Mechanical/Molecular Mechanical MethodsAmber has had a long tradition of QM/MM methods and implementations,70 with the most recent additions being the QUICK/sander QM/MM implementation in AmberTools23.67,71−73QUICK/sander has been extensively updated, and its performance has been significantly improved. QUICK, as distributed with AmberTools23, can also be used as a standalone QM program for single point calculations or geometry optimizations.7.1Performance Improvements/AMD ImplementationWith the second-generation electron repulsion integral code and other performance enhancements recently introduced into QUICK,67,71−73 higher ps/day can be obtained in QM/MM simulations.73 For instance, with respect to AmberTools21,74 up to 2× speedups have been observed for benchmark simulations with different QM regions of photoactive yellow protein on NVIDIA V100 GPU.73 Furthermore, support for AMD GPUs has been enabled. Users can now make use of AMD data center cards such as MI50, MI100, MI200, and MI250 for simulations. According to benchmark studies, the performance on the MI100 is similar to that of NVIDIA V100.73 The implementation runs properly on MI200 and MI250 cards; however, the performance is not yet optimized for these cards. The recommended AMD GPU for the current version is MI100. An optimized version for MI2XX will be available to users in the next AmberTools release.7.2Long-Range ElectrostaticsFor the treatment of long-range electrostatics in QM/MM, the ambient-potential composite Ewald method (CEw) developed by Giese and York75 has been integrated. The performance penalty for turning on CEw in the GPU version is <25% for Hartree–Fock (HF) and <10% for density functional theory (DFT) in comparison to standard QM/MM with 8 Å electrostatic cutoff. This allows users to carry out more accurate simulations at a slightly higher computational cost.7.3DispersionAmong other minor features introduced into QUICK, dispersion corrections in DFT and data exporting capability into Molden format are notable. Grimme’s dispersion corrections (D2, D3 with different damping)76 can be used in QM/MM with appropriate functionals. Users can also export Cartesian coordinates, molecular orbitals, etc. of the QM region into Molden format for visualization purposes.8Automated Building of Membrane-Protein–Lipid-Bilayer SystemsPACKMOL-Memgen is a simple-to-use command line implementation of a generalized workflow for the automated building of membrane-protein–lipid-bilayer systems based on open-source tools including Packmol, memembed, pdbremix, and AmberTools.77 It allows for setting up multiple configurations of a system in a user-friendly and efficient manner, which can serve as starting configurations in MD simulations under periodic boundary conditions. Since its introduction, support was added for additional lipid headgroups and to include solutes in the water or membrane phase and generate curved membrane surfaces or double bilayer systems. Additionally, SIRAH78 coarse-graining routines can be used, and non-membrane systems (water or mixed-solvent simulations) can now be set up.79 In the AmberTools23 release, PACKMOL-Memgen now handles all Amber-supported ions and the OPC3 water model as well as allows generating HMR systems, providing control for pmemd.cuda, and using pdb2pqr for protonating the protein.9mdgxThe mdgx program, which began as a denovo reimplementation of the basic features needed for molecular dynamics and stayed in service for its uncommon capability of storing multiple topologies and coordinate sets in the space of a single runtime instance, has gained two noteworthy features. First, it can postprocess Amber topology files to add pmemd-compatible representations of the GROMACS virtual sites.80 While mdgx itself can perform limited MD simulations with such models, the performant pmemd GPU implementation can now incorporate massless sites into its free energy methods. Virtual sites require parameters to be useful, but the mdgx program itself has tools for fitting their charges as well as bonded parameters in the context of these extra monopoles. Virtual site force fields are a logical extension of popular fixed-charge models, entailing incremental updates to the dynamics engine and incremental increases in the cost of the simulations. Second, through its ability to calculate multiple systems at once, mdgx has an exploratory feature for running simple implicit solvent dynamics on many replicas of different topologies on one GPU. By running independent trajectories on each GPU multiprocessor, mdgx scales simulations of small peptides and drug molecules to modern GPUs with tens of times the throughput of other GPU MD implementations when tasked with small systems. This capability has been applied to docked pose refinement.8110The Amber Web Site and TutorialsThe Amber Web site ( supports the user community with new release information, manuals, tutorials, and information on force fields. Users are directed to the most recent manual version to learn about technical usage and appropriate literature references to communicate best practices in the field. The Amber tutorials have also been reorganized and span topics ranging from initial system setup to advanced methods (Figure 2). A tutorial overview page guides new users through the process of building, running, and analyzing a system and points them to key initial case studies. The recent tutorials overall are more modular, and learning objectives are given. New tutorial development has focused on building different system types and tutorials for creating stable systems through relaxation of system positions for both explicit and implicit solvent as well as a tutorial covering advanced thermodynamic integration methods such as using smoothstep softcore potentials,68 enhanced sampling for softcore ligand energies, and methods such as ACES.69Figure 2Overview of the Amber Tutorials. Tutorials are modular, cover the basic steps of a typical molecular dynamics simulation, introductory case studies, advanced methods, and some tools that are commonly employed by Amber users.Modeling software is not useful without compatible force fields. Included in the release of AmberTools are the force fields developed by the Amber community. The main force fields page contains a list of recommended force fields, and each type of molecule/ion has a separate page outlining nuances in choosing an appropriate force field.ConclusionsThe most significant additions to AmberTools23 are briefly summarized. AmberTools is freely available at Full details on licensing, distribution, and hardware supported can be found at

PMC

Annals of Dermatology

38061721

PMC10727879

11-01-2023

10.5021/ad.21.293

The First Case of Cutaneous Acanthamoebiasis Caused by

Choi Mi Soo, Myong Na Hye, Seo Min, Jang Sukbin, Yun Dae Kwan, Yeom Kyujin, Chung Dong-Il, Park Byung Cheol, Hong Yeonchul, Kim Myung Hwa

A 62-year-old man with multiple myeloma visited our clinic with multiple painful erythematous to purpuric nodules on his whole body. He received a skin biopsy which showed septal and lobular inflammation with vasculitis, and multiple amoebic organisms were found. Polymerase chain reaction and culture were performed and an Acanthamoeba triangularis infection was diagnosed. This is the first report on cutaneous acanthamoebiasis caused by A. triangularis, suggesting that A. triangularis should be regarded as a clinical pathogen that can cause ocular as well as disseminated infection.

INTRODUCTIONAcanthamoeba causes granulomatous amebic encephalitis (GAE), pneumonitis, cutaneous infection leading to disseminated disease, and amoebic keratitis (AK)1. Disseminated Acanthamoeba infections are rare and are self-limiting in healthy individuals but principally occur in immunocompromised individuals, including patients with AIDS or those who have undergone organ transplant, which may lead to fatal consequences2345678. Herein, we report the first case of cutaneous Acanthamoeba triangularis infection in Korea. We received the patient’s wife’s consent form about publishing all photographic materials.CASE REPORTA 62-year-old man with relapsed multiple myeloma who underwent chemotherapy presented with multiple painful erythematous to purpuric indurated papulonodules, pustules on his whole body in January 2021 (Fig. 1). He was hospitalized in the department of internal medicine due to general weakness and motor weakness in both legs.He was diagnosed with multiple myeloma and received an autologous peripheral stem cell transplantation in 2009. But the multiple myeloma relapsed, and he was treated with pomalidomide and dexamethasone since August 2020. He also had endoscopic sinus surgery for chronic rhinosinusitis in September 2020 and was under oral antibiotic therapy for septal abscess in December 2020. He suffered from bilateral optic neuritis since September 2020 and had been treated with oral methylprednisolone until December 2020.During hospitalization, the chest computed tomography (CT) showed multiple solid nodules in both lungs, and physicians considered them as lung involvement of the known multiple myeloma. On a second chest CT, bilateral diffuse airspace consolidation with surrounding ground-glass opacification was found in both lungs and was considered to be pneumonia. On initial blood examination, his white blood cell count was 12.5×103/µl (neutrophil 93.9%), red blood cell count was 11.2×103/µl, platelet count was 82×103/µl, C-reactive protein level was 13.37 mg/dl. On blood culture, Candida albicans was detected, and micafungin was added to his treatment.A skin biopsy was done on the right thigh and chin. Histopathologic examination showed septal and lobular inflammatory infiltrates predominantly composed of neutrophils with vasculitis (Fig. 2A, B). In addition, 20 to 50 pm-sized round amoebic organisms with eccentrically placed nuclei and central karyosomes were found (Fig. 2B). Amoebic organisms were also detected in the Giemsa stain (Fig. 2C). Entamoeba histolytica IgG was negative, and protozoa on routine stool examination were negative. The skin lesion did not respond to metronidazole treatment, and some of the mass lesions progressed into black crusts and ulcers (Fig. 3). Therefore, we sent a biopsy sample to the Department of Parasitology and Tropical Medicine in Kyungpook National University School of Medicine for further analysis.Acanthamoeba culture was performed using the biopsy samples as previously described9. Briefly, a part of the biopsy sample was chopped into small pieces using forceps and a scalpel and directly transferred onto 1.5% non-nutrient agar plates covered with heat-treated Escherichia coli (ATCC 25922; Washington D.C., USA). The plates were sealed, incubated at 27℃, and examined daily using inverted microscopy until amoebic growth was observed (Fig. 4A).According to the manufacturer’s instructions, DNA from the tissue biopsy samples was extracted using the QIAamp DNA Mini kit (Qiagen GmbH). Acanthamoeba DNA extracted from Acanthamoeba castellanii (ATCC 50374) and human genomic DNA (Promega) were used as positive and negative controls, respectively. A PCR was performed using three previously reported primer pairs10; ACARNA (forward: 5′-TCCCCTAGCAGCTTGTG-3′, reverse: 5′-GTTAAGGTCTCGTTCGTTA-3′)11, Nelson (forward: 5′-GTTTGAGGCAATAACAGGT-3′, reverse: 5′-GAATTCCTCGTTGAAGAT-3′)12, and JDP1-JDP2 (JDP1: 5′-GGCCCAGATCGTTTACCGTGAA-3′, JDP2: 5′-TCTCACAAGCTGCTAGGGAGTCA-3′)1314 in a final volume of 50 µl containing 25 µl of 2×GoTaq G2 Hot Start Green Master Mix (Promega Co.), 2 pmol of each primer, and an aliquot of the DNA. DNA was amplified at 30 cycles; 1 minutes at 58℃ and 40 seconds at 72℃ after a denaturation step at 94℃ for 7 minutes, 30 seconds at 94℃, followed by a final extension of 72℃ for 15 minutes. For Acanthamoeba species identification, the amplified PCR products obtained with JDP primer pairs were purified using a QIAquick PCR Purification kit (Qiagen GmbH) and cloned into pGEMT-easy plasmids (Promega Co.). The cloned DNA was analyzed for nucleotide sequences using Macrogen, Inc ( and subjected to basic local alignment search tool sequence analysis at the NCBI databases ( The nucleotide sequences obtained from this study were submitted to the Gen-Bank database (GenBank accession no. MZ310461).Direct microscopy of the skin biopsy sample showed the eukaryotic cells with morphological features resembling amoebic trophozoites. To improve the accuracy of diagnosis, PCR results using the three different primer sets previously reported revealed that the amoebae belonged to Acanthamoeba (Fig. 4B). Furthermore, 18S rRNA sequence analysis was performed using the extracted Acanthamoeba DNA from the culture of skin tissue samples and revealed 99% sequence identity with those of A. triangularis KA/E23 and KA/E2.There was no evidence of amoebiasis on the brain magnetic resonance imaging and cerebrospinal fluid test. A lung biopsy was considered to determine the presence of Acanthamoeba but could not be done because of the patient’s thrombocytopenia. Combination therapy of bactrim, metronidazole, and fluconazole was administered, but it was not effective. Miltefosine was recommended, but his wife declined. A few days later, the patient expired due to septic shock.DISCUSSIONAmoebiasis is the second leading cause of death from parasitic diseases. Only a few amoebas are pathogenic to humans. Within the genus Entamoeba, E. histolytica is a pathogenic amoeba that can cause intestinal and extraintestinal diseases15. Pathogenic and opportunistic free-living amoebas (FLAs) such as Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Sappinia pedata can infect humans5. Acanthamoeba are ubiquitous FLAs known to thrive in soil, air, and water, including sewage, swimming pools, flower pots, water tubs, humidifiers, aquaria, eyewash solutions, and hospital environments (e.g., dialysis and dental treatment units)5, as both cyst and trophozoite forms25. The cyst form is potentially infectious in humans because of its ability to convert to trophozoites2. Acanthamoeba causes GAE, pneumonitis, cutaneous infection leading to disseminated disease, and AK. The portal of entry is usually the nasal mucosa, so they can reach the central nervous system. They can also invade through a skin break or respiratory tract, and show subsequent hematogenous dissemination15. Disseminated Acanthamoeba infections are rare and are self-limiting in healthy individuals but principally occur in immunocompromised individuals, including patients with AIDS or those who have undergone an organ transplant, which may lead to fatal consequences2345678.Up until now, nine species of Acanthamoeba; A. castellanii, Acanthamoeba culbertsoni, Acanthamoeba divionensis, Acanthamoeba griffinii, Acanthamoeba hatchetti, Acanthamoeba healyii, Acanthamoeba lenticulata, Acanthamoeba polyphaga, and Acanthamoeba rhysodes have been associated with human disease. T4 genotype, the most pathogenic group of Acanthamoeba species, has been the most commonly identified in AK, GAE5,16, and cutaneous acanthamoebiasis16. In addition, A. triangularis, belonging to the T4 genotype, was originally isolated in France from human feces17 and had not been reported to be a keratopathogen. However, we previously determined the epidemiologic significance of A. triangularis as a keratopathogen from the case reports of AK and surveys for Acanthamoeba contamination in the contact lens care system in Korea9181920.Cutaneous acanthamoebiasis present with Acanthamoeba trophozoites and cysts, and the face and extremities are commonly involved. Multiple widely distributed papulonodules, pustules, cellulitis, non-healing ulcers, and eschars, have been reported. These lesions are typically 0.1 to 3.0 cm in diameter and can be tender. Lymphadenopathy and fever can be accompanied15. In our case, histopathologic findings showed dermal and/or subcutaneous inflammation, including predominantly neutrophils, lymphocytes, with vasculitis, and granulomatous changes. The identification of 20- to 30-µm trophozoites with abundant vacuolated cytoplasms and central nuclear karyosomes is diagnostic but may be difficult because of their macrophage-like appearance. Diagnostic methods include skin biopsy, indirect immunofluorescence assays, smears of tissue with Giemsa and trichrome staining, culture, and PCR. The comparatively high mortality rate between infected individuals and the lack of well-established treatment recommendations is of great concern for Acanthamoebic infections. Early diagnosis and treatment using a combination of intravenous pentamidine and oral fluconazole, sulfadiazine, isethionate, or 5-fluorocytosine may improve outcomes. HIV-positive patients may benefit from antiretroviral therapy2515.Interestingly, previous cases of A. triangularis infection and surveys have been observed only in the southeastern province of South Korea (Daegu and Busan). However, this case occurred in the western part of the central region of South Korea (Cheonan, Chungnam), indicating that the distribution of A. triangularis is gradually spreading throughout South Korea.We could not confirm whether it is primary cutaneous acanthamoebiasis or a secondary infection derived from another origin. We supposed that abnormal CT findings in the lung might be associated with acanthamoebiasis, but we could not perform further evaluations to confirm this because of the patient’s poor general condition. To our knowledge, this is the first report on cutaneous acanthamoebiasis caused by A. triangularis and suggests that A. triangularis should be regarded as a clinical pathogen that causes ocular as well as disseminated infection. This case can serve to inform physicians of a rare but fatal infection of Acanthamoeba in an immunocompromised patient with multiple myeloma.

PMC

Gastroenterology and Hepatology From Bed to Bench

PMC10520390

Jan-01-2023

10.22037/ghfbb.v16i2.2773

Gastric cavernous hemangioma in 48-years male patient: a rare case presenting upper gastrointestinal bleeding manifestations

Sukmagautama Coana, Asaduddin Aiman Hilmi, A’malia Ulya, Putri Desy Puspa

Gastric hemangioma (GH) is a rare benign tumor that may cause to upper gastrointestinal bleeding. Furthermore, this condition could lead life-threatening conditions thus should be recognized sooner to minimize unnecessary invasive surgical intervention, and accident. We reported a 48 years old man which came to emergency room (ER) with the chief complaint of hematemesis and black stool accompanied by abdominal pain, cold sweat, body weakness and enlarger stomach. Physical examination showed slightly icteric eye, and conjunctival pallor. On palpation, the epigastric and right upper quadrant was tender, and occult blood was detected in the excrement. A minor microcytic hypochromic anemia, absolute neutrophilia, hypoalbuminemia, and an increase in urea and creatinine were determined by laboratory tests. Moreover, the esophagogastroduodenoscopy was performed, and showed broad mass with dilated blood vessels. The histopathological examination result showed gastric mass with the histological erythrocyte extravasation. The diagnosis was hematemesis melena owing to cavernous GH with differential diagnosis of hematoma, and other gastric mass, with anemia gravis. For the treatment, patient received fluid resuscitation, omeprazole, tranexamic acid, somatostatin, and antibiotics. He received two kolfs transfusion of packed red cell. Gastric hemangiomas are benign vascular tumors that can lead to severe gastrointestinal bleeding. These benign tumors are lesions that develop as a result of endothelial cell proliferation, and concomitant pericytic hyperplasia, which leads to a collection of dilated vessels. The cavernous subtype of GHs often comprises of bigger blood-filled areas and larger blood vessels. It is more likely for the cavernous GH to rupture, leading to substantial bleeding. Endoscopic assessment is important in the patients with upper GI bleeding, and GH appear as well-circumscribed vascular submucosal mass. Although this disease is benign with a lower recurrence, we suggest for further surgical treatment and the requirement for long-term follow-up to assess the outcome.

IntroductionUpper gastrointestinal bleeding can be danger if not managed timely or properly. Serious effects include respiratory distress, myocardial infarction, infection, shock, and, worst of all, fatality might result . In the third year of following admission, the mortality rates from all causes are close to 37% . Hematemesis and melena are both often seen clinical signs of upper gastrointestinal hemorrhage. Hematemesis is the process of having blood mingled with stomach contents or regurgitated. Melena is a term used to describe black, dark, and tarry feces. Its distinctive odor is brought on by the action of intestinal bacteria and digestive enzymes on hemoglobin .Gastric hemangioma (GH) is a rare benign tumor that usually develops from congenital abnormalities of the mesenchymal tissues, particularly angioblastic cells, and only accounts for 1.6% of benign tumors of stomach. It is one of the uncommon disorders that may lead to upper gastrointestinal bleeding (3, 4). It may be very difficult to detect the condition because of its rarity and unique features, which might pose a significant diagnostic problem and perhaps need surgical excision. Among the most common symptoms are epigastric discomfort, dyspepsia, and upper gastrointestinal bleeding, which can be gradual and subtle or acute and life-threatening conditions . However, this case should be recognized immediately to minimize unnecessary invasive surgical intervention. Herein, we describe an upper gastrointestinal bleeding caused by GH.Case reportA 48 years old man came to the emergency room (ER) with the chief complaint of hematemesis and black stool accompanied by abdominal pain, cold sweat, body weakness, and stomach seemed enlarger for past weeks. He confirmed that he routinely consumed herbal medicine and alcohol as a youth. He had no history of intestinal hemorrhage, black stools, or bowel habit changes in the past. Previously, he had been confined to the hospital due to a biliary disorder. Moreover, patient denied for history of hypertension, trauma, diabetes, or using antiplatelet, anticoagulant, and steroid. Figure 1Esophagogastroduodenoscopy showed broad mass with dilated blood vessels extended from the gastric fundus via the angulusOn the arrival at ER, the patient was hemodynamically stable with the blood pressure (BP) was 121/78 mmHg, pulse 126 beats per minute (bpm), respiration rate (RR) 20x/minutes, SPO2 92%, body weight 70 kg, height 158 centimeter. A visual examination revealed paler conjunctiva and a little icterus. No murmurs or other abnormalities were detected; only normal cardiac sounds were audible. When the right upper quadrant and the epigastrium were palpated, there was soreness. During a rectal examination, feces that tested positive for occult blood were found. For the laboratory test at ER, the result was slight microcytic hypochromic anemia (Hb 12.7 g/dL, MCV 84.5/UM, MCH 29.3 pg), absolute neutrophilia (neutrophil 83.4%, leucocytes 23.85 x 103/µl), hypoalbuminemia (3.2 g/dL), and elevation of urea (66 mg/dl) and creatinine (1.38 mg/dL) with the normal liver function test, and normal prothrombin time test. An upper esophagogastroduodenoscopy (EGD) was performed (Figure 1). The lower esophageal sphincter (LES) was not in a good condition. The entire area of gastric mucosa was hyperemic, and there was a gaping pylorus, and a broad mass with dilated blood vessels (hemangioma) that extended from the gastric fundus via the angulus. Duodenum was shown hyperemic. The results of the histopathological investigation showed a stomach mass with histological erythrocyte extravasation, which is a characteristic of hematomas and hemangiomas. The biopsy revealed tissue pieces without any evidence of cancer (Figure 2), including extensive extravasations of erythrocytes, necrotic tissue, lymphocytes, and macrophages. Thus, the patient was diagnosed as hematemesis melena owing to gastric cavernous hemangioma with differential diagnosis of hematoma and other gastric mass, with anemia gravis.In the ER, the patient was given injection of omeprazole 40 mg, ondansetron 8 mg, tranexamic acid 500 ml, supportive therapy, and intravenous fluid resuscitation with the streamed nasogastric tube (NGT). Because of stability hemodynamic, he was transferred to the hospital. Ondansetron 8 mg/12 hours, tranexamic acid 500 mg/8 hours, omeprazole 40 mg in normal saline 25 cc on syringe pump, vitamin K 1 amp/18 hours, somatostatin 1 vial diluted in 50 cc normal saline, and injections of antibiotics ceftriaxone 2 gr/24 hours and metronidazole 500 mg/8 hours were all administered to him at the ward. Before there are no longer any blood products in NGT, patients are recommended to fast. At day 6 at the ward hospital, the patient still developed hematemesis and intermittent black stool, and the hemoglobin dropped to 5.9 g/L with MCV 86.4/UM, MCH 29.8 pg and absolute neutrophilia (neutrophil 81.6%, leucocytes 21.94 x 103/µl). Regarding these conditions, patient received two kolfs transfusion of packed red cell and emergently transferred to gastroenterology and hepatology sub-specialist for further treatment.Figure 2Histopathological examination result revealed gastric mass with the histological erythrocyte extravasation that could be found in hemangioma (Hematoxylin-Eosin Staining; Left 40x, Right 100x)DiscussionHemangiomas may occur in all organs, especially on the body surface as isolated or multiple lesions. But, hemangioma rarely observed in visceral organs, except the liver . Gastric hemangiomas, one of visceral hemangioma, are benign vascular tumors that can lead to severe gastrointestinal bleeding . These benign tumors are lesions that arise from endothelial cell proliferation together with concurrent pericytic hyperplasia or aberrant pericyte division, which results in a group of dilated vessels . Gastrointestinal hemangiomas were pathologically classified by Kaijser as Multiple phlebectasia, cavernous hemangioma, capillary hemangioma, and angiomatosis . Between 40% and 60% of all gastrointestinal hemangiomas have multiple phlebectasia. Rarely, the muscularis propria, and the subserosa have also been the site of these 1–5 mm lesions, which typically affect the submucosa. An autosomal dominant form found in Osler-Rendu-Weber illness and a non-hereditary variety were identified. Both methods carry a considerable risk of bleeding . The cavernous subtype, on the other hand, often consists of bigger blood vessels with broader gaps (caverns) or sinuses that are coated with one or more layers of endothelal cells . Thus, the cavernous GH are more prone to rupture, which resulting in significant bleeding . Besides, capillary hemangioma, which represent fewer than 10% of all hemangiomas, is a proliferation of small capillaries composed of thin-walled, blood-filled spaces lined by endothelial cells . This capillary subtype is usually solitary, well-circumscribed intraluminal growths around 1 cm in diameter . Another hemangioma is linked to hemangiomatosis, which frequently associated with extragastrointestinal hemangioma. It consisted between 2% and 12% of all hemangiomas and cannot be distinguished from diffuse cavernous hemangioma grossly . In this instance, the EGD examination revealed a wide mass that matched a gastric cavernous hemangioma and had dilated blood vessels extending from the fundus through the angulus.GHs are frequently found asymptomatic, and mostly identified accidentally advance imaging techniques. Epigastric pain and concomitant GI bleeding are the characteristic signs of symptomatic GH, with heavy sudden onset, occasionally recurrent bleeding from cavernous-type tumors presenting with anemia, hematemesis, and/or melena symptoms (4, 6, 10, 11). In our case, the patient present all of those symptoms, and planned for EGD examination. Therefore, USG, endoscopic USG, CT, abdominal magnetic resonance imaging (MRI), and angiography are essential components of the imaging modality. As non-invasive preoperative evidence of resectability , radiological examinations are advantageous for demonstrating the location and structure. The combination between imaging and endoscopic assessment may provide more effective, especially with the occurrence of phleboliths as pathognomonic presentation of GHs in CT-scan . Thus, our patient was not examined for imaging modality because there was gastrointestinal warning sign, which were hematemesis and melena. Endoscopic assessment is importance in the patients with upper GI bleeding. In most cases, GH manifests as well-defined, vascular submucosal masses that range in color from bluish-black to brilliant red, with or without calcification (6, 12). We only found vascular submucosal mass ranging from gastric fundus through the angulus. The diagnosis of this case was acquired by histopathology examination, which showed histological erythrocyte extravasation without sign of malignancy. Previous study described that extravasated red blood cells are frequently identified in anastomosing hemangioma . The submucosal placement and dense vascular character of such lesions, however, restrict the significance of endoscopic biopsy investigation for the histological identification of hemangiomas . Endoscopic biopsy was thus unable to provide a firm diagnosis. Severe hemorrhage may occur in terms of rupture of GH, which lead to rapid hemodynamic decompensation and should be treated emergently . Our patient showed signs of hemodynamic compromise, which was treated with adequate fluid resuscitation and blood transfusion. The combination of somatostatin and omeprazole has been suggested in a prior research as a potentially effective therapy option for the management of acute upper gastrointestinal bleeding . Somatostatin analogue was given to decrease the portal pressure and antibiotics should be started to reduce the risks of infection . However, surgical involvement is considered as the main curative treatment following supportive pharmacotherapy in adult patients with no reports of recurrence after complete resection .ConclusionWe reported a case of gastric cavernous hemangioma in 48-years male patient originating from the gastric fundus via the angulus. Despite biopsy examination is limited because of the submucosal localization and dense vascular nature of such lesions, our assessment showed histological erythrocyte extravasation without sign of malignancy, which could be found in hemangioma or hematoma. Although the current condition is benign and has a low recurrence rate, we recommend further surgical treatment and the need for long-term follow-up to evaluate the results.Conflict of interestsAuthors have no potential conflicts of interest to disclose.

PMC

Journal of Inflammation Research

37663759

PMC10473432

8-28-2023

10.2147/JIR.S413994

Differential Expression Profiles of Plasma Exosomal microRNAs in Rheumatoid Arthritis

Yang Xiaoke, Wang Zhixin, Zhang Mingming, Shuai Zongwen

AimDifferential expression maps of microRNAs (miRNAs) are connected to the autoimmune diseases. This study sought to elucidate the expression maps of exosomal miRNA in plasma of rheumatoid arthritis (RA) patients and their potential clinical significance.MethodsIn the screening phase, small RNA sequencing was performed to characterize dysregulated exosome-derived miRNAs in the plasma samples from six patients with RA and six healthy patients. At the independent verification stage, the candidate plasma exosomal miRNAs were verified in 40 patients with RA and 32 healthy patients by using qRT-PCR. The correlation of miRNA levels and clinical characteristics was tested in patients with RA. The value of these miRNAs in diagnosing RA was assessed with the receiver operating characteristic curve.ResultsDuring the screening phase, 177 and 129 miRNAs were increased and decreased in RA patients and healthy controls, respectively. There were 10 candidate plasma exosomal miRNAs selected for the next identification. Compared with the healthy controls, eight plasma exosomal miRNAs (let-7a-5p, let-7b-5p, let-7d-5p, let-7f-5p, let-7g-5p, let-7i-5p, miR-128-3p, and miR-25-3p) were significantly elevated in RA patients, but miR-144-3p and miR-15a-5p expression exhibited no significant changes. The let-7a-5p and miR-25-3p levels were linked to the rheumatoid factor-positive phenotype in RA patients. For the eight miRNAs, the area under the subject work characteristic curve (AUC) is 0.641 to 0.843, and their combination had a high diagnostic accuracy for RA (AUC = 0.916).ConclusionOur study illustrates that novel exosomal miRNAs in the plasma may represent potential noninvasive biomarkers for RA.

IntroductionRheumatoid arthritis (RA) belongs to the autoimmune disease that exhibits chronic systemic symptoms that primarily impair cartilage and bone, leading to joint swelling, pain, impaired movement, and decreased quality of life.1 Approximately 0.5–1.0% of the population are affected by RA worldwide, disproportionately affecting females at a 3:1 ratio to males.2,3 It has been proved that genetic, epigenetic and environmental factors were involved in RA development.4 Many genes have been found to be related to RA susceptibility via genome-wide association studies. Over 80% of the human genome is transcribed with little or no protein-coding capability.5 It was reported that microRNAs (miRNAs) played a key role in the development and progression of autoimmune diseases such as RA, and circulating levels of miRNA may represent potential biomarkers for these diseases.6,7Cells could secret various extracellular vesicles, including exosomes, and are ubiquitously distributed in bodily fluids.8,9 With the characteristics of stability and biocompatibility, exosomes play essential roles in autoimmune diseases.10 Moreover, exosomes can also serve as carriers through which to contain and deliver a variety of functional substances to assist in intercellular communications.11–13 MiRNAs are defined as single-stranded noncoding RNAs consisting of ~22 nucleotides that participate in regulating post-transcriptional gene expression.14 Therefore, utilizing RNA sequence technology, growing evidence indicates that dysregulated exosomal miRNAs were promising biomarkers for diagnosis and treatment of tumor and autoimmune diseases.15–19 However, there are limited studies investigating the differential expression profiles of miRNAs derived from plasma exosomes in RA.Exosomes that could be released by various cells have been reported, including inflammatory and immune cells responsible for RA development.20 Growing results suggest that miRNAs contained in exosomes are critically involved in inflammation and immunity.21 Therefore, it is important to identify additional dysregulated miRNAs as novel biomarkers and potential targets for RA therapy. Herein, we investigated expression maps of exosomal miRNAs in plasma of patients with RA versus healthy controls, and analyzed their potential clinical significance.Materials and MethodsPatients and Data CollectionA two-stage analysis was conducted. For the screening phase, 6 RA patients (1 male and 5 females; 45.17 ± 6.65 years) and 6 healthy age- and sex-matched patients (1 male and 5 females; 40.00 ± 12.63 years) were included. In the validation phase, 40 patients with RA (4 males and 36 females; 47.12 ± 10.72 years) and 32 healthy age- and sex-matched patients (2 males and 30 females; 42.72 ± 10.49 years) were included.We recruited the patients with RA and healthy patients from the First Affiliated Hospital of Anhui Medical University, and diagnosed in accordance with the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) 2010 classification criteria for RA.22 Any RA patients with other autoimmune conditions, serious infections, or pregnancy were eliminated. The healthy patients did not have RA or other autoimmune diseases.We collected demographic and clinical parameters through questionnaires or medical records. The informed consent was supplied by all participants on the basis of the Helsinki Declaration. The Ethics Committee of Anhui Medical University approved the protocols used in this study.Extraction of PlasmaPlasma samples were obtained from EDTA-anticoagulant venous blood (10 mL) by centrifugation at 4000 rpm for 10 min and then stored at −80°C in a fresh RNase-free tube preparing for extracting exosomes.Isolation of ExosomesIn the screening phase, ultra-centrifugation was conducted to isolate exosomes from the plasma samples collected from six RA patients and six healthy controls. During the independent validation phase, we used an exoEasy Maxi Kit (Qiagen, Germany) to extract plasma exosomes from 40 RA patients and 32 healthy controls in accordance with the manufacturer’s protocol. The study subject characteristics in both stages are summarized in Table 1.Table 1Clinical Characteristics of RA Patients and Healthy ControlsCharacteristicsScreening StageValidation StageHealthy controls632 Age (years), mean±SD40.00±12.6342.72±10.49 Sex, female/male5/130/2RA640 Age (years), mean±SD45.17±6.6547.12±10.72 Sex, female/male5/136/4 Disease Duration (years), M(P25, P75)1.60(0.20, 6.40)5.45(1.80, 10.05) DAS28, M(P25, P75)3.28(1.86, 5.26)2.83(2.30, 4.11) ESR (mm/h), M(P25, P75)23.00(9.75, 56.25)20.50(12.25, 27.75) CRP (mg/L), M(P25, P75)6.60(0.68, 37.13)10.73(3.30, 26.98) RF (IU/mL), M(P25, P75)69.50(0.10, 210.23)73.35(29.48, 108.23) Anti-CCP (RU/mL), M(P25, P75)225.00(8.00, 1305.00)401.60(2.00, 1094.75) GC, no. (%)1(16.67)17(42.50)Abbreviations: ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, rheumatoid factor; Anti-CCP, anticyclic citrullinated peptide; GC, glucocorticoid. Identification of ExosomesTransmission Electron Microscopy (TEM)TEM was performed to observe the morphology of exosomes. Briefly, 10 µl diluted exosomes were dripped onto the copper mesh with precipitating for 1 minute. The float was removed by filter paper. Then, 2% phosphotungstic acid was dripped onto the copper mesh with precipitating for 1 minute. After removing the float, the sample was dried at room temperature for a few minutes. The images of exosomes were taken on TEM (HITACHI, HT-7800, Hitachi, Ltd, Japan) at a 80-kV acceleration voltage.Nano-Flow Cytometry (NanoFCM)Thirty microliters of diluted exosomes were used to obtain the size and concentration information. The size distribution of isolated exosomes was detected by NanoFCM using High-Sensitivity Flow Cytometry Flow NanoAnalyzer (Fuliu Biotechnology, Ltd, China).Western Blot (WB)BCA Protein Assay (Pierce, Thermo Scientific) was used to detect the protein concentrations before WB analysis. Then, the protein samples were configured into 10% gel of sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS PAGE) for electrophoresis. After the proteins were transferred to PVDF membranes, the primary antibody CD9 (1:1000, Abcam, ab92726), CD81 (1:1000, Abcam, ab109201), TSG101 (1:1000, Abcam, ab125011) and Calnexin (1:1000, Abcam, ab22595) were used to incubate proteins at 4°C overnight. Then, the proteins were incubated with secondary antibody. Finally, the proteins were visualized by a chemiluminescence imager (BioRad, China).Total RNA ExtractionIn the small RNA sequencing screening phase, the total RNA was extracted from plasma exosomes with an miRNAeasy Plasma Kit (Qiagen, Germany). In validation phase, total RNA in exosome was abstracted by a miRNAcute miRNA isolation kit (Tiangen Biotech, Beijing, China). All operations were performed in accordance with the manufacturer’s protocol. We used NanoDrop™ 2000 spectrophotometer (Thermo Scientific, USA) to detect the RNA concentrations.Small RNA Sequencing and Data AnalysisBGI (China) conducted small RNA library preparation, sequencing, and bioinformatic analyses. Library sequencing was carried out with the BGISEQ-500 platform. The sample miRNAs were analyzed using DESeq2 software.A threshold P value of 1.5 was used to identify the upregulated and downregulated miRNAs. The clustering hierarchy and volcano plot were used to display the differences in exosomal miRNAs expression patterns between the samples. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were carried out to describe the roles of target mRNAs.miRNA Verification by qRT-PCRmiRcute Plus miRNA First Strand cDNA Synthesis Kit (Tiangen Biotech, Beijing, China) was used to independently verify the data according to the instructions. Subsequently, qRT-PCR assay was conducted with miRcute Plus miRNA qPCR Kit (SYBR Green, Tiangen Biotech, Beijing, China) to measure the candidate miRNAs selected based on the P value and fold change. The following reaction conditions were used: 95°C for 15 min, 5 cycles at 94°C for 20 s, 63°C for 30 s and 72°C for 34 s, followed by 40 cycles at 94°C for 20 s and 60°C for 34 s. All of the primers used for 10 miRNAs (let-7a-5p, let-7b-5p, let-7d-5p, let-7f-5p, let-7g-5p, let-7i-5p, miR-128-3p, miR-25-3p, miR-144-3p, miR-15a-5p) and U6 (as an internal standard) were obtained from Tiangen Biotech. The data were presented as the relative level of target miRNA expression normalized to U6. Each sample was detected in triplicate. Relative quantification of candidate miRNAs expression was according to the 2−ΔΔCt method, with ΔCt = Cttarget − CtU6 and −ΔΔCt = −(sample ΔCt − control ΔCt).Statistical AnalysisSPSS 23.0 (SPSS Inc., Chicago, IL, USA) software was used to calculate all of the statistical analyses, mean ± SD, or median. The distribution of the continuous variables was described according to the interquartile range. Categorical data was presented as a frequency (percentage). Quantitative data were compared by using Mann–Whitney U-test or Student’s t-test. Mann–Whitney U-test, Student’s t-test, and Spearman's rank correlation analysis were used to analyze the correlation between miRNA expression levels and clinical characteristics of RA. According to the results of univariate analysis and binary logistic regression analysis, the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) were performed to indicate the diagnostic power of these exosomal miRNAs. Bilateral P-value 1.5 plus a P value of 0.05). Then, RA patients were divided into active and stable RA subgroups. However, there was no significant difference between active RA and stable RA groups (all P > 0.05) (Figure 6).Table 210 Candidate Exosomal miRNAs Detected Using RNA SequencingExosomal miRNAsFold Change (RA vs HC)P (RA vs HC)let-7a-5p1.79511806983619<0.001let-7b-5p1.88682438980247<0.001let-7d-5p2.12796920044394<0.001let-7f-5p2.20236686026957<0.001let-7g-5p2.10234694158634<0.001let-7i-5p2.14912033540066<0.001miR-128-3p2.34503224590021<0.001miR-25-3p2.47566008042918<0.001miR-144-3p2.63516794800077<0.001miR-15a-5p2.53623348007171<0.001 Table 3The Expressions of 10 Candidate Exosomal miRNAs in the Preliminary Validation PhaseExosomal miRNAHealthy ControlsRAZPlet-7a-5p0.24(0.14, 0.36)0.73(0.43, 0.92)−4.975<0.001let-7b-5p0.14(0.07, 0.25)0.36(0.17, 0.60)−3.796<0.001let-7d-5p3.36(2.24, 5.57)10.67(4.74, 16.25)−3.972<0.001let-7f-5p0.20(0.15, 0.46)0.59(0.28, 0.81)−3.649<0.001let-7g-5p0.18(0.10, 0.30)0.49(0.27, 1.15)−4.720<0.001let-7i-5p25.00(11.68, 47.88)45.08(21.86, 56.70)−2.0460.040miR-128-3p1.54(1.02, 3.35)3.65(1.76, 5.39)−2.9290.003miR-25-3p28.84(16.14, 46.77)43.44(28.27, 52.90)−2.3910.016miR-144-3p5.75(2.19, 7.77)6.28(4.19, 9.47)−1.5190.128miR-15a-3p42.16(26.69, 63.12)56.39(31.64, 77.72)−1.5870.112 Figure 5(a–j) Comparison of relative levels of 10 plasma exosomal miRNAs between RA patients and control subjects.Figure 6(a–j) Comparison of relative levels of 10 plasma exosomal miRNAs between the stable and active RA patients.Correlation Between Significant Plasma Exosomal miRNAs and RA Clinical ParametersThe exosomal let-7a-5p and miR-25a-3p expressed in RA patients were positively associated with RF (Z = 0.363, P = 0.022; Z = 0.317, P = 0.046). However, there was no significant correlation of the eight upregulated exosomal miRNAs with disease duration, DAS28, ESR, CRP and anti-CCP (Table 4). Furthermore, no significant difference in the eight exosomal miRNAs between GC treatment RA group and GC non-treatment RA group was found (all P > 0.05).Table 4Correlation Between Clinical Parameters and 8 Upregulated Exosomal miRNAs in RA PatientsCharacteristicslet-7a-5plet-7b-5plet-7d-5plet-7f-5plet-7g-5plet-7i-5pmiR-128-3pmiR-25-3prsPrsPrsPrsPrsPrsPrsPrsPDisease Duration−0.0500.7610.0480.7670.0430.7910.1030.529−0.2360.1420.1830.2580.0430.790−0.0860.596DAS280.2180.1760.1080.5060.1540.3440.1890.2420.1170.471−0.1220.4550.2140.186−0.0520.750ESR0.1410.3870.0420.7970.1120.4930.0750.6450.0770.637−0.1150.4810.1870.248−0.0830.611CRP0.2130.1870.0170.9160.1340.4090.2830.0770.1600.3240.0470.7720.2140.185−0.0760.640RF0.3630.022*0.0450.7830.1430.3790.2870.0720.1420.383−0.0100.9510.1490.3600.3170.046*Anti-CCP0.1130.486−0.0340.837−0.0060.9690.1660.3070.2360.143−0.2200.1720.0930.5680.0940.563Note: *P<0.05. The Diagnostic Power of Significant Plasma Exosomal miRNAsThe AUC values (95% confidence intervals) of the eight exosomal miRNAs elevated in RA patients were 0.843 (0.749, 0.937), 0.762 (0.652, 0.871), 0.774 (0.662, 0.886), 0.752 (0.632, 0.872), 0.825 (0.731, 0.919), 0.641 (0.510, 0.772), 0.702 (0.578, 0.826), and 0.665 (0.536, 0.794), respectively, and that of the eight exosomal miRNAs in combination was 0.916 (0.853, 0.980) (Table 5 and Figure 7).Table 5Diagnosis Value of 8 Upregulated Exosomal miRNAs Distinguishing RA Patient and Healthy SubjectsExosomal miRNAAUCP95% CISensitivitySpecificitylet-7a-5p0.843<0.001(0.749, 0.937)80.0%81.2%let-7b-5p0.762<0.001(0.652, 0.871)62.5%84.4%let-7d-5p0.774<0.001(0.662, 0.886)82.5%71.9%let-7f-5p0.752<0.001(0.632, 0.872)87.5%62.5%let-7g-5p0.825<0.001(0.731, 0.919)70.0%84.4%let-7i-5p0.6410.0408(0.510, 0.772)65.0%71.9%miR-128-3p0.7020.0034(0.578, 0.826)72.5%65.6%miR-25-3p0.6650.0168(0.536, 0.794)72.5%65.6%Combined miRNAs0.916<0.001(0.853, 0.980)82.5%90.6%Abbreviation: CI, confidence interval. Figure 7ROC curves of eight plasma exosomal miRNAs and combined miRNAs for RA diagnosis.DiscussionThis study applied small RNA sequencing technology to elucidate a comprehensive miRNA expression map of exosomes in plasma of RA patients and healthy controls. The results revealed that, 306 miRNAs in the plasma exocrine of RA patients were abnormally expressed compared with the healthy control group, including 177 upregulated and 129 downregulated miRNAs. A total of 10 candidate miRNAs were further identified by using RT-qPCR. The levels of eight miRNAs (let-7a-5p, let-7b-5p, let-7d-5p, let-7f-5p, let-7g-5p, let-7i-5p, miR-128-3p, and miR-25-3p) were elevated in plasma exosomes of RA patients, consistent with the small RNA sequencing results. These findings support the reliability of the small RNA sequencing analysis. Additionally, the let-7a-5p and miR-25-3p levels in our study were associated with a rheumatoid factor-positive phenotype in RA patients. Finally, the value of these eight miRNAs was explored as a potential biomarker of RA.The let-7 family is a novel miRNA, which can regulate the immune killing effect of the immune system by participating in the metabolism, maturation and activation process of immune cells, and is considered to be a new target molecule for immunotherapy.23 Numerous studies have suggested that multiple members of the let-7 family may be involved in the pathogenesis of RA. Recently, a study demonstrated that elevated plasma let-7a-5p could serve as diagnostic marker of RA.24 Another study indicated that let-7a-5p was downregulated in peripheral blood mononuclear cells (PBMCs) of RA.25 Zhu suggested that the expression of let-7a in synovial fluid macrophages was significantly lower in RA patients than in osteoarthritis (OA) patients.26 Besides, let-7a downregulated macrophage activation induced by anti-CCP through targeted binding to HMGA2 and was associated with RA severity. Lai showed that anti-CCP inhibited monocyte let-7a expression in RA patients and promoted inflammation in RA.27 It was reported that the elevated level of exosomal let-7b in the synovial fluid of RA could induce arthritis by combining with Toll-like receptor 7 (TLR-7).28 It has previously been demonstrated that the level of let-7d-5p were increased in the serum and CD8+T cells of RA patients.29,30 The level of let-7f-5p has been identified as a potential biomarker in various autoimmune diseases such as systemic lupus erythematosus (SLE), osteoarthritis (OA), type 1 diabetes (T1D), multiple sclerosis (MS) and myasthenia gravis (MG).31–35 Yang suggested that let-7g-5p was decreased in CD4+T cells and let-7g-5p could alleviate arthritis by inhibiting Th17 cell differentiation.36 Besides, the level of let-7i-5p was increased in the plasma of T1D and the serum of systemic sclerosis (SSc).32,37 The above evidence indicated that the let-7 family is closely associated with the occurrence of multiple autoimmune diseases including RA. Our study is the first study to investigate the association between plasma exosomal let-7 (let-7a-5p, let-7b-5p, let-7d-5p, let-7f-5p, let-7g-5p, let-7i-5p) and RA.It has been reported that the levels of miR-128-3p were significantly elevated in the plasma, T cells and RA-FLS of RA patients.38–42 Our study first discovered increased level of plasma exosomal miR-128-3p in RA patients. Recently, a bioinformatics analysis showed that miR-25-3p is possibly associated with the regulation of potential RNA regulatory pathway by targeting GZMA in RA.43 Rodríguez-Muguruza found that the serum exosomal miR-25-3p was related to the early diagnosis of RA, which is compatible with our current study results.44 Intriguingly, Rodríguez-Muguruza verified increased levels of miR-144-3p and miR-15a-5p expression in the serum exosomes of early RA patients.44 In contrast, our study revealed no significant differences in exosomal miR-144-3p and miR-15a-5p expression between the RA patients and healthy controls. The reason for this difference may be due to differences sample sizes, methods of measurement, clinical heterogeneity of the patients and the conditions of drug use.This study was associated with some limitations. First, this study had a relatively small sample size. Therefore, the correlation requires further verification in a larger cohort. Second further studies should be performed to verify whether the changes of these miRNAs in RA patients can be distinguished from other autoimmune diseases. Finally, the detailed mechanism of these miRNAs in plasma exosomes was not explored with respect to the underlying RA development and progression. Thus, additional fundamental studies on miRNAs of interest should be conducted.The findings of this study reveal the potential value of differentially expressed plasma exosomal miRNA profiles and provide novel insight into the diagnostic biomarkers and promising RA therapeutic targets. However, further experimental studies are necessary to validate the functional mechanism between these identified exosomal miRNAs and RA.ConclusionOur study illustrates that novel exosomal miRNAs in the plasma may represent potential noninvasive biomarkers for RA. The let-7a-5p and miR-25-3p levels are correlated with a certain parameter of RA. The potential mechanism between these exosomal miRNAs and RA needs to be verified.

PMC

Blood Advances

36689726

PMC10248041

1-25-2023

10.1182/bloodadvances.2022009382

Acalabrutinib and high-frequency low-dose subcutaneous rituximab for initial therapy of chronic lymphocytic leukemia

Wallace Danielle S., Zent Clive S., Baran Andrea M., Reagan Patrick M., Casulo Carla, Rice Geoffrey, Friedberg Jonathan W., Barr Paul M.

Key Points•Acalabrutinib and HFLD subcutaneous rituximab was assessed as a potential fixed-duration initial therapy for chronic lymphocytic leukemia.•This home-administered combination had a 100% response rate and can be a backbone for regimens aiming to achieve limited duration therapy. Visual Abstract AbstractBruton tyrosine kinase inhibitors are an effective therapeutic agent for previously untreated patients with chronic lymphocytic leukemia but require indefinite treatment that can result in cumulative toxicities. Novel combinations of agents that provide deep remissions could allow for fixed duration therapy. Acalabrutinib, unlike ibrutinib, does not inhibit anti-CD20 monoclonal antibody-dependent cellular phagocytosis, making it a suitable partner drug to rituximab. Using standard dosing (375 mg/m2) of rituximab causes loss of target membrane CD20 cells and exhaustion of the finite cytotoxic capacity of the innate immune system. Alternatively, using high-frequency, low-dose (HFLD), subcutaneous rituximab limits loss of CD20 and allows for self-administration at home. The combination of HFLD rituximab 50 mg administered twice a week for 6 cycles of 28 days with the addition of acalabrutinib starting in week 2 was evaluated in a phase II study of 38 patients with treatment naive chronic lymphocytic leukemia. Patients achieving a complete response with undetectable minimal residual disease after 12 or 24 cycles of acalabrutinib could stop therapy. All patient responded, including one with a complete response with undetectable minimal residual disease in the peripheral blood and bone marrow at 12 months who stopped therapy. At a median follow-up of 2.3 years 2 patients with high-risk features have progressed while on acalabrutinib monotherapy. We conclude that HFLD rituximab in combination with acalabrutinib is an effective and tolerable self-administered home combination that provides a platform to build upon regimens that may more reliably allow for fixed-duration therapy. This trial was registered at www.clinicaltrials.gov #NCT03788291.

IntroductionChronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) is the most prevalent mature B-cell neoplasm in the United States.1 Although the introduction of targeted small molecules has improved clinical outcomes, CLL remains incurable, and conventional therapies that are often continued as long as being tolerated and are effective. Further improvement in treatment outcomes will likely require development of multidrug targeted therapies with the potential to achieve deep remissions with undetectable minimal residual disease (MRD).2 These regimens could then be used for limited duration therapy and provide the basis for future development of potentially curative treatments.The anti-CD20 monoclonal antibodies (mAb), rituximab and obinutuzumab, have previously been used with limited efficacy as monotherapy in patients with CLL.3 In contrast, addition of anti-CD20 mAb to chemotherapy significantly improves clinical outcomes.4 Regimens combining targeted therapies with anti-CD20 mAb have been developed and are widely used to treat CLL. The combination of Bruton tyrosine kinase inhibitors (BTKi) with anti-CD20 mAb has the possible advantage of combining the ability of BTKi to mobilize CLL cells from lymphoid tissue into the circulation where they are especially susceptible to clearance by anti-CD20 mAbs.5 However, 2 randomized clinical trials testing the addition of standard dose of rituximab to the inhibitor ibrutinib did not show any improvement in progression-free survival (PFS).6,7 On the basis of emerging data on the mechanisms of action of anti-CD20 mAb and the effect of ibrutinib on these mechanisms, we developed a novel combination of BTKi and rituximab regimen for patients with CLL.Rituximab cytotoxicity in CLL is primarily mediated by antibody-dependent cellular phagocytosis (ADCP) with lesser roles for complement dependent cytotoxicity and antibody-dependent cellular cytotoxicity.8 The authors and others have previously shown in vitro that macrophage ADCP is significantly decreased by ibrutinib, but not by the more targeted BTKi, acalabrutinib.8,9 Subsequently, The ELEVATE-TN trial showed an improvement in PFS when obinutuzumab was added to acalabrutinib compared with acalabrutinib monotherapy (however, the study was not powered to formally compare these groups.)10 Our combination therapy regimen thus used acalabrutinib.Original dose (375 mg/m2) IV rituximab has been previously shown to cause rapid onset and durable decreases in CD20 levels in circulating CLL cells which could limit treatment efficacy.11,12 The authors and others have previously shown that this potential cause of drug resistance could be obviated though use of a lower dose of rituximab (20 mg/m2) which can be administered every 48 hours without causing sustained loss of CD20 from circulating CLL cells (NCT00669318).12,13On the basis of the above rationale that acalabrutinib might be a more ideal partner to rituximab than ibrutinib, our clinical trial used acalabrutinib in combination with high-frequency low-dose (HFLD) rituximab. Rituximab was selected over obinutuzumab because it is available for subcutaneous (SQ) at-home administration.14,15 Our primary goal was to determine if the use of this combination could allow for limited duration therapy in those who achieved a complete response (CR) with no detectable MRD after either 12 or 24 cycles of therapy.MethodsStudy design and objectivesThe primary objective of this single center phase 2 trial was to estimate the rate of complete remissions after 1 year of therapy as defined by the 2018 iwCLL (International Workshop for Chronic Lymphocytic Leukemia) criteria.16 Secondary objectives were to assess the rate of undetectable measurable residual disease (defined as <1 in 10 000 clonal B cells via 6-color flow cytometry), PFS, and safety of the regimen. Correlative studies on the cytokine responses to the first infusion, effect of treatment on CLL-cell CD20 expression, and in vitro sensitivity of CLL cells to ADCP, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity will be reported separately. The institutional review board approved the protocol, and informed, written consent was obtained from all patients before enrollment. All authors had access to the primary clinical trial data. The study was registered before enrolling patients (ClinicalTrials.gov NCT03788291).Eligibility criteriaPatients aged ≥18 years were eligible if they had a diagnosis of CLL that was previously untreated and warranted therapy on the basis of the 2018 iwCLL criteria.16 An Eastern cooperative oncology group performance status of ≤2 was required, unless a performance status of ≤3 was attributable to their CLL. An absolute neutrophil count of ≥0.5 ×109/L, platelet count of ≥30 × 109/L, CrCl >30 mL per minute, and adequate hepatic function were required. Patients with Richter’s transformation, ongoing systemic infections or uncontrolled immune cytopenia were excluded. As per typical requirements to receive acalabrutinib, patients had to avoid strong CYP3A inhibitors, such as warfarin, and proton pump inhibitors before an August 2022 amendment, using the tablet formulation of acalabrutinib that allows for coadministration with acid-reducing agents.Treatment protocol and clinical protocol assessmentsBaseline evaluation included history and physical examination, laboratory evaluations, and imaging by computed tomography. Risk factor assessment was performed before treatment. Abnormalities detected by standard fluorescent in situ hybridization using probes for detection of deletion 17p13, 11q22.3, and 13q14 and trisomy 12 are reported using the standard hierarchical method.17 Next generation sequencing was used to detect deleterious mutations in TP53, NOTCH1, and SF3B1. IGHV somatic hypermutation status was measured with Sanger sequencing with those reads deviating from wild type by ≥2% reported as mutated.Rituximab was given biweekly at 50 mg per dose on the same 2 days of the week with a 48-hour interval between doses for six 28-day cycles. The initial dose was given IV at an infusion rate of 25 mg per hour starting 30 minutes after premedication with oral diphenhydramine (50 mg) and acetaminophen (650 mg) to ensure that patients could tolerate the drug based on standard practice. If IV therapy was completed, all subsequent rituximab doses were administered SQ. After training, patients who tolerated SQ rituximab could then self-administer treatment at home with optional use of premedication (supplement 1). To study the effect of the first doses of IV and SQ rituximab on CLL cells, the trial was designed to give these initial doses in the first week of therapy before the initiation of acalabrutinib therapy. Acalabrutinib 100 mg oral twice daily was started on day 8 of the first cycle. Dose modifications were advised for grade 4 neutropenia or thrombocytopenia, and drug discontinuation for grade 4 infusion-related reactions or colitis.Adverse events to evaluate safety as a secondary outcome were assessed at baseline, throughout treatment, and during the 30-day period after treatment discontinuation and were graded by the National Cancer Institute Common Terminology Criteria for Adverse Events Version 5.0.Response assessments, including computed tomography and MRD testing by flow cytometry, were performed just before the completion of cycle 12 and 24. If patients achieved a CR by iwCLL criteria and had undetectable MRD in the blood and bone marrow, they were able to terminate therapy and be followed until disease progression. Because the intent of the study was only to stop acalabrutinib if patients had undetectable MRD disease status, bone marrow biopsies were not routinely performed in patients who had detectable MRD in the peripheral blood. Patients who did not achieve a CR with undetectable MRD remained on acalabrutinib and had repeat response assessments as above, performed after 24 cycles of therapy. In the absence of a CR with undetectable MRD, acalabrutinib could be continued until disease progression or unacceptable toxicity at the patient and physician discretion.Statistical analysisPatient and disease characteristics were summarized using count and proportions for categorical variables and medians and ranges for continuous variables. The primary objective was to estimate the CR rate of acalabrutinib and HFLD rituximab in patients with previously untreated CLL. The planned sample size of 40 participants had produced a 95% 2-sided exact binomial confidence interval (CI) with a maximum width of 32.4%. The final evaluable sample size of 38 participants did not appreciably reduce the precision of the CR rate estimate. The resulting CI based on 38 participants had a maximum width of 33.2%.CR rate was defined as the proportion of patients with a best overall response of CR with or without marrow recovery. Associated 95% 2-sided exact binomial CIs were calculated for all response rates. PFS was defined as the time from treatment initiation until the date of first documentation of definitive disease progression or date of death from any cause, whichever occurred first. Patients who did not progress, die, or were failed to follow-up were censored at the day of their last clinical trial clinic visit. All analyses were descriptive in nature, and no hypothesis testing was performed. SAS v9.4 was used for all analyses (SAS Institute Inc, Cary, NC).ResultsPatientsThirty-nine patients were enrolled from April 2019 to July 2021. One patient was found to have a non-CLL B-cell non-Hodgkin lymphoma and was excluded from analyses. Demographics for the 38 evaluable patients are included in Table 1. The median age was 67 years, 60.5% were male, and 92.1% had an ECOG (Eastern Cooperative Oncology Group) performance status of ≤1. At time of treatment, most patients were Rai stage 2. Most (68.4%) patients had at least 1 high-risk feature: TP53 mutation, 17p13 deletion, 11q22.3 deletion, NOTCH1 mutation, or IGHV unmutated disease (Table 1).Table 1.DemographicsCharacteristicTreated patients (n = 38), n (%)Age (y) Median66.5 (40-78) RangeMale23 (60.5)ECOG PS 018 (47.4) 117 (44.7) 23 (7.9)Rai stage 00 I3 (7.9) II19 (50.0) III11 (28.9) IV5 (13.2)β2 microglobulin (mg/L) Median3.7 (2, 11.4) RangeHemoglobin Median11.9 (7.8, 15.4) RangePlatelet count Median138 (69, 438) RangeCytogenetics Del (17p)5 (13.2) Del (11q)6 (15.8) Trisomy 126 (15.8) Del (13q)15 (39.5) No cytogenetic abnormalities6 (15.8)IGHV mutation status Mutated15 (39.5) Unmutated23 (60.5)Mutated TP53 Yes8 (21.1) No30 (78.9)Mutated NOTCH1 Yes9 (23.7) No29 (76.3)Mutated SF3B1 Yes4 (10.5) No34 (89.5)At least 1 high-risk feature (TP53mut/del(17p)/del(11q)/ NOTCH1 mut/IGHV unmutated) Yes26 (68.4) No12 (31.6)Cytogenetics are reported via Döhner hierarchical method.17ECOG, Eastern Cooperative Oncology Group; PS, performance status.EfficacyMedian follow-up was 2.3 years. At the time of cycle 12 response assessment, all 38 evaluable patients had responded with 1 patient (2.6%; 95% CI, 0.07-13.8) having a CR based on iwCLL 2018 criteria with undetectable MRD in the blood and bone marrow. This patient was able to terminate the use of acalabrutinib. Twenty-six patients (68.4%; 95% CI, 51.4-82.5) had a partial response and 11 (28.9%; 95% CI, 15.4-45.9) had a partial response with lymphocytosis (Table 2). Of the 32 patients who had gone through a cycle 24 response assessment, 23 patients (71.9%; 95% CI, 53.3-86.3) had a partial response, and 7 (21.9%; 95% CI, 9.3-40.0) had a partial response with lymphocytosis (Table 2; Figure 1). Nodal responses are described in Figure 2. Two patients with TP53 disruption (del17p and TP53 mutation) had progressive disease at 1.1 and 1.9 years. Median PFS was not reached (Figure 3). No patients had died during the study period; 6 patients had not yet reached their cycle 24 response assessment at time of data cutoff. Notably, no patients had COVID-19 infection or received COVID-19 vaccines, which have the potential to affect absolute lympocyte count or adenopathy, proximal to efficacy assessments.Table 2.Response by 2018 iwCLL criteria and MRD status via flow cytometry undetectable is defined as <1 in 10 000 CLL cells detected via flow cytometryResponseCycle 12 (n = 38)Cycle 24 (n = 32)MRD statusCR1 (2.6%; 95% CI, 0.07-13.8)-Undetectable in PB and BMPR26 (68.4%; 95% CI, 51.4-82.5)23 (71.9%; 95% CI, 53.3-86.3)PR lymphocytosis11 (28.9%; 95% CI, 15.4-45.9)7 (21.9%; 95% CI, 9.3-40.0)Progressive disease-2 (6.3%; 95% CI, 0.8-20.8)Detectable is ≥1 in 10 000 CLL cells.BM, bone marrow; CI, confidence interval; PB, peripheral blood; PR, partial response.Figure 1.Swimmer plot: bars represent each study patient’s time on treatment through time of last follow-up (triangle) or progression (x). Response to therapy was assessed at cycle 12 and 24 (diamonds represent partial responses, squares partial response with lymphocytosis, and stars CRs).Figure 2.Waterfall plots representing change in the sum of the products of the dimensions of target lymph nodes from baseline following cycle 12 (top) and cycle 24 (bottom). Cycle 24 data is shown for the 30 subjects who had a cycle 24 assessment without previous progression.Figure 3.PFS. Kaplan-Meier curve of PFS from treatment initiation (N = 38). Two subjects progressed (1.1 and 1.9 years), with no deaths observed.SafetyThe most common all-grade, all cause adverse events (Table 3) included headache (68.4%), myalgias (50%), COVID-19 infection (39.5%), arthritis or arthralgias (39.5%), bruising (39.5%), fatigue (36.8%) and rash (23.7%). IV rituximab infusion reactions occurred in 63.2% of the patients (all grade 2), and 31.6% of the patients experienced low-grade injection site reactions related to SQ rituximab.Table 3.All-causality adverse events, listed by organ system, including all-grade events occurring in ≥20% of patients, and grade 3 or 4 adverse events which occurred in 1 or more patientsAdverse event (all causality)N = 38All grades, (≥20%) n (%)Grade 3/4 (≥ 1 patient) n (%)Hematologic toxicities Anemia2 (5.3)2 (5.3) Neutropenia1 (2.6)1 (2.6)Infections COVID-1915 (39.5)5 (13.2) Cryptococcus lymphadenitis1 (2.6)1 (2.6) Febrile neutropenia1 (2.6)1 (2.6) Joint infection1 (2.6)1 (2.6) Pneumonia (non-COVID)5 (13.2)1 (2.6) Dental infection2 (5.3)2 (5.3) Cellulitis3 (7.9)0 Upper respiratory infection12 (31.6)0 Urinary tract infection8 (21.1)3 (7.9)Musculoskeletal/Skin Arthritis/arthralgias15 (39.5)0 Bruising15 (39.5)0 Hematoma4 (10.5)1 (2.6) Myalgias19 0 Rash9 (23.7)0Gastrointestinal Constipation8 (21.1)0 Diarrhea9 (23.7)0 Nausea11 (28.9)0 Rectal bleeding1 (2.6)1 (2.6)Other A-fib1 (2.6)1 (2.6) Cough10 (26.3)0 Dizziness8 (21.1)0 Edema12 (31.6)0 Fatigue14 (36.8)0 Headache26 (68.4)0 Infusion reaction24 (63.2)0 Injection site reaction12 (31.6)0 Sinus bradycardia2 (5.3)2 (5.3) CVA1 (2.6)1 (2.6) Urinary tract obstruction1 (2.6)1 (2.6)CVA, cerebral vascular accident,Grade 3 or 4 events occurring in ≥1 patient included COVID-19 infection (13.2%) with no patients requiring intubation for respiratory failure, urinary tract infection (7.9%), dental infections (5.3%), anemia (5.3%), and sinus bradycardia (5.3%) (Table 3). No patients had a new diagnosis of atrial fibrillation during the trial and 1 patient had a new diagnosis of hypertension. One patient had paraesophageal Cryptococcus neoformans lymphadenitis requiring short-term interruption of the acalabrutinib during diagnostic evaluation and subsequent acalabrutinib dose was decreased to 100 mg daily while on treatment with isavuconazole, which resolved the infection. Acalabrutinib administration was briefly paused during a COVID-19 infection, a rectal hemorrhage, and a urinary tract obstruction that required hospitalization. Rituximab administration was delayed for patients with COVID-19 and urinary tract infections. No patients discontinued therapy owing to adverse events.DiscussionAcalabrutinib and HFLD rituximab was an effective initial treatment for progressive CLL. The response rate was 100%, and with a median follow-up of 2.3 years, only 2 high-risk patients with TP53 disruptions have progressed. With 1 patient achieving a CR with undetectable MRD, the combination did not allow for early treatment discontinuation for the majority of the patients. However, this combination could serve as a platform for the addition of other agents in an attempt to deepen responses in fixed duration therapy, as has been demonstrated with the combination of ibrutinib and venetoclax in treatment-naïve CLL.18Although previous trials have shown no improvement in PFS when rituximab is added to ibrutinib vs ibrutinib alone,6 there was an improvement in depth of response in patients who received the combination,7 which is relevant in a field that is moving toward MRD-guided therapy. The results of these trials of ibrutinib based regimens are not necessarily informative as to the combination of acalabrutinib and rituximab. Ibrutinib is known to inhibit antibody-dependent cellular cytotoxicity and ADCP8,19 as an off-target effect9 and also downregulates expression of CD20.20,21 In contrast, acalabrutinib does not significantly decrease ADCP in vitro suggesting that this important mechanism of action of rituximab remains intact.8,9,22 In our study, the overall response rate of 100% compares favorably to the response rate to acalabrutinib monotherapy (86%) or acalabrutinib in combination with obinutuzumab (94%)10 as well as ibrutinib in combination with rituximab,23 (95.8%). We could not compare the quality of responses in our trial with the ECOG 1912 trial because bone marrow biopsies were deferred in our study in patients with detectable MRD in the peripheral blood.Given our “real-world” approach to deferring marrow biopsies that would not change management, we sought to compare our data to a recently presented real-world analysis of patients who received a BTKi in the frontline setting.24 This analysis included 373 patients who had received acalabrutinib as first-line treatment and set out to define the time to next treatment as a surrogate for PFS. Patients who had switched to a different BTKi were excluded to account for intolerability owing to adverse effects and attempt to focus solely on patients in which treatment was altered because of progression of disease. They found that at 12 months, 91.2% of patients remained on acalabrutinib therapy, compared with our study in which all patients remained on treatment at 12 months. With the caveats of comparing real-world and clinical trial populations, we perceive that this does provide a reasonable estimate of the efficacy of our combination regimen compared with acalabrutinib monotherapy. These efficacy data support further investigation of the acalabrutinib and HFLD rituximab regimen as a component of future multidrug therapy regimens.The treatment regimen was tolerable. Despite slow infusion of a small dose of rituximab, most patients experienced an infusion reaction, but none were more than that of grade 2, and all were able to complete the infusion. Although 12 patients had grade 1 or 2 injection site reactions to the SQ rituximab, all were able to continue. This study demonstrated that HFLD SQ rituximab can be safely and effectively self-administered. No patients needed to terminate treatment owing to drug toxicities.This study design is feasible as a fully home-administered regimen that would limit the cost and inconvenience of infusion centers for patients. Access to infusion chair time has become increasing more limited with the current healthcare staffing crisis, and our method of patient delivered anti-CD20 mAb could help address this. This regimen also has implications for administration of rituximab in rural areas, or global administration of rituximab in areas with limited resources. This combination also used less dose of rituximab than the standard dose, which also affects total drug cost to patients and healthcare systems. When one considers the vast number of patients who receive rituximab,25 use of home dosing with lower cumulative amount of drug has major implications for these patients.Limitations of the study include relatively short follow-up duration because with longer assessment there may be additional undetectable MRD remissions. Future endeavors could include comparing this combination to acalabrutinib and rituximab with standard dose of rituximab, given our hypothesis that HFLD rituximab might be more effective in CLL, and discussions of a randomized trial to address this are ongoing. This can serve as a platform for adding additional agents such as BCL2 inhibitors which might result in more undetectable MRD responses, while retaining the ability of at-home administration.In summary, we present a single-center experience using HFLD SQ rituximab in combination with acalabrutinib in previously untreated patients with CLL. This effective and tolerable regimen allowed patients to receive anti-CD20 monoclonal antibody treatment at home amidst the COVID-19 pandemic and could be the basis for designing future multidrug regimens aimed at achieving more effective targeted therapy combinations.Conflict-of-interest disclosure: C.S.Z. discloses research funding from Acerta/AstraZeneca and TG Therapeutics; P.M.R. discloses research funding from Roche/Genentech, consulting for Kite Pharma/Gilead and Caribou Biosciences; C.C. discloses research funding from SecuraBio, Gilead, Genentech, and Bristol Myers Squibb (BMS); P.M.B. discloses consulting for AstraZeneca, AbbVie, Gilead, Genentech, BMS, Adaptive, BeiGene, Janssen, and GSK. The remaining authors declare no competing financial interests.

PMC

BMJ Open

37197822

PMC10192581

5-17-2023

10.1136/bmjopen-2022-070203

Sociodemographic and institutional determinants of zinc bundled with oral rehydration salt utilisation among under-five children with diarrhoeal diseases in East Wallaga zone, western Ethiopia: a community-based cross-sectional study

Terefa Dufera Rikitu, Shama Adisu Tafari, Kenea Abdi Kebede

ObjectiveThis study aimed to assess the sociodemographic and institutional determinants of zinc bundled with oral rehydration salt (ORS) utilisation among under-five children with diarrhoeal diseases in East Wallaga zone, western Ethiopia, in 2022.MethodsA community-based cross-sectional study was conducted among 560 randomly selected participants from 1 to 30 April 2022. Data were entered into EpiData V.3.1, then exported to the Statistical Package for Social Science (SPSS) V.25 for analysis. An adjusted OR (AOR) along with a 95% confidence level was estimated to assess the strength of the association, and a p value <0.05 was considered to declare the statistical significance.ResultsAbout 39.6% of the participants had used zinc bundled with ORS for their children with diarrhoea at least once in the last 12 months. Being aged 40–49 years for mothers or caregivers (AOR 3.48, 95% CI 1.41, 8.53); merchant (AOR 4.11, 95% CI 1.73, 8.12); mothers or caregivers able to read and write (AOR 5.77, 95% CI 1.22, 11.67); visited secondary level (AOR 2.82, 95% CI 1.30, 6.10) and tertiary level health facilities (AOR 0.016, 95% CI 0.03, 0.97); degree and above (AOR 0.06, 95% CI 0.03, 0.12) and doctorate (AOR 0.13, 95% CI 0.04, 0.44) holder healthcare professionals were statistically associated with utilisation of zinc bundled with ORS.ConclusionThe study found that about two in five of the participants had used zinc bundled with ORS for their under-five children with diarrhoeal diseases. Age, occupation, educational status, level of health facilities visited and level of health professionals provided care were determinants of zinc bundled with ORS utilisation. So, health professionals at different levels of the health system have to enhance the maximisation of its bundled uptake.

STRENGTHS AND LIMITATIONS OF THIS STUDYThis is the first study in the study setting and even in Ethiopia to assess the sociodemographic and institutional determinants of zinc bundled with oral rehydration salt (ORS) utilisation among under-five children with diarrhoea at the community level.The cross-sectional nature of the study made it difficult to show the cause-and-effect relationship.There might be a probability of recall bias.Unable to generalise these findings to the whole under-five children with diarrhoeal disease treatment with zinc and ORS bundling because there might be patients admitted at health facilities.It is difficult to compare these findings with those of other studies due to lack of literature.BackgroundGlobally, diarrhoea is both a disease and an economic burden each and every year, with sub-Saharan African countries disproportionately affected by the illness and disease.1Currently, nearly 1.7 billion cases of childhood diarrhoeal diseases account for one in nine child deaths, making diarrhoea the second leading cause of death and the leading cause of malnutrition in children under 5 years old across the globe.2 It kills more than 5.2 million under-five children globally, and around 800 000 children die of diarrhoea and dehydration each year in Africa.3 Of all child deaths from diarrhoea, 78% occur in the African and South-East Asian regions, which are also disproportionately burdened with infant and childhood HIV infections.4 Sub-Saharan African countries share a significant proportion (42%), of which Ethiopia ranks fifth globally, as diarrhoea causes about one-fourth (20%–27%) of child deaths.3Sub-Saharan African countries have made the least progress in the reduction of infant and child mortality. The two leading causes of mortality among children under 5 years of age in sub-Saharan Africa are pneumonia and diarrhoea, which accounted for 18% and 15% of deaths, respectively.5Although Ethiopia has already achieved remarkable progress in reducing under-five mortality in recent decades, studies done in different parts of Ethiopia have shown that diarrhoea is still a major public health problem.6 According to the Ethiopian Demographic and Health Surveys (EDHS) 2016, 12% of children under age 5 had diarrhoea.7Different countries have incorporated zinc and oral rehydration salt (ORS) as an effective treatment combination in their policies since July 2019, when WHO added ORS-bundled zinc to its core Model List of Essential Medicines for Children and encouraged countries to prioritise the bundle in their expenditures, procurement and supply, and training of healthcare providers.8 This recommended regimen of zinc bundled with ORS, along with continued feeding, is a safe, effective and inexpensive treatment for children, and 50% of diarrhoea deaths can be prevented.9 In addition, Ethiopia’s health policy stated that the Federal Ministry of Health included zinc as an essential drug that should be available at local health facilities and prescribed free of charge at the health post level in order to be easily accessible and given to the community since 2013.10Despite these important benefits, access to ORS and zinc remains a challenge in low-resource settings, and the rate of bundling of both products was extremely low. Globally, about 55% of the highest burden countries had ORS and zinc coverage levels of 2% or less.11It is recommended that under-five children should receive 10–14 days of zinc treatment for diarrhoea, and full coverage and utilisation of ORS and adjunct zinc supplementation could avert over 75% of all diarrhoea-related deaths. However, the study showed that the level of adherence to zinc supplementation was low.12Bundling (copackaging) zinc with ORS may encourage their combined use and improve access to and utilisation of the treatment in children under the age of 5, but different studies around the world have found low utilisation. Hence, a study conducted on ORS use and its correlates in low-level care of diarrhoea among children under 36 months old in rural Western China indicated that the therapy rate of ORS was 34.62%.13Of East African countries, studies conducted on zinc utilisation and associated factors indicated that Uganda had the highest prevalence of zinc utilisation (40.51%), whereas the Comoros had the lowest (0.44%). This study also revealed that utilisation of zinc was 18% in Tanzania, 10% in Nigeria, 15% in Sudan and 21.5% in Ethiopia.14 This indicates that much needs to be done to increase its utilisation and reduce the impact of diarrhoea, a preventable cause of under-five mortality in the region.Also, another study conducted in Ethiopia’s Addis Ababa city showed that slightly over two-thirds (67.1%) of caretakers used zinc bundled with ORS during the recent diarrhoeal attack. This was higher than a study conducted in Nigeria (8.3%) and comparable to the Kenyan findings (67%).15 The proportion of children under the age of 5 who received treatment for diarrhoea has risen from 13% in 2000 to 22% in 2005, 32% in 2011 and 44% in 2016, whereas the percentage of children who received no treatment has decreased from 42% in 2011 to 38% in 2016. According to the Ethiopian EDHS 2016 report, one in three children (33%) under age 5 with diarrhoea received zinc, and 17% received a combination of ORS and zinc. Antibiotics were given to 9% of children with diarrhoea, and two in five (38%) children with diarrhoea did not receive any treatment.7In general, age, occupation, caregiver relationship with the child, type and level of health facilities visited, distance from health facilities and community-based health insurance membership were some of the determinants of zinc bundled with ORS utilisation among under-five children with diarrhoeal diseases studied.12 15 16 Although these studies showed some variations, there were gaps in identifying sociodemographic and institutional determinants in this area specifically because these determinants are more vital in assessing utilisation of these bundled products than assessing these variables with other determinant factors together.Moreover, to the best of the authors’ knowledge, no prior studies have been conducted on the sociodemographics and institutional determinants of the utilisation of zinc-bundled ORS among under-five children with diarrhoeal diseases in the East Wallaga zone, western Ethiopia. Also, studies that have been conducted elsewhere have mostly revealed specific interventions, either on zinc or ORS only,14 rather than focusing on the recently implemented copackaged zinc and ORS and particularly giving attention to its sociodemographics and institutional determinants. Therefore, to fill these gaps, this study aimed to assess the sociodemographics and institutional determinants of zinc bundled with ORS utilisation among under-five children with diarrhoeal diseases in the East Wallaga zone, western Ethiopia, in 2022.Methods and materialsStudy setting and periodThe study was conducted in East Wallaga zone, Oromia region, western Ethiopia, from 1 to 30 April 2022. The zonal town, Nekemte, is located 333 km west of Addis Ababa, which is the capital city of Ethiopia. East Wallaga zone has an area of 21 980 million km2 and is geographically bounded in the east by West Shewa and Jimma zones, in the west by West Wallaga zone, in the north by Horo Guduru Wallaga zone and Amhara regional state, and in the south west by Buno Bedele zone. Administratively, the zone has a total of 17 districts, and the total population of the zone in 2021/2022, as projected from 2007, was 1 585 215 with a male to female ratio of 1.1:1.Study design and populationStudy designA community-based cross-sectional study design was employed to assess the sociodemographic and institutional determinants of zinc bundled with ORS utilisation among under-five children with diarrhoeal diseases in East Wallaga zone, western Ethiopia.PopulationSource populationAll households among selected districts whose under-five children had diarrhoea in the last 1-year period were the source population.Study populationAll selected under-five children who had diarrhoea in the last 12 months and their caregivers were the study population.Eligibility criteriaInclusion criteriaAll households whose under-five children had diarrhoea in the last year and who had stayed for more than 1 year in the area were included in the study.Exclusion criteriaUnder-five children’s mothers or caregivers who were sick at the time of data collection were excluded from the study.Sample size and sampling techniqueSample size determinationThe sample size was determined using a single population proportion formula by considering the following assumption: where the proportion of zinc bundled with ORS utilisation among under-five children of 67.1%15 was taken. Also, by considering 5% margin of error, a design effect of 1.5 and a 10% potential non-response rate, the final sample size became 560.Sampling techniques and proceduresA multistage sampling procedure was carried out. In the first stage, four districts (40%) were randomly selected using a lottery method from nine nutrition international project-supported districts in the zone.17 In the second stage, all Kebles were listed for each selected district, and among them, a total of 12 (3 Kebles per district) were selected for the study as representative of the Kebles using a simple random sampling technique for each district based on WHO health facility assessment tool.18 19 Then, after the selection of the Kebles to be included in the study, records of diarrhoeal diseases from each health facility in the catchments for each Keble and diarrhoeal disease data for under-five children were obtained from health extension workers. Keble is Ethiopia’s smallest administrative division or unit, which is a subsection of the district. Households that could fulfil the inclusion criteria from these records were listed from the Master Family Index and family folders of the Community Health Information System registration books, and the households’ numbers were obtained and used as a sampling frame. The sample size was then distributed to each Keble in proportion to the size of their household in each district. Finally, to obtain the final sample size, simple random sampling techniques were used to select households based on the allocated sample size of each Keble, and the data were collected from mothers or caregivers.Study variablesUtilisation of zinc bundled with ORS was the outcome variable and the independent variables were: sociodemographic and economic-related variables (age of the caregivers or mothers, sex of caregivers or mothers, marital status, family size, educational status, occupation, residence, age of the child, sex of the child, caregiver relationship with child and household’s monthly income); and institutional-related variables (place of treatment, types of health facility visited, level of health facilities, perceived quality of care by health professional, status of health professionals, availability of drugs or supplies in the facilities, perceived affordability of drugs, distance from nearby health facilities and health insurance membership status).Operational definitionsZinc and ORS bundling: a bundle containing zinc sulfate and ORS which can be prepared in different forms for supplementation, such as:Central bundling: prebundled zinc and ORS using a pouch that had an instructional message intended for improving the rational use of zinc-ORS treatment, distributed to health facilities.Facility-level bundling: zinc and ORS bundling pouch that had instructional messages distributed to the health facilities; bundling was made by the health workers while administering the treatment.Status quo: zinc and ORS are coadministered without bundling.12Utilisation of zinc bundled with ORS: use of services by under-five children, at least one child in the household, at least once, from health facilities for the purpose of preventing and curing health problems, promoting health and well-being or obtaining information about one’s health status and prognosis, regarding diarrhoeal disease treatment with zinc and ORS copacked in the previous year, which was answered by a closed-ended binary question (Yes/No). Based on this, if they had received the drugs from health facilities, it was answered as ‘yes’, and if not, it was answered as ‘no’.15Level of health facilities: healthcare facilities that provide various levels of care in accordance with Ethiopia’s current health tier system (three-tier system) (health posts, health centres, hospitals).20Data collection instrument and proceduresData were gathered through face-to-face interviews with mothers or child caregivers using a semistructured, interviewer-administered, pretested questionnaire. It was adapted from a review of different literature9 15 and modified to fit the local context. The tool was first prepared in English, translated to Afan Oromo, and then back translated to English by Afan Oromo and English language bachelor’s degree holders to check for consistency. It consists of sociodemographic and institutional-related factors.Data quality managementTo maintain the quality of the data, different measures were undertaken before, during and after data collection. A preliminary translation and retranslation of the questionnaire was made to check for its consistency before the actual data collection. Training was given for all data collectors and supervisors on the objective of the study, the contents of the questionnaire, issues of maintaining confidentiality, informed verbal consent and interview techniques.Data analysis procedureData were entered into EpiData V.3.1 and exported to SPSS V.25 for statistical analysis. A descriptive analysis was used to describe the percentages and number of distributions of the respondents. A binary logistic regression analysis was performed on the independent variables and their proportions, and a crude OR was computed against the outcome variable. Finally, independent variables with a p value 62 (0.4)Age of children (months)6–11162 (30.0)12–23270 (50.0)24–59108 (20.0)Sex of childMale335 Female205 Relationship with childMother452 (83.7)Father16 Grandmother11 Grandfather16 Auntie16 Sister/brother29 (5.4)Household’s headMale headed506 (93.7)Female headed34 (6.3)Place of residenceUrban86 (15.9)Rural454 (84.1)Monthly income (ETB)1651–3200317 (58.7)3201–5250149 (27.6)5251–780052 (9.6)7801–10 90010 (1.9)>10 90012 (2.2)*Catholic, Wakefata.†Students, government employees.ETB, Ethiopian birr; ORS, oral rehydration salt.Institutional-related factorsIn the previous 12 months, 195 (36.1%), 262 (48.5%) and 83 (15.4%) of them received treatment during their illness at home, public health facilities and private health facilities, respectively.In terms of facilities visited, approximately 187 (34.6%), 319 (59.1%) and 34 (6.3%) of them had visited a health post, health centre and hospital, respectively. Also, the majority (84.3%) of the facilities they have visited were primary level health facilities. Most of the studied households (277, 51.3%)had a distance greater than or equal to 10 km from the nearby health facilities.About 356 (65.9%) of the respondents were satisfied with the quality of care provided by healthcare professionals (HCP). Also, about 171 (31.7%) and 430 (79.5%) of them perceived that drugs were always available and affordable, respectively (table 2).Table 2Institutional-related factors for the study on sociodemographic and institutional determinants of zinc bundled with ORS utilisation among under-five children with diarrhoeal diseases in the East Wallaga zone, western Ethiopia, 2022 (n=540)VariablesCategoriesFrequency (%)Place of treatment during child illnessHome195 (36.1)Public health facility262 (48.5)Private health facility83 (15.4)Distance from health facilities (km)<10263 (48.7)≥10277 (51.3)Types of health facilities visited during recent episodeHealth post187 (34.6)Health centre319 (59.1)Hospital34 (6.3)Level of health facilities visited during recent episodePrimary level455 (84.3)Secondary level69 (12.8)Tertiary level16 (3.0)Perceived quality of healthcare by healthcare professionalsGood390 (72.2)Poor150 (27.8)Level of healthcare professionals provided careDiploma133 (24.6)Degree and above340 (63.0)Doctor (GP)31 (5.7)Paediatrician (specialist)36 (6.7)Perceived satisfaction from quality of care by HCPsSatisfied356 (65.9)Not satisfied184 (34.1)Perceived availability of drugs during facility visitAlways available171 (31.7)Sometimes available270 (50.0)Write prescription to outside99 (18.3)Perceived affordability of drugs for treatmentAffordable430 (79.6)Not affordable110 (20.4)Status of CBHI membershipMember413 (76.5)Not member127 (23.5)CBHI, Community Based Health Insurance; GP, general practitioner; HCP, healthcare professional; ORS, oral rehydration salt.Utilisation of zinc bundled with ORSThe study showed that about 214 households (39.5%) of the participants had used zinc bundled with ORS for their under-five children with diarrhoea at least once in the last 12 months (table 1). Regarding duration of treatment or utilisation of the drugs, about 10 (4.7%), 54 (25.2%) and 150 (70.1%) of them had copack supplements for less than 7, 7–10 and 10–14 days, respectively. This implies that only 150 (70.1%) of them had received full doses of the copacks or bundles, and the majority of them (50, 78.2%) reported that diarrhoea had stopped being the reason for not taking the full dose.Concerning the form of supplementation of zinc bundled with ORS, about 152 (71.0%), 27 (12.6%) and 35 (16.4%) were received from central bundling, facility-level bundling and status quo, respectively (figure 1). The duration of diarrhoea stopped after bundled supplementation was reported as immediately (65, 28.8%), after 1–3 days (138, 61.1%)and after 4–6 days (23, 10.2%)(table 3).Figure 1Forms of bundled supplementation among under-five children with diarrhoeal diseases in East Wallaga zone, western Ethiopia, 2022 (n=540).Table 3Utilisation of zinc bundled with ORS among under-five children with diarrhoeal diseases in the East Wallaga zone, western Ethiopia, 2022 (n=540)VariablesCategoriesFrequency (%)Received zinc bundled with ORS at least once in the last 12 monthsYes214 (39.6)No326 (60.4)Reason for not receiving bundleDon’t know where to obtain56 (17.3)Don’t know it should be given together232 (71.8)Unsure how to administer24 (7.4)Don’t think it is effective11 (3.4)Duration of supplementation (days)<710 (4.7)7–1054 (25.2)10–14150 (70.1)Bundled utilisation for your childNot full dose64 (29.9)Full dose150 (70.1)Reason for not giving full doseVomiting14 (21.8)Diarrhoea stopped50 (78.2)Duration of diarrhoea after bundled supplementationImmediately65 (28.8)After 1–3 days138 (61.1)After 4–6 days23 (10.2)ORS, oral rehydration salt.Determinants of zinc bundled with ORS utilisationSome sociodemographic variables, such as the respondents’ age, occupation, educational status, family size, the child’s age in months and residence, as well as institutional variables, such as distance from a nearby health facility, level of health facility visited, level of HCPs providing care and status of Community Based Health Insurance (CBHI) membership, were associated with zinc-bundled ORS use among children with diarrhoea in the study.After controlling for confounders, a multivariable logistic regression analysis revealed that respondents’ age, occupation, educational status, level of health facility visited and level of health professionals providing care were significantly associated.From this multivariable logistic regression analysis, age was associated with the utilisation of zinc bundled with ORS (AOR 3.48, 95% CI 1.41, 8.53). This implied that the probability of using zinc bundled with ORS among household respondents whose ages ranged from 40 to 49 years was almost three times more likely to be used than that of those whose ages ranged from 18 to 29 years.Regarding the occupation of the respondents, being a merchant was statistically strongly associated with zinc bundled with ORS (AOR 4.11, 95% CI 1.73, 8.12). This indicated that those who were merchants were four times more likely to use zinc bundled with ORS for their children than those who were farmers.The educational status of the respondents was also strongly associated with zinc bundled with ORS for those who were able to read and write (AOR 5.77, 95% CI 1.22, 11.67). This revealed that respondents’ households whose educational status was able to read and write were 5.7 times more likely to use zinc and ORS bundling than those whose educational status was unable to read and write.The study indicated that respondents who had visited secondary level health facilities were 2.8 times (AOR 2.82, 95% CI 1.30, 6.10) more likely to use zinc bundled with ORS than those who had visited primary level health facilities, but the probability of using zinc bundled with ORS among respondents who had visited tertiary level health facilities was reduced by 98.4% (AOR 0.016, 95% CI 0.03, 0.97) as compared with their counterparts.This study also found that for the degree and above (AOR 0.06, 95% CI 0.03, 0.12) and doctorate (general practitioners) (AOR 0.13, 95% CI 0.04, 0.44) holders, the level of health professionals providing care was statistically strongly associated with the utilisation of zinc bundled with ORS. This indicated that the likelihood of using zinc bundled with ORS was reduced by 40% and 87%, respectively, among respondents treated by bachelor’s degree and above and doctorate holder health professionals, when compared with diploma holder health professionals (table 4).Table 4Factors that influence zinc combined with ORS utilisation among under-five children with diarrhoeal diseases in the East Wallaga zone of western Ethiopia in 2022 (n=540)VariablesZinc bundled with ORS utilisationOR (95% CI) and P valueNon-used n (%)Used n (%)CORAORAge of the respondent 18–29162 (49.7)98 (45.8)11 30–39142 (43.6)90 (42.1)1.04 (0.72, 1.50)1.46 (0.81, 2.63) 40–4922 (6.7)26 (12.1)1.9 (1.05, 3.63)3.48 (1.41, 8.53)**Occupation Farmer148 (45.4)42 (19.6)11 Housewife136 (41.7)94 (43.9)2.43 (1.581, 3.75)2.49 (0.27, 4.87) Merchant21 (6.4)66 (30.8)11.07 (6.08, 20.15)4.11 (1.73, 8.12)*** Labourer5 (1.5)6 (2.8)4.22 (1.22, 14.54)2.79 (1.14, 14.45) Others†16 (4.9)6 (2.8)2.11 (0.72, 6.15)2.71 (1.33, 11.18)Educational status Unable to read and write49 (15.0)39 (18.2)11 Read and write20 (6.1)34 (15.9)2.13 (1.06, 4.276)5.77 (1.22, 11.67)* Primary education (Grades 1–8)163 (50.0)81 (37.9)0.62 (0.37, 1.02)0.29 (0.08, 1.04) Grades 9–1279 (24.2)43 (20.1)0.68 (0.39, 1.199)0.14 (0.032, 0.65) Diploma10 (3.1)12 (5.6)1.50 (0.59, 3.85)0.32 (0.03, 3.33) Degree and above5 (1.5)5 (2.3)1.25 (0.339, 4.65)0.02 (0.00, 0.06)Family size <5152 (46.6)161 (75.2)3.47 (2.38, 5.08)1.75 (0.08, 6.76) ≥5174 (53.4)53 (24.8)11Age of children (months) 6–1191 (27.9)71 (33.2)4.48 (2.42, 8.29)1.91 (0.33, 13.87) 12–23143 (43.9)127 (59.3)5.10 (2.85, 9.14)2.07 (0.24, 19.72) 24–5992 (28.2)16 (7.5)11Place of residence Urban41 (12.6)45 (21.0)11 Rural285 (87.4)169 (79.0)1.85 (1.16, 2.94)1.89 (0.38, 5.23)Level of health facility visited Primary Level290 (89.0)165 (77.1)11 Secondary level31 (9.5)38 (17.8)2.15 (1.29, 3.59)2.82 (1.30, 6.10)** Tertiary level5 (1.5)11 (5.1)0.44 (0.32, 0.86)0.016 (0.03, 0.97)*Level of health professionals provided care Diploma27 (8.3)106 (49.5)11 Degree and above264 (81.0)76 (35.5)0.07 (0.04, 0.12)0.06 (0.03, 0.12)*** Medical doctor15 (4.6)16 (7.5)0.27 (0.11, 0.61)0.13 (0.04, 0.44)*** Paediatrician20 (6.1)16 (7.5)0.20 (0.09, 0.44)0.40 (0.13, 1.24)CBHI membership status Member295 (90.5)118 (55.1)11 Non-member31 (9.5)96 (44.9)3.03 (4.89, 12.23)2.24 (0.43, 11.71)1=reference.*P<0.05; **p<0.01; ***p<0.001.†Students, government workers.AOR, adjusted OR; COR, crude OR; ORS, oral rehydration salt.DiscussionThis study aimed to address sociodemographic and institutional determinants of zinc bundled with ORS utilisation among under-five children with diarrhoeal diseases. Based on this, the study showed that 39.6% (35.6%–43.9%) of the studied participants’ households had used zinc bundled with ORS at least once in the last 12 months. This finding was higher than the study findings from different corners of the world, as in Nigeria, 8.3%21; in Sudan, where only 18.9% and 14.8% of the children had received ORS and zinc supplements, respectively22; and in Ethiopia, where the EDHS 2016 reported that 17% of them received a combination of zinc and ORS.7 The discrepancy might be due to sociocultural differences, study area differences and the time of study considered for those of the studies and surveys conducted even in the study area. Also, for the current study, we have considered primarily rural and semiurban households, whereas these studies have assessed primarily urban residences, and they were institutional-based studies. However, this study’s finding was lower than the study conducted in Kenya, 75%.15 Furthermore, the finding was lower than in both Ethiopian studies: on the effectiveness of bundling zinc with ORS, 67%12; and on the occurrence of diarrhoea and utilisation of zinc bundled with ORS among caregivers of children under the age of 5 in Addis Ababa, where it accounted for two-thirds.15 The probable differences might be due to differences in the study design used, in which some of them have used randomised controlled trials, and differences in the study settings.Also, this study’s finding was in line with the study conducted in rural China at 34.6%.13 This similarity might be due to the fact that in both of the studies, the majority of the cases were considered low-level care for diarrhoea among children under 5 years old.Zinc bundled with ORS utilisation was statistically associated with variables such as respondent age, occupation, educational status, level of health facility visited and level of health professionals providing care.The age of the mothers or caregivers was an important variable significantly associated with the utilisation of zinc bundled with ORS. This finding was in contradiction with a study conducted on ORS use and its correlates in low-level care of diarrhoea among children under 36 months old in rural Western China that indicated children in families with several preschool-aged children or those of the smaller age groups were less likely to receive ORS therapy against diarrhoea.13 This could be due to sociocultural differences and differences in study settings, as well as the ages considered, with the Chinese study focusing on children under the age of 36 months.Regarding the occupation of the respondents, merchants were more likely to use zinc bundled with ORS for their children with diarrhoea than those who were farmers. This might be explained by different reasons; the exhaustive nature of the work of the farmer in the study setting might force them to not give the drugs on time as needed and even to forget the drugs. Also, the opportunities that they might have to move from place to place, especially to urban areas, were minimal for farmers compared with merchants, which might have an influence on the utilisation of these drugs.The educational status of the respondents was also strongly associated with the bundled utilisation. This revealed that the likelihood of using zinc combined with ORS was six times higher among those whose educational status allowed them to read and write than among those whose educational status did not allow them to read and write. A study conducted in Kebri Dehar town, Somali Region, Ethiopia, supported this finding.23 Also, the higher the educational level, the more likelihood of utilisation of zinc was reported according to a study conducted in East Africa.14 This could be due to the fact that education is the way of gaining knowledge, which could lead to an understanding of the utilisation of health services.The level of health facility visited played a vital role in the utilisation of zinc with bundled ORS. A study conducted in Addis Ababa, Ethiopia, revealed that 56.6% of the respondents visited health facilities, and nearly all (93.9%) of the mothers or caregivers using public health facilities used health centres (ie, primary level health facilities). However, only 11.9% of them obtained ORS plus zinc supplementation.5 This indicated that utilisation of zinc bundled with ORS was low at primary level health facilities, which was consistent with our study findings, which revealed that respondents who visited secondary level health facilities used zinc and ORS bundle three times more than those who visited primary level health facilities. This might be due to the similarity of the health system, and the higher the level of the health facility, the greater the capacity and skill of HCPs to understand and determine healthcare needs. However, in our study findings, at the tertiary level, health facility utilisation of zinc with bundled ORS showed a reduction. This might be due to the low number of referral cases associated with the specified disease. Finally, this study showed that a greater reduction in the utilisation of zinc bundled with ORS was observed among respondents who had been treated by bachelor’s degree and above and doctorate holder health professionals than diploma holder health professionals. This could be due to alignment, utilisation and the ability to follow clinical treatment guidelines that might prevent higher level HCPs from providing services to service seekers.Conclusion and recommendationThe study found that about two in five of the respondents had used zinc bundled with ORS for their under-five children with diarrhoeal diseases. Age of the respondents, occupation, educational status, level of health facilities visited and level of health professionals provided care were the variables statistically associated with utilisation of zinc bundled with ORS. So, health professionals at different levels of the health system have to enhance the maximisation of its bundled uptake. Additionally, health education and information dissemination services and awareness for the community to improve its utilisation should have to be strengthened at different levels of health facilities across different levels of HCPs.Supplementary MaterialReviewer commentsAuthor's manuscript

PMC

BMJ Open Respiratory Research

38262669

PMC10806566

1-23-2024

10.1136/bmjresp-2023-002053

Effects of indoor air pollution on clinical outcomes in patients with interstitial lung disease: protocol of a multicentre prospective observational study

Yoon Hee-Young, Kim Sun-Young, Song Jin Woo

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrosing interstitial lung disease with a poor prognosis. While there is evidence suggesting that outdoor air pollution affects the clinical course of IPF, the impact of indoor air pollution on patients with IPF has not been extensively studied. Therefore, this prospective multicentre observational study aims to investigate the association between indoor air pollution and clinical outcomes in patients with IPF.Methods and analysisThis study enrolled 140 patients with IPF from 12 medical institutes in the Seoul and Metropolitan areas of the Republic of Korea. Over the course of 1 year, participants visited the institutes every 3 months, during which their clinical data and blood samples were collected. Additionally, indoor exposure to particulate matter ≤2.5 µm (PM2.5) was measured using MicroPEM (RTI International, Research Triangle Park, North Carolina, USA) in each participant’s house for 5 days every 3 months. Lung function was assessed using both site spirometry at each institution and portable spirometry at each participant’s house every 3 months. The study will analyse the impact of indoor PM2.5 on clinical outcomes, including mortality, acute exacerbation, changes in lung function and health-related quality of life, in the participants. This study represents the first attempt to evaluate the influence of indoor air pollution on the prognosis of patients with IPF.Ethics and disseminationThis study has received approval from the institutional review board of all participating institutions, including Asan Medical Center, Seoul, Republic of Korea (2021-0072).Trial registration numberKCT0006217.

WHAT IS ALREADY KNOWN ON THIS TOPICSeveral previous studies reported the association between air pollution and clinical outcomes in idiopathic pulmonary fibrosis (IPF), including mortality, acute exacerbation and disease progression. However, the most of these studies have focused on the effects of outdoor air pollutants, so there is a lack of comprehensive data on health effects of indoor air pollutants in patients with IPF.WHAT THIS STUDY ADDSOur study presents a comprehensive research protocol investigating the effects of exposure to indoor particulate matter ≤2.5 µm on patients with IPF. In addition, we are collecting a dataset that includes on-site and home lung function measurements, health-related quality of life and key clinical outcomes (such as death, acute exacerbation and hospitalisation) over a 1-year period, with regular blood sampling to assess biomarkers.HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYThis study can provide a basis for evidence-based policy-making, potentially reducing medical costs and improving the health of IPF patients by comprehensively assessing the impact of indoor air pollution on their health.Background and rationaleIdiopathic pulmonary fibrosis (IPF) is a specific form of chronic progressive fibrosing interstitial pneumonia with unknown aetiology. It has a poor prognosis, with a median survival of 3–5 years without treatment.1–3 Several studies have demonstrated the harmful effects of air pollution on IPF prognosis, including incidence,4 5 acute exacerbation (AE),6 7 hospitalisation,8 9 changes in lung function10–12 and mortality.13 14 Particulate matter (PM) refers to the mixture of solid particles and liquid droplets found in the air. It is categorised into PM10 (≤10 µm) or PM2.5 (≤2.5 µm) based on their aerodynamic diameter.15 PM2.5 is small enough to penetrate deeply into the lung parenchyma, leading to various harmful effects such as increased reactive oxygen stress, release of inflammatory cytokines and activation of inflammatory cells.16 In both in vitro and in vivo models, PM2.5 exposure has been shown to promote the development of pulmonary fibrosis.17–19 Specifically, chronic exposure of human bronchial epithelial cells to PM2.5 results in the activation of the transforming growth factor-beta 1 (TGF-β1)/SMAD3 pathway, TGF-β1 excretion and epithelial–mesenchymal transition,18 which are known to promote the proliferation and activation of fibroblast. Additionally, exposure to PM2.5 has been shown to increase the levels of profibrotic cytokines in the lungs, including TGF-β1 and connective tissue growth factor, in a bleomycin-induced pulmonary fibrosis model.17 Previous clinical studies have provided evidence that long-term exposure to PM2.5 increases the mortality13 of IPF, whereas short-term exposure to PM2.5 has been linked to the occurrence of AE7 or hospitalisation9 in patients with IPF. However, most previous investigations on the effects of air pollution have primarily focused on outdoor air pollution, overlooking the potential impact of indoor air pollution.The concentration of indoor PM can differ significantly compared with that of outdoor PM due to various factors; these include the presence of indoor emission sources, infiltration rate of outdoor PM, ventilation system, use of air purifiers and seasonal factors such as window-opening tendencies during certain times of the year.20 21 Since patients with IPF are predominantly elderly individuals and have reduced exercise capacity, they tend to spend a significant amount of time indoors. Therefore, many IPF patients may be susceptible to the effects of indoor air pollution. Nevertheless, the impact of indoor air pollution on clinical outcomes in patients with IPF remains poorly understood. Therefore, the primary objective of our study is to examine the association between indoor PM2.5 levels and clinical outcomes in patients with IPF who lives in Seoul and Metropolitan areas. We hypothesise that indoor PM2.5 levels significantly affect the health outcomes in patients with IPF; here we describe the protocol of our study.Methods and analysisAim and study designThis study is a prospective multicentre observational study to investigate the association between indoor PM2.5 exposure and clinical outcomes in patients with IPF. Given the lack of previous studies on indoor PM2.5 exposure in patients with IPF, the appropriate sample size required to achieve sufficient statistical power was uncertain. In order to ensure a robust analysis with a minimum of 100 participants, we enrolled 140 participants, considering the potential 30% drop-out rate observed in our preliminary study. This enrolment strategy guarantees the inclusion of over 100 participants in the final analysis. Patients with IPF were enrolled from 12 medical institutions located in Seoul and the Metropolitan areas of Republic of Korea, as shown in figure 1. After enrolment, the study participants were regularly followed up at 3-month intervals for a duration of 1 year. This study aimed to recruit patients from June 2021 to September 2022 and follow them for 1 year, with the final visit scheduled for October 2023.Figure 1Distribution of participating institutions. (A) Map of South Korea, (B) map of the metropolitan (Seoul, Incheon and Gyeonggi) area. Each institution is represented by a symbol, with Seoul and Metropolitan areas highlighted in green.Study populationThe inclusion criteria for study participants were as follows: patients diagnosed with IPF according to the American Thoracic Society (ATS)/European Respiratory Society (ERS)/Japanese Respiratory Society/Latin American Thoracic Association statement in 201822; those residing in the metropolitan area and those with forced vital capacity (FVC) ranging 40%–80% predicted, or diffusing capacity for carbon monoxide (DLco) ranging 30%–80% predicted. Exclusion criteria were as follows: current smokers, as their exposure to cigarette smoke could influence PM2.5 measurements and confound the analysis and patients aged <50 years, as the likelihood of IPF diagnosis in individuals below this age range is relatively low.Clinical data collectionBaseline clinical dataAt enrolment (visit 1), demographic data, including smoking status, comorbidities, medication history (use of antifibrotics, steroids or immunosuppressants), occupational history and address, were collected through medical records or patient interviews (table 1). The Charlson Comorbidity Index was calculated.23 Information on participant’s socioeconomic status (marital status, household income, education, employment) was collected through questionnaires and will be used as covariates in the health impact analysis. In addition, a survey was conducted during visit 1 to collect information on factors that may affect indoor air pollutant concentrations, including the distance from the residence to roads, traffic volume, cooking facilities, presence of mould, pets, year of construction and length of residency, chemicals used, ventilation methods, air purifiers, humidifiers, air conditioners, dehumidifiers and dryers.Table 1Study protocol for the assessment of health effect of air pollution on patients with interstitial lung diseaseTimeVisit 1Visit 2Visit 3Visit 4Visit 50 month3 months6 months9 months12 monthsEnrolment Screening and eligibilityx Informed consentxAssessment Baseline clinical datax Site lung functionxxxxx Portable lung functionxxxxQuestionnaire K-BILD, EQ-5D-5L, EQ-VASxxxxx Socioeconomic statusx Indoor environment and activities related to air pollutionx Clinical outcomesxxxxxMeasurement of indoor exposure (PM2.5)xxxxCollection of blood samplexxxxxEQ-5D-5L, the five-level EuroQol five-dimension; K-BILD, The King’s Brief Interstitial Lung Disease; PM2.5, particulate matter ≤2.5 μm; VAS, Visual Analogue Scale.Lung functionFVC and DLco were measured every 3 months at each institution according to the ATS/ERS recommendations.24–26 Additionally, FVC was measured using a portable spirometer (SPROLENIS, JNBIO, Chuncheon-si, Republic of Korea) every 3 months at each participant’s house, synchronised with the indoor air pollution measurement period. The measurement of FVC using portable spirometry was performed twice daily at 8:00 and 20:00 hours for 5 days to account for diurnal variability. All lung function results were expressed as percentages of the normal predicted values (% predicted).Questionnaire for health-related quality of lifeTo assess the health-related quality of life (HRQL) of patients with IPF, two questionnaires were used: the King’s Brief Interstitial Lung Disease (K-BILD) and five-level EuroQol five-demensional version (EQ-5D-5L). K-BILD is a brief, valid, specific questionnaire designed to measure the HRQL of patients with ILD.27 It consists of 15 items covering domains including breathlessness and activities, psychological and chest symptoms. K-BILD scores are weighted using logit transformation and range from 0 to 100. A Korean version of K-BILD, translated by a certified translator company (Mapi Language Services, Lyon, France), was used in this study.EQ-5D-5L is a health status measurement questionnaire developed by the EuroQol Group28 and is widely used for several conditions, including IPF.29 It consists of the EQ-5D descriptive section and the Visual Analogue Scale (VAS) section. The descriptive section assesses five dimensions (mobility, self-care, usual activities, pain/discomfort and anxiety/depression) on a five-point Likert scale. The results from the five dimensions are converted into a single index value between 0 and 1.30 VAS is a simple and easy-to-use tool that can provide visual information about a person’s subjective experience or phenomena, such as pain, quality of life and overall health. It consists of a vertical line with a fixed end point at each end, representing the minimum and maximum values. Higher VAS values indicate better health status. Korean versions of EQ-5D and EQ-VAS, translated using standardised protocols that comply with internationally recognised guidelines from the EuroQol Research Foundation (Rotterdam, Netherlands), were used in this study.Clinical outcomesThe primary outcomes of this study were death, hospitalisation, AE, as well as changes in lung function and HRQL. AE of IPF was defined as acute worsening of dyspnoea typically within 30 days, accompanied by new bilateral lung infiltration that is not fully explained by heart failure or fluid overload and no identified extraparenchymal causes (pneumothorax, pleural effusion, pulmonary embolism).31 A decline of ≥10% in FVC is generally considered a surrogate marker for IPF mortality in clinical trials32–34 as it has been strongly related to mortality in IPF.35 36 In this study, disease progression was defined as a relative 10% decline in FVC predicted, calculated as ([FVCfollow-up − FVCbaseline] × 100)/FVCbaseline.37Sample collectionBlood samples (plasma, serum and DNA) were collected from participants using ethylenediaminetetraacetic acid (EDTA) tubes (BD Vacutainer EDTA Tubes, Becton, Dickinson and Company, New Jersey, USA) and serum separator tubes (SST) (BD Vacutainer SST Tubes, Becton, Dickinson and Company). Samples were stored frozen at −80°C until the measurement of biomarkers, including Krebs von den Lungen-6 (Nanopia KL-6 assay, SEKISUI MEDICAL, Tokyo, Japan), matrix metalloproteinase-7 (R&D Systems, Minneapolis, USA), surfactant protein-D (Biovender Laboratory Medicine, Karasek, Czech Republic) and chemokine ligand-18 (R&D Systems).Measurement of indoor air pollutionThe MicroPEM (RTI International, Research Triangle Park, North Carolina, USA) device is a lightweight PM exposure monitor that weighs only 240 g and has three batteries. Figure 2A,B depicts the external and internal structures of MicroPEM, respectively. It has been widely used in previous clinical studies.38–40 MicroPEM combines real-time nephelometry and integrated referee filters for PM measurements. It includes an impactor and a light-scattering particle detector, with an airflow of 0.5 L/min. For real-time measurements, the MicroPEM samples PM2.5 at a rate of 0.5 L/min using light-scattering nephelometry every 10 s. The concentration of PM2.5 measured by the nephelometer is automatically corrected by embedded temperature and relative humidity sensors.41 Gravimetric and chemical analyses are conducted on the integrated filter to quantify the concentrations of PM2.5 and its various chemical components. Gravimetric analysis can estimate the average PM2.5 exposure concentration over a 24 hours period. Additionally, the filter is used to measure the mass of black carbon and brown carbon, as well as environmental tobacco smoke using a multiwavelength optical absorption method.42 The 10 s nephelometry PM2.5 data are corrected and then integrated into a 1 min average to be comparable to the average equals with the corresponding gravimetric concentration.43Figure 2MicroPEM device. (A) External configuration, (B) Internal architecture. Quarters (25-cent coins) placed for size comparison.Participants visited each medical institution, and indoor air pollution at their homes was measured every 3 months for a 1-year period to assess seasonal variations in indoor air quality. Our measurement team visited each participant’s home and positioned the MicroPEM at the height of an adult’s waist in the space where participants spend most of their time, such as the living room or bedroom (figure 3A,B). During each season, indoor PM2.5 measurements and home spirometry were performed on five consecutive days within a month of the patient’s clinic visit. In addition, we measured outdoor PM2.5 in 20% of the homes of our study participants (n=24), who were randomly selected, living on the fifth floor or lower for safety reasons, to assess the infiltration of outdoor PM2.5 into the indoor environment (figure 3C,D). We will estimate the infiltration using the ratio of indoor to outdoor concentrations of sulphur in PM2.5, which has negligible indoor sources.44Figure 3Examples of MicroPEM installation locations in patients’ homes. (A, B) Indoor installation. (C, D) Outdoor installation. The MicroPEM is indicated with a red circle in each (A, B).Statistical analysisThe PM2.5 concentration will be analysed as a continuous variable and also categorised into quartiles due to the lack of specific indoor PM2.5 concentration thresholds. The continuous variables will be expressed as mean±SD, while categorical variables will be expressed as number (percentages). Student’s t-test or Mann-Whitney U test will be used for continuous data, and the χ2 test or Fisher’s exact test for categorical data. Pearson’s or Spearman’s correlation analyses will be used to assess the correlation between PM2.5 concentrations and the health outcomes after testing of normality assumption. Paired t-test or Wilcoxon signed rank test will also be used to compare changes in clinical parameters between each visit. We will analyse repeated measurements using a linear mixed model, adjusting for covariates such as age, sex, smoking status, baseline FVC and DLco, while including a random intercept for each participant to account for intraindividual variability. Kaplan-Meier survival curves will be used to evaluate the occurrence of clinical outcomes. The Cox proportional hazards analysis will be used to identify risk factors of time-to-event clinical outcomes (death, AE, disease progression), and variables with a p<0.1 in the unadjusted analyses will be included in the multivariable analysis. For subgroup analysis, we will consider the severity of IPF according to the Gender-Age-Physiology index.45 In addition, we will perform a further analysis in which patients will be divided into two different groups: those affiliated to specialised tertiary hospitals for ILD and those from other healthcare institutions. A p<0.05 will be considered significant (two tailed). All statistical analyses will be performed using Statistical Package for the Social Sciences V.23.0 (IBM).Regulatory aspectsParticipant safetyAny potential risks to the study participants were carefully managed by ensuring that all aspects of recruitment and data collection were overseen by fully trained, experienced and competent research staff. The recruitment team included pulmonologists and research nurses with a strong background in both research methodology and clinical practice. The data collected were securely stored and individual details will be documented in a centrally monitored electronic case report form ( go. kr/) under the supervision of the Korea National Institute of Health. Personally identifiable information was encrypted, and access was strictly limited to authorised individuals with research approval, ensuring the confidentiality and security of personal information.Unexpected findings during examinationsDuring the informed consent process, participants were informed that the research team has a duty of care to inform or raise the issue if required by law, or if there are health concerns that require urgent attention during routine appointments.DisseminationThe findings from the data analysis will be disseminated in a variety of ways, including abstracts, posters and presentations at conferences, and the publication of research manuscripts in peer-reviewed journals. In addition, these findings will be communicated to government agencies at the local levels, thereby contributing to policy formulation. Detailed reports will also be provided to the funding agencies, institutes and hospitals that supported and participated in the cohort study. Members of the study team will be granted the right to publish and claim authorship in accordance with the principles outlined in the ethical guidelines and authorship criteria.

PMC

Revista de Neurología

36973888

PMC10478112

4-01-2023

10.33588/rn.7607.2021447

X-linked myotubular myopathy: a clinical report and a review of the mild phenotype

Barreto-Mota Ricardo, Figueirinha Joana, Quental Rita, Fonseca Jacinta, Melo Cláudia, Sampaio Mafalda, Sousa Raquel

Introduction.X-linked myotubular myopathy is a rare centronuclear myopathy that affects approximately 1 in 50,000 male newborns caused by pathogenic variants in the myotubularin 1 gene (MTM1). The clinical severity varies, however the need for ventilatory support occurs almost invariably.Case report.We report the case of a 4-year-old boy presenting mild muscle hypotonia at 12 months-old, expressive language disorder, global developmental delay, and a sensory processing disorder. Clinical exome sequencing identified the hemizygous variant c.722G>A p.(Arg241His) in exon 9 of the myotubularin 1 gene (NM_000252.2). The mother is a heterozygous carrier of the same variant. A diagnosis of a mild form of maternal inherited X-linked myotubular myopathy was established. The child presented significant improvement with speech, occupational, and physical therapies, with no respiratory intercurrences or ventilator dependency.Conclusion.The presentation of a mild form of this myotubular myopathy, being less commonly reported, added challenge to the diagnosis. The combination of mild hypotonia, feeding difficulties and expressive language disorder should raise suspicion of a neuromuscular disease. There is a lack of verified motor or developmental scores specific to this myopathy to further determine prognosis and need of other therapies. While currently the severity myotubular myopathy is classified according to ventilator dependency, this may be insufficient and unapplicable to milder cases. There is an evident need for a grading system for mild and moderate cases assessing muscle weakness and fatigue, daily life limitations, motor developmental delay, early phenotypical scores, or recurrent respiratory infections.

IntroductionX-linked myotubular myopathy is a rare centronuclear myopathy that affects approximately 1 in 50,000 male new-borns. This myopathy was first reported by A. Spiro in 1966 in a 12 year old boy with progressive generalized motor weakness and, histologically, myotubes with central nuclei and lack of myofibrils . It is caused by pathogenic variants in the myotubularin 1 gene (MTM1), on chromosomal region Xq28, responsible for encoding ubiquitous phosphatase myotubularin. A recent multicentre retrospective analysis revealed that loss-of-function variants due to frameshift and nonsense variants are the most common type of change . The clinical severity varies and is classified by the degree of ventilatory support. Most patients present a severe phenotype (55-79%), while 6-16% are moderate and 15-29% have a mild form of the disease . Patients with most severe forms require early ventilator support and frequently die within the first year of life, while long term survivors have chronic respiratory failure with ventilator dependency and are non-ambulant . Mild forms have been described in patients with no need for ventilatory or feeding support and little to none motor compromise. We report the case of a 4-year-old boy with a developmental delay history and a past of lower tract infections, who was otherwise healthy.Case reportA 12 months-old male child was admitted to the child neurology outpatient clinic due to developmental delay. He was a full-term baby, born from an uneventful pregnancy, with healthy non consanguineous parents and two older healthy brothers. The boy had an history of two viral lower tract respiratory infections since he was 6 months-old, with two hospital admissions, without need of ventilatory support, and was followed in pneumology outpatient clinic due to recurrent wheezing. On physical examination he presented brachycephaly, mild hypotonia, normal osteotendinous reflexes and a bilateral 2-3 toes syndactyly. Head circumference was between the 85th and 97th percentiles. Stature and height were above the 97th percentile. He did not show ptosis or ophthalmoplegia. At 12 months-old, he was able to sit unassisted, but he could not get to sitting position. He seemed to understand simple orders but was not able to vocalize. Parents were also concerned with feeding; while describing that he had no problems with liquids, they reported apparent difficulty with chewing. Speech therapy, occupational therapy and physical therapy were started, with significant improvement. From born until the age of 4 years old he presented a slow but positive psychomotor evolution. He started independent walking at 18 months-old while expressive language was significantly delayed with words starting at 3 years old and sentences with 4 years-old. Social skills were always preserved; however, communication was affected. Developmental assessment revealed a global developmental delay (global developmental quotient 70) and a sensory processing disorder. He has no respiratory problems since the age of 3 years and 6 months. Cardiology assessment revealed no structural heart anomaly. Ear, nose, and throat assessments, including evoked auditory potentials, were normal. A brain magnetic resonance imaging at the age of 2 years-old revealed brachycephaly, normal brain parenchyma, adequate myelination for age, enlargement of the subarachnoid spaces in the frontal-parietal brain, with reduction of parenchymal volume, a wide posterior fossa with slight counter-clockwise rotation of the vermis and an arachnoid cyst compressing left cerebellum hemisphere. An array comparative genomic hybridization showed nonrelevant variants and a metabolic study work up was normal. Clinical exome sequencing identified the hemizygous variant c.722G>A p.(Arg241His) in exon 9 of the MTM1 gene (NM_000252.2), associated with myotubular myopathy. This variant was classified as likely pathogenic according to the American College of Medical Genetics and Genomics variant classification guidelines and, to our knowledge, has never been reported before. Muscle biopsy revealed atrophic myofibers, some rounded myofibers with internally located nuclei and, even though not frequent, accumulation of centrally located staining with oxidative stains some with nuclear centralization, commonly related with MTM1 gene myopathies. Genetic testing in patient’s mother showed that she is a heterozygous carrier of the same variant in MTM1 gene, which is not unexpected since only 10-20% of patients carry de novo mutation . In addition, we found out that she presents mild neuromuscular symptoms since youth that were not valued before.DiscussionOur patient never presented respiratory deficiency with ventilation dependency nor severe hypotonia, the most severe, hence most common, presenting symptoms of this condition. The fact that he presents a mild form of this condition, being less commonly reported, added challenge to the diagnosis. We consider relevant to highlight the high prevalence of motor developmental delay in mild cases reported in the literature, as well as the muscle weakness in older patients, since this could give clues regarding our patient’s prognosis and evolution. Our case fits most of the most common clinical features, including mild hypotonia, swallowing difficulties, and lack of ventilator dependency. Interestingly, he lacks some almost ubiquitous phenotypical features, namely myotonic facies, high arched palate, ophthalmoplegia or shoulder gridle weakness. This case highlights that the presentation of mild hypotonia, feeding difficulties and expressive language disorder should also raise suspicion of a neuromuscular disease. Our patient presented expressive language disorder. Amburgey et al , in a review describing the natural history of this condition, reported a high incidence of learning disability (43%), likely associated with speech abnormalities including hypophonic voice, speech articulation difficulties and late first words and sentences. Intellectual disability, however, has conflicting reports in the literature. While it has been described in neuromuscular disorders such as myotonic dystrophy type 1, in patients with myotubular myopathy, cognitive impairment has been described as secondary to perinatal hypoxic ischemic encephalopathy . In 1999, Herman et al described no cognitive impairment in long term survivors; it reported clinical seizures in four patients that were likely due to metabolic encephalopathies . Despite myotubularin being expressed in the brain, there are no reported central nervous system malformations in animals, but research on this topic is lacking . Adequate cognitive development and normal creatinine-kinase should steer the diagnosis towards a myopathic condition. Despite the phenotypical variation, this is a progressive disease that inexorably leads to regression of motor development, including in patients with milder phenotypes . The maximum development acquired appears to be determined by genotype . Currently, the severity myotubular myopathy is solely and variably classified according ventilator dependency . While respiratory function tests are useful to determine the need of respiratory therapies to improve this outcome , it is an incomplete way to characterize this condition. There is a lack of verified motor or developmental scores specific to this myopathy to further determine prognosis and need of other therapies. We believe that there is an evident need for a grading system for mild and moderate cases other than ventilator dependency, such as muscle weakness and fatigue, daily life limitations, motor developmental delay (especially independent walking and head control), early phenotypical scores (high arched palate, myotonic facies, hypotonia) or recurrent respiratory infections. The management of this condition is proposed in a standard care consensus, which describes supportive treatment, since there are, currently, no curative therapies for congenital myopathies . Patients benefit from close a multidisciplinary follow-up guided by a neuromuscular specialist, who should monitor for speech, swallowing and respiratory problems. Despite not being a common presentation, heart and rhythm abnormalities must be excluded. Gene therapy is currently being investigated for X-linked myotubular myopathy. In both double blinded randomized and open label non-randomized trials, the administration of recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM) resulted in dose-dependent improvement in survival, strength and muscle structure in dogs without adverse effects . In another study, an AAV vector was used to knockdown DMN2 expression in Mtm1 knock-out mice, based on the hypothesis that dynamin 2 GTPase activity leads to a centronuclear myopathy-like phenotype. This resulted in improved muscle force and histology . The ASPIRO trial (NCT03199469) is an ongoing a phase 1/2, multinational, open-label, clinical study in which an AAV8 vector carrying the MTM1 gene (AT132) is being administered in individuals with X-linked myotubular myopathy aged less than 5 years old.Although carrier females are generally asymptomatic, affected women have been described with symptoms ranging from mild to severe. Proposed mechanisms to explain this feature include skewed inactivation of the X chromosome and /or genetic variants in additional genes that can modulate the phenotype . X inactivation analysis in our patient’s mother showed a slightly skewed pattern (70:30). Even when the mother is asymptomatic, knowing her carrier status is important in other to offer proper genetic counselling to the family.We report the diagnosis of a child with a neuromuscular condition presenting with non-classic features, such as global developmental delay and expressive language disorders, while having milder hypotonia and motor delay. In the presence of global developmental delay with milder motor features, a neuromuscular disorder must be considered in the differential diagnosis.

PMC

Angewandte Chemie (International Ed. in English)

36253337

PMC10099152

12-23-2022

10.1002/anie.202211559

Catalytic Biosensors Operating under Quasi‐Equilibrium Conditions for Mitigating the Changes in Substrate Diffusion

Muhs Anna, Bobrowski Tim, Lielpētere Anna, Schuhmann Wolfgang

AbstractDespite the success of continuous glucose measuring systems operating through the skin for about 14 days, long‐term implantable biosensors are facing challenges caused by the foreign‐body reaction. We present a conceptually new strategy using catalytic enzyme‐based biosensors based on a measuring sequence leading to minimum disturbance of the substrate equilibrium concentration by controlling the sensor between “on” and “off” state combined with short potentiometric data acquisition. It is required that the enzyme activity can be completely switched off and no parasitic side reactions allow substrate turnover. This is achieved by using an O2‐independent FAD‐dependent glucose dehydrogenase embedded within a crosslinked redox polymer. A short measuring interval allows the glucose concentration equilibrium to be restored quickly which enables the biosensor to operate under quasi‐equilibrium conditions. We propose a catalytic quasi‐equilibrium biosensor concept in which an O2‐insensitive enzyme is wired with a redox polymer to the electrode. This allows the enzymatic conversion to be switched off by applying a low potential, the redox polymer can be charged with a short positive potential pulse, and the glucose concentration can be determined potentiometrically with minimal disturbance of the concentration equilibrium. This concept is opening the door for mitigating any impact of the foreign‐body reaction in implantable biosensors.

IntroductionLong‐term implantable biosensors play a key role in the monitoring and treatment of metabolic diseases, particularly for applications in continuous glucose monitoring (CGM).[ 1 , 2 , 3 , 4 ] To date, there are several commercial CGM systems available that mainly rely on two different detection principles. Most of the through‐skin operating CGM systems are based on amperometric catalytic biosensors detecting current responses proportional to the conversion of the glucose concentration at the active site of an enzyme which in most cases is immobilised on the electrode surface.[ 3 , 5 ] Besides their simple detection principle, the specific substrate affinity is another advantageous feature of enzyme‐based sensors, which allows highly selective and reliable substrate determination in complex body fluids. Importantly, the enzyme‐catalysed conversion of the substrate followed by product release is a self‐cleaning process which makes the active site of the enzyme again available for the conversion of the substrate. However, the continuous conversion of the substrate leads to the most significant disadvantage of these types of sensors for their application in long‐term implantable systems. In principle, a catalytic biosensor can be seen as a technical chemical reactor with a reaction volume (the sensing layer) which is separated from the external solution by a permeable barrier through which the substrate can enter the reaction volume and the product can leave it. Within the sensing layer, the substrate is consumed at a constant reaction rate if sufficient enzyme activity is available, leading to a difference in substrate concentration between the enzyme layer and the external environment and, thus, to the formation of a diffusion gradient. Consequently, the apparent concentration of the substrate (c app) within the sensing layer and therefore the substrate turnover depends on the diffusional flux into the reaction volume. Under continuous sensor operation of an assumed long‐term implantable sensor, the so‐called foreign‐body reaction is adding encapsulation layers around the sensor and lowers by this the diffusional flux of the substrate into the sensor. This decrease in diffusional flux in turn decreases c app within the sensor layer and lowers the recorded signal (Scheme 1). Importantly, the signal varies even at the same external substrate concentration and the same reaction rate of the enzymatic conversion (Scheme 1).Scheme 1Schematic representation of a typical concentration profile of a catalytic enzymatic sensor under continuous operation without (left) and with an additional layer as formed after implantation due to the foreign‐body reaction (right). The substrate conversion depends on its local concentration at the enzyme which is determined by the reaction rate and the diffusional flux. The gradient changes with time with the increase of the diffusion layer, which leads to a more pronounced difference between c app inside the sensor layer and the external substrate concentration which leads to a signal change at unchanged external substrate concentration.Since it is impossible to prevent the formation of additional diffusion barriers by fibrous encapsulation of implanted sensors,[ 6 , 7 , 8 ] it is unfeasible to obtain a long‐term stable sensor output in the case of implanted catalytic biosensors with implantation times above several months.To circumvent this issue, there are basically two opportunities. On the one hand, there is the possibility of frequent recalibration using a direct measurement of the external substrate concentration to readjust the calibration graph using a one‐point calibration. Alternatively, one would have to avoid the building up of a concentration gradient between the external substrate reservoir and the sensor layer irrespective of the permeability of the layers formed by the foreign‐body reaction, i.e., c ext=c app.One commercial CGM system is available on the market which follows an affinity interaction principle on modified hydrogels which is governed by an equilibrium concentration of the substrate inside and external of the sensor layer, however, in this case, no substrate conversion occurs. The operational lifetime of this sensor amounts to 90 days whereby recent studies also show a possible usability of up to 180 days. The sensor concept is based on affinity interactions between substrate and recognition matrix using an optical detection system coated with a boronic‐acid modified hydrogel. In contrast to catalytic biosensors, these affinity‐based sensors determine the glucose concentration level without chemical conversion of the glucose. Instead, glucose is reversibly bound to the recognition matrix followed by a modulation in the detected fluorescence signal. Since affinity sensors are less dependent on substrate diffusion, encapsulation by the foreign‐body reaction has a minor effect on the functionality of such sensors. While recalibration is still required, the changes in the diffusional properties due to the foreign‐body reaction have less impact on the sensor signal. However, non‐catalytic sensors face a dilemma of binding constant versus response time and accuracy, while they have the advantage of a non‐enzymatic and hence possibly more robust recognition site. The binding constant of the sensor matrix must be precisely adjusted for the prevailing conditions. If it is too strong, the detachment of the substrate from the recognition matrix will be very slow and changes in glucose concentration in the sensor environment can be determined only very slowly. If the binding constant is too weak, the accuracy of the detection will be compromised. Additionally, not only glucose molecules but also other sugars and diols bind to the boronate groups resulting in decreased measurement accuracy or false results. Some advances to overcome the limitations of dynamic electrochemistry have been made in the field of potentiometry. Bakker's group showed a triple‐pulse measurement sequence for ion selective electrodes that provides a reference electrode‐independent signal with higher sensitivity than predicted by Nernst equation. Ishige et al. developed a field‐effect transistor (FET)‐based enzyme sensor that measures the changes in the interfacial potential depending on the ratio of a redox compound participating in an enzymatic reaction. First, second and third[ 15 , 16 ] generation open‐circuit potential (OCP) biosensors have been demonstrated for the detection of glucose[ 13 , 14 , 15 , 16 ] or other analytes,[ 14 , 15 ] and the performance of the sensors demonstrate the size‐ and interference‐independence advantage of potentiometry. However, continuous monitoring of the OCP can still result in a concentration gradient because the enzyme may not be completely switched‐off, and third‐generation sensors with the enzymes in the direct‐electron transfer regime show disadvantages at low substrate concentrations due to undefined mixed potentials.The question therefore arises if it is possible to conceive a self‐cleaning catalytic biosensor operating under substrate concentration equilibrium conditions, i.e., with c ext=c app? We propose three prerequisites to design such a sensor: 1. The catalytic turnover reaction can be switched off. 2. Uncontrollable parasitic reactions, such as oxygen reduction at the enzyme or the electrode, need to be avoided. 3. During the measurements, the disturbance of the substrate concentration equilibrium must be minor to guarantee a fast reestablishment of the equilibrium after enzymatic substrate conversion.We introduce a redox hydrogel‐based catalytic microbiosensor concept for the detection and monitoring of glucose with minimal disturbance of the substrate equilibrium concentration which overcomes the issues imposed by sensor encapsulation as expected by the foreign‐body reaction upon implantation. Specifically, the signal becomes independent of any diffusional flux into the sensing layer provided a sufficient waiting time to fully establish concentration equilibrium. The proposed strategy is the first demonstration of a catalytic substrate equilibrium biosensor for the reliable determination of glucose based on an enzyme embedded in a redox polymer.Results and DiscussionThe proposed sensor concept is based on a switching mechanism which allows the control of the enzymatic substrate conversion by applying predefined potentials (Scheme 1). In the “off” state, the enzymatic conversion must be fully stopped which requires an enzyme which can be on the one hand wired to the electrode by means of a redox polymer but is completely insensitive to accept molecular oxygen as an alternative electron acceptor. This ability to completely switch‐off the enzymatic turnover allows the glucose concentration in the sensor layer to equilibrate with the external glucose concentration. The time of applying the “off”‐state potential depends on the thickness and permeability of encapsulating layers. For switching the enzyme to the “off”‐state a potential must be applied which is at least 250 mV cathodic of the formal potential of the redox mediator tethered to the redox polymer, which will make the oxidised mediator species negligible according to the Nernst equation. We utilised a FAD‐GDH which is known to have a low sensitivity towards oxygen. Glomerella cingulata FAD‐dependent glucose dehydrogenase (FAD‐GDH) expressed in Pichia pastoris was used as oxygen‐insensitive FAD‐GDH embedded in an Os‐complex modified redox hydrogel (see Figure S1 left), namely P(VI‐AANH2 )‐Os (poly(1‐vinylimidazole‐co‐acryl hydrazide)‐[Os(bpy)2Cl]+, with bpy=2,2′‐bipyridine).[ 20 , 21 ] Besides its function as an immobilisation matrix, Os‐complex modified redox polymers are known to serve as efficient relays for the mediated electron transfer between enzymes and electrode.[ 17 , 22 ]Potentiostatic control enables to adjust the redox state of the Os‐complexes within the redox hydrogel correlating to a specific concentration ratio of oxidised and reduced Os‐complexes according to the Nernst equation.Additionally, the proximity between the Os‐complexes and the enzymes within the recognition layer together with the only accessible electron‐transfer pathway for the enzyme enables to control the catalytic activity of the enzyme by the applied potential.A potential pulse from the “off”‐state (‐250 mV of E 0 (Os3+/Os2+)) to the “on”‐state (+250 mV of E 0 (Os3+/Os2+)) allows charging of the redox polymer film and enables electron transfer from the enzyme via the polymer‐bound Os‐complexes by means of electron hopping to the electrode (Figure S2). To reliably turn the enzymatic turnover activity off, parasitic side reactions by oxygen reduction must be prevented. Otherwise, reoxidation of the Os‐complexes or the enzyme itself may occur resulting in substrate consumption and, thus, in the formation of a glucose diffusion gradient. It was shown that O2 can be reduced to H2O2 by Os‐complexes with a redox potential more negative than +0.07 V versus Ag/AgCl/3 M KCl (pH 7). Therefore, the redox potential of the redox polymer should be sufficiently positive to prevent catalytic reduction of O2 but concomitantly low enough to avoid co‐oxidation of interfering compounds, such as ascorbic acid or uric acid directly at the electrode surface. Furthermore, as already pointed out above, the enzyme itself must be insensitive to oxygen.Since the substrate is consumed under enzymatic turnover leading to a concentration gradient between the sensing layer and the external volume evoking diffusion, it is essential to keep the “on”‐time as short as possible, however long enough, that the Os3+/2+ ratio is sufficiently changed to allow wiring of the embedded enzyme. The disturbance of concentration equilibrium must be kept to a minimum. For this, electrodes with dimensions at the micro‐ to nanometre scale are utilised because their intrinsic properties, such as the low capacitive charging current, make them ideal for measurements at short timescales.[ 21 , 25 ] Since the redox polymer acts as a pseudo‐capacitive element, the time to charge the film depends on the film thickness. Hence, homogeneous and thin polymer/enzyme films have to be immobilised on the microelectrode surface.We utilised carbon microelectrodes for the sensor fabrication (Figure 1). The immobilisation of the enzyme embedded in the P(VI‐AANH2 )‐Os matrix was performed by a layer‐by‐layer dipping approach which enables the total thickness of the active layer to be controlled by the number of immersion processes (for details see Supporting Information and Table S1). Following, the measurement sequence in Scheme 2, the enzymatic turnover is switched between the “off” and “on” state according to a predefined time regime, and after a short period in the “on” state data acquisition is invoked by opening the electrochemical circuit and following the change of the sensor potential by measuring the OCP. In a typical measurement sequence, the sensor is switched “off” for 90 s by applying a potential 250 mV more negative than the E 0 of P(VI‐AANH2 )‐Os which is corresponding to a potential of −50 mV vs. Ag/AgCl/3 M KCl. Subsequently, a short oxidative pulse to a potential of +450 mV vs. Ag/AgCl/3 M KCl was applied for 1 s. While applying the short potential pulse, the redox hydrogel is charged by the formation of an excess of Os3+ as defined by the Nernst equation. It is essential that the duration of the “on” phase lasts long enough to sufficiently charge the polymer film to be able to wire the enzyme productively allowing the enzyme to oxidize the glucose present at equilibrium concentration in the sensing layer. However, the charging process must be short enough to avoid the generation of a diffusion gradient due to substrate depletion within the film. This charging pulse is followed by switching off the electrical circuit and measuring the course of the OCP potentiometrically. The enzymatic turnover will consume Os3+ and discharge the redox polymer in dependence of the available glucose concentration in the sensing layer. Hence, the measured change in potential relies solely on the enzymatic glucose oxidation causing reduction of Os3+ to Os2+ and the discharge of the Os‐complex modified redox polymer film, respectively. Figure 1Schematic representation of the sensor fabrication. The surface of a freshly prepared carbon electrode is initially functionalised with amino groups by means of grafting followed by the immobilisation of PEGDGE crosslinker via dipping the electrode into a 5 mg mL−1 PEGDGE solution for 5 minutes. Subsequently, the electrode is coated with the active enzyme layer using a layer‐by‐layer approach. Herein, the electrode was alternately immersed in a polymer solution and in a mixture of polymer and enzyme solution. In between, the electrode was kept in the air to dry. This coating step was carried out a total of 3 times but can be varied in dependence on the desired total film thickness.Scheme 2Schematic representation of the proposed measuring principle. According to the Nernst equation, the redox polymer and with it the embedded enzyme is either switched “on” or “off” depending on the applied potential with respect to E 1/2 of the Os‐complex tethered to the redox polymer. After switching to a short “on”‐phase to charge the redox polymer and with this to invoke enzymatic turnover, the circuit is interrupted by switching to open‐circuit potential (OCP) and following the decay of the potential which is governed by the glucose concentration in the sensor layer.Depending on the time for the charging pulse and the potentiometric measurement the decay of the potential is solely governed by the glucose concentration in the sensing layer and only to a minor extent modulated by a diffusional flux of glucose into the sensing layer. It seems to be advantageous to make the potentiometric data acquisition as short as possible before the enzymatic turnover is again disabled by applying the “off” state potential.During this phase, the glucose concentration inside the sensor layer (c app) is regenerating to c ext and this regeneration time must be sufficiently long to allow the concentration equilibrium to be fully attained. Figure 2 shows individual potential transients recorded after a 1 s charging of the redox polymer film in the presence of different concentrations of glucose in the range of 0 mM and 7.5 mM. Figure 2Potential transients recorded during the OCP measurement for different glucose concentrations in the range of 0 mM (PBS) and 7.5 mM. Before the potentiometric data acquisition, the sensor was kept at −50 mV vs. Ag/AgCl/3 M KCl for 90 s followed by an oxidative potential pulse to 450 mV vs. Ag/AgCl/3 M KCl for 1 s.Figure S3 shows the results of a similar experiment in which each potential discharge curve was measured 4 times at each glucose concentration demonstrating the reproducibility of subsequent measurements with the same sensor. As suggested by the Nernst equation, the slope of the potential decay decreased significantly within the first 500 ms. After this, it becomes quasi stationary, and in this time regime the glucose within the sensing layer was mainly consumed and is replenished by diffusion from the external reservoir. These measurements not only confirm the proposed sensing strategy, but show that the charging pulse and the potentiometric data acquisition has to be performed during a very short time to avoid complete depletion of glucose in the sensing layer.One of the key aspects for a substrate‐consuming implantable sensor that functions reliably over a long period of time is its behaviour in the presence of an increased diffusion barrier (such as the encapsulation imposed by the foreign‐bode reaction).[ 1 , 6 , 7 , 8 ] Ideally, the implanted sensor is unaffected by the continuous increase of the diffusion barrier. We conducted a control experiment and varied the regeneration time (“off” state) using a sensor modified with an additional thick layer of a redox‐inactive polymer, P(SS‐GMA‐BA) (see Figure S1, right) to simulate encapsulation by the foreign‐body reaction. This additional layer decreases the diffusion of glucose into the sensing layer. For short regeneration times, we observed a depletion of the glucose concentration within the catalytic recognition layer indicated by the shift of the diffusion‐dependent potentials towards higher values compared to the first potentiometric transient (Figure 3a). However, the depletion of glucose within the sensing layer is decreasing with increasing regeneration time. For a regeneration time of above 30 s, the obtained transients coincided again (Figure 3b, c) and make the sensor response independent from the additional polymer layer thus completely mitigating the effect of the encapsulation. Figure 3Comparative measurements of a sensor modified with a high diffusion barrier to affect the mass transfer of glucose into the catalytic sensing layer. The duration of the charging pulse was set to 2 s. For short regeneration times of 2 s between the individual measurements (a), a depletion of glucose in the film is observed between the first measurement (black trace) and the last measurement (blue trace). The depletion of glucose in the film is decreasing with increasing regeneration times (b). From a regeneration time of 30 s (c), no differences between the individual measurements can be detected .A series of measurements, using a sensor without an additional diffusion barrier, was carried out yielding similar results (Figure S4). The glucose depletion is though less distinct compared to the results for a sensor with additional diffusion layer and it almost vanished for a regeneration time of 10 s. In the case of the sensor without the additional layer the in‐diffusion of glucose during the regeneration time is substantially faster and glucose concentration equilibrium is faster attained. Based on these results, we suggest operating such a catalytic equilibrium sensor with a typical measurement frequency of ca. 35 s. The measurement frequency, which is mainly determined by the “off” phase, may be adjusted if needed.The procedure was further optimised by reducing the duration of the potentiometric data acquisition. The depletion of glucose in the sensing layer is substantially reduced, the overall measurement interval is shortened, and the replenishment of the glucose within the sensing layer by diffusion is facilitated. To detect the substrate concentration present in the sensing layer without any influence of substrate diffusion, we evaluated the slope in the linear range of the potential transient recorded directly after charging the sensing layer (Figure 4a). Figure 4a) Slopes of individual potential transients vs. their initial potential for increasing glucose concentration with determination of the respective straight line slope in the range between 20 ms and 45 ms. b) Calibration curve consisting of the current, potential and slope of potential transient values for the same sensor in dependence on the glucose concentration.The slopes of the potential transients clearly show differences in dependence from the glucose concentration in the range of 20–45 ms with an increased negative slope at higher substrate concentrations which is in accordance with our predictions. To validate the dependence of the potential slopes (Figure 3a) on the glucose concentration, we compared the results with steady‐state amperometric data for glucose concentrations in the range of 0 mM and 7.5 mM (Figure 4b). Corresponding potentiometric and amperometric calibration curves are shown in Figure 4b and Figure S5, respectively. The correlation confirms that the newly suggested measuring sequence leads to robust data within only a hundred of milliseconds. The short data acquisition time and with this the short time during which the enzyme is switched on and depletes the glucose concentration within the sensing layer accelerates the re‐adjustment of the substrate equilibrium within the sensing layer after deriving a glucose concentration value. This provides the basis for a substantial decrease of the total measurement interval between two measured values. The potentiometric and amperometric responses measured for different individual sensors differ substantially (Figure S6), which is due to differences in the electrode geometry and variations in the manual sensor fabrication. We are confident that this can be mitigated using automatic non‐manual sensor fabrication procedures in the future.A dynamically changing glucose concentration profile was recorded over 4 h to simulate the rapid response of the proposed sensor with respect to changes in c ext (Figure 5). We frequently measured the change in the OCP values after short oxidative pulses during controlled exchange of the solution via an in‐ and outlet. In addition, chronoamperometry was performed at a constant potential of 450 mV vs. Ag/AgCl/3 M KCl for 180 s after applying each 20 potentiometric measuring pulses to validate the measured glucose concentrations which varied between 1 mM and 7.5 mM (Figure S6). The amperometric measurements fit almost seamlessly into the course of the determined slopes of the potentiometric measurements demonstrating that the sensor concept is capable to rapidly detect fluctuations in the external glucose level over time. Figure 5Series of multiple measurements while dynamically changing the external glucose concentration in the range between 1 mM and 7.5 mM. The individual measurement values (black crosses) were determined from the slope of the corresponding potential transients between 10 ms and 20 ms after charging the film for 1 s. The red squares show independently performed amperometric measurements correlated to the pulse potential transients. The plotted blue trace was obtained from the moving average of 4 individual pulse measurements.ConclusionIn summary, we proposed a proof‐of‐principle procedure of a catalytic biosensor operating under quasi‐equilibrium conditions. Its feasibility was demonstrated for an example of a glucose micro‐biosensor employing completely oxygen‐independent FAD‐GDH immobilised within an Os‐complex modified redox polymer on the electrode surface. By means of potentiostatic control, the sensor was repetitively switched between catalytic conversion and regeneration of the substrate's concentration equilibrium. To ensure the reliable control over the enzymatic turnover state, parasitic side reactions were prevented by choosing a redox polymer with a specifically tailored potential and by employing an oxygen‐independent highly selective enzyme. To invoke just a minimal disturbance of the concentration equilibrium between sensor and analyte solution, the actual potentiometric measurement after charging the sensor film was kept extremely short to a few milliseconds. The sensors could reliably detect glucose concentrations regardless of the microelectrode size. The evaluation of the slopes of the potential transients at different glucose concentrations showed a good correlation with steady state control measurements using the same sensors. Even in the presence of an artificially enlarged diffusion barrier for simulation of the effect of the foreign‐body reaction the same quality of measurement has been achieved. The short measurement intervals could be beneficial to identify trends during fast changing glucose concentrations in the human body. The sensor concept offers the opportunity to combine the advantages of the competing glucose sensor technologies currently dominating the market and concomitantly to overcome the previous disadvantages, mainly the dependence of the signal from diffusional fluxes of the substrate towards the enzyme under non‐equilibrium conditions. Moreover, the presented strategy is applicable for other enzyme classes and can be extended for applications detecting other important analytes, such as lactate or glutamate,[ 21 , 27 ] by exchanging the biorecognition element within the redox hydrogel.Conflict of interestThe authors declare no conflict of interest.1Supporting informationAs a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.

PMC

Journal of Diabetes Investigation

36811237

PMC10119925

2-22-2023

10.1111/jdi.13997

Low serum dehydroepiandrosterone levels are associated with diabetic retinopathy in patients with type 2 diabetes mellitus

Zhang Xinxin, Huang Yadi, Xu Ning, Feng Wenli, Qiao Jingting, Liu Ming

ABSTRACTAimsThis cross‐sectional study assessed the association of serum dehydroepiandrosterone levels with the risk of diabetic retinopathy in patients with type 2 diabetes mellitus in China.Materials and MethodsPatients with type 2 diabetes mellitus were included in a multivariate logistic regression analysis to assess the association of dehydroepiandrosterone with diabetic retinopathy after adjusting for confounding factors. A restricted cubic spline was also used to model the association of serum dehydroepiandrosterone level with the risk of diabetic retinopathy and to describe the overall dose–response correlation. Additionally, an interaction test was conducted in the multivariate logistic regression analysis to compare the effects of dehydroepiandrosterone on diabetic retinopathy among age, sex, obesity status, hypertension, dyslipidemia, and glycosylated hemoglobin level subgroups.ResultsIn total, 1,519 patients were included in the final analysis. Low serum dehydroepiandrosterone was significantly associated with diabetic retinopathy in patients with type 2 diabetes mellitus after adjustment for confounding factors (odds ratio [quartile 4 vs quartile 1]: 0.51; 95% confidence interval: 0.32–0.81; P = 0.012 for the trend). Additionally, the restricted cubic spline indicated that the odds of diabetic retinopathy decreased linearly as the dehydroepiandrosterone concentration increased (P‐overall = 0.044; P‐nonlinear = 0.364). Finally, the subgroup analyses showed that the dehydroepiandrosterone level stably affected diabetic retinopathy (all P for interaction >0.05).ConclusionsLow serum dehydroepiandrosterone levels were significantly associated with diabetic retinopathy in patients with type 2 diabetes mellitus, suggesting that dehydroepiandrosterone contributes to the pathogenesis of diabetic retinopathy. Low serum dehydroepiandrosterone (DHEA) was found to be statistically associated with diabetic retinopathy (DR) in patients with type 2 diabetes mellitus after adjustment for traditional risk factors. The restricted cubic spline further indicated that the odds of diabetic retinopathy decreased linearly with the increase of DHEA concentrations. The subgroup analysis stratified according to age, sex, obesity, hypertension, dyslipidemia, and HbA1c, also showed stable effect of DHEA on diabetic retinopathy.

INTRODUCTIONDiabetic retinopathy (DR), a common microvascular complication of diabetes mellitus (DM), remains the leading cause of vision impairment and blindness in adults 1 . Worldwide, the number of adults with diabetic retinopathy was estimated to be 103.12 million in 2020 and is expected to increase to 160.50 million by 2045 2 . In China, 18.45% of patients with diabetes mellitus have diabetic retinopathy 3 , and the independent risk factors for diabetic retinopathy are younger age, higher systolic blood pressure (SBP), longer duration of diabetes mellitus, and poor glycemic control 4 . However, despite these traditional indicators of risk, wide variations in the development and severity of diabetic retinopathy exist which cannot be completely explained by these known factors 5 . Therefore, diabetic retinopathy risk factors are not fully understood.Dehydroepiandrosterone (DHEA), an androgen precursor, is an abundant steroid hormone in human circulation. Previous studies have reported that DHEA improves endothelial cell function, inhibits inflammation, and reverses vascular remodeling 6 , 7 . Low DHEA levels are associated with the risk of coronary heart disease (CHD) in the general population and individuals with type 2 diabetes mellitus 8 , 9 . Furthermore, our previous study demonstrated an inverse relationship between low serum DHEA and diabetic kidney disease (DKD) in men with type 2 diabetes mellitus 10 . In recent years, the point of a unifying mechanism has been raised in the pathogenesis of micro‐ and macrovascular complications of diabetes mellitus. These common pathogenic pathways included the production of reactive oxygen species, oxidative stress, and chronic low‐grade inflammation 11 . In animal experiments, DHEA attenuated the adverse effects of hyperglycemia on bovine retinal pericytes 12 . However, the association of DHEA with the risk of diabetic retinopathy remains unclear in patients with diabetes mellitus.Therefore, this cross‐sectional study assessed the association of DHEA with the risk of diabetic retinopathy in patients with type 2 diabetes mellitus in China.MATERIALS AND METHODSStudy designThis cross‐sectional study was conducted at the Department of Endocrinology and Metabolism, Tianjin Medical University General Hospital in Tianjin, China. Hospitalized patients with type 2 diabetes mellitus were enrolled and DHEA measured between October 12, 2020, and June 30, 2022. The patients were admitted for glycemic control and evaluation of diabetic complications. The type of diabetes mellitus was determined by physicians based on clinical features, including the onset age, acute or chronic onset, body mass index (BMI), fasting or post glucose‐challenge insulin and C‐peptide levels, insulin dependence, and pancreatic beta cell autoantibodies if necessary. If multiple medical records for one patient existed, only one of the patient's records was included. The exclusion criteria were: age 27.5 kg/m2 13 . The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation 14 .The serum DHEA concentration was quantified using liquid chromatography–tandem mass spectrometry as described previously 10 , 15 . Briefly, fasting blood samples were collected in the morning after admission and immediately sent to the Laboratory of Endocrinology and Metabolism at the Tianjin Medical University General Hospital. Two professionals pre‐treated the samples following uniform standards and then loaded them into a Jasper™ HPLC system coupled to an AB SCIEX Triple Quad™ 4500MD mass spectrometer (AB SCIEX, Framingham, MA, USA) to measure DHEA.DefinitionsDiabetes mellitus was defined as a fasting blood glucose level ≥7.0 mmol/L, a 2 hour plasma glucose level ≥11.1 mmol/L, an HbA1c level ≥6.5%, a self‐reported history of diabetes mellitus, or the use of hypoglycemic medications 16 . Hypertension was defined as an SBP ≥140 mmHg, a DBP ≥90 mmHg, a self‐reported history of hypertension, or the use of hypotensive medications 17 . Dyslipidemia was defined as a total cholesterol (TC) level ≥6.2 mmol/L, a triglyceride (TG) level ≥2.3 mmol/L, a low‐density lipoprotein cholesterol (LDL‐C) level ≥4.1 mmol/L, a high‐density lipoprotein cholesterol (HDL‐C) level <1.0 mmol/L, or the use of lipid‐lowering medications 18 . Diabetic kidney disease was defined as an albumin to creatinine ratio value >30 mg/g or an eGFR level <60 mL/min/1.73 m2 19 .Experienced and trained specialists took standard non‐mydriatic fundus photographs to evaluate diabetic retinopathy. Diabetic retinopathy was diagnosed based on microaneurysms, hard exudates, cotton wool spots, intraretinal hemorrhages, venous beading changes, intraretinal microvascular anomalies, neovascularization, vitreous hemorrhage, or tractional retinal detachment 20 . The International Classification of Diabetic Retinopathy Scale 20 defines sight‐threatening diabetic retinopathy as severe non‐proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR) or diabetic macular edema. Thus, the patients were divided into non‐diabetic retinopathy, non‐sight‐threatening diabetic retinopathy (i.e., mild or moderate NPDR), and sight‐threatening diabetic retinopathy (i.e., severe NPDR, PDR or diabetic macular edema) groups.Statistical analysesNormally distributed continuous variables were presented as mean ± standard deviation, and between‐group comparisons were performed using Student's t‐tests or one‐way analysis of variance. Non‐normally distributed variables were expressed as medians with interquartile ranges, and between‐group comparisons were conducted using Mann–Whitney U or Kruskal–Wallis tests. Categorical variables were described as numbers with percentages, and between‐group comparisons were performed using the chi‐squared tests.Multivariate logistic regression analyses were used to evaluate the association of DHEA with diabetic retinopathy after adjusting for confounding factors. Potential confounders were determined according to univariate findings (including the insurance type, BMI, duration of diabetes mellitus, SBP, DBP, and diabetic kidney disease [yes/no], the HDL‐C, FBG, and HbA1c levels, the use of metformin, α‐glucosidase inhibitors, and insulin [all P < 0.05]) and literature reports (including age) 4 , 21 . The serum DHEA levels were equally categorized into quartiles, and the lowest quartile was used as a reference. The restricted cubic spline with four knots (5th, 35th, 65th, and 95th percentiles) was used to model the association of DHEA with diabetic retinopathy and to depict the overall dose–response correlation.Moreover, subgroup analyses were performed to determine the relationship between DHEA and diabetic retinopathy based on the following subgroups: age (<65 or ≥65 years), sex, obesity status, hypertension, dyslipidemia, and HbA1c (<7.0 or ≥7.0%). An interaction test in the logistic regression analyses was used to compare the effects of DHEA on diabetic retinopathy between the analyzed subgroups. The DHEA data were log‐transformed with the base natural constant in the logistic regression analyses and restricted cubic spline. A two‐sided P‐value less than 0.05 was considered statistically significant. Analyses were performed using SPSS for Windows (version 25.0; Armonk, NY, USA) and R software (version 4.1.3; R Foundation, Vienna, Austria).RESULTSClinical characteristics of participantsTable 1 presents the clinical characteristics of the participants based on the DHEA quartiles. During the study period, 2,107 patients with type 2 diabetes mellitus were hospitalized, and 1,519 participants were included in the analysis; 826 (54.4%) were men, and the mean overall age was 55.60 ± 14.17 years. The prevalences of non‐sight‐threatening diabetic retinopathy and sight‐threatening diabetic retinopathy were 20.3% and 2.4%, respectively. The median duration of diabetes mellitus was 7.00 (1.00–15.00) years. The mean fasting blood glucose and HbA1c levels were 7.77 ± 2.89 mmol/L and 8.68 ± 2.21%, respectively. Participants in the higher quartiles were younger, had a shorter duration of diabetes mellitus, and had fewer instances of diabetic kidney disease, cardiovascular disease, hypertension, dyslipidemia, and medication use (hypotensive and lipid‐lowering medications, sulfonylureas, metformin, α‐glucosidase inhibitors, dipeptidyl peptidase 4 inhibitors, and insulin) than those in the lower quartiles (all P < 0.05). Furthermore, BMI, DBP, eGFR, and the TC, TG, LDL‐C, FBG levels significantly trended upward, whereas the ACR significantly trended downward (all P < 0.05).Table 1Characteristics of patients categorized by quartiles of DHEA levelVariablesOverallQuartiles of DHEA level P Quartile 1Quartile 2Quartile 3Quartile 4Participants1,519380308379380–Age, years55.60 ± 14.1763.14 ± 11.3258.57 ± 12.2653.69 ± 13.4146.99 ± 14.25<0.001Male sex, %826 (54.4)196 (51.6)230 (60.5)201 (53.0)199 (52.4)0.048BMI, kg/m2 27.15 ± 5.4626.33 ± 4.3626.79 ± 4.9627.39 ± 6.1628.04 ± 6.00<0.001Current smoking, %432 (28.5)88 (23.3)126 (33.2)117 (31.0)101 (26.6)0.012Current drinking, %407 (26.9)83 (22.0)113 (29.7)105 (27.8)106 (28.0)0.084Insurance type, %Urban workers1,262 (83.1)310 (81.6)330 (86.8)325 (85.8)297 (78.2)0.013Non‐working urban residents179 (11.8)48 (12.6)36 (9.5)42 (11.1)53 (13.9)Self‐pay78 (5.1)22 (5.8)14 (3.7)12 (3.2)30 (7.9)Duration of type 2 diabetes, year7.00 (1.00, 15.00)10.00 (3.00, 20.00)10.00 (2.00, 17.00)7.00 (1.00, 13.00)3.00 (0.16, 10.00)<0.001DR status, %Non‐DR1,173 (77.2)267 (70.3)297 (78.2)291 (76.8)318 (83.7)0.002Non‐sight‐threatening DR309 (20.3)100 (26.3)74 (19.5)77 (20.3)58 (15.3)Sight‐threatening DR37 (2.4)13 (3.4)9 (2.4)11 (2.9)4 (1.1)DKD, %447 (31.9)157 (44.0)120 (33.7)84 (23.9)86 (25.6)<0.001CVD, %334 (22.0)130 (34.2)96 (25.3)70 (18.5)38 (10.0)<0.001Hypertension, %911 (60.0)259 (68.2)254 (66.8)213 (56.2)185 (48.7)<0.001Dyslipidemia, %1,122 (74.7)310 (82.4)270 (72.4)282 (75.0)260 (68.8)<0.001Use of hypotensive medications, %759 (50.2)238 (62.8)217 (57.4)173 (46.0)131 (34.6)<0.001Use of lipid‐lowering medications, %389 (25.7)145 (38.3)101 (26.6)77 (20.4)66 (17.5)<0.001Glucose‐lowering medicationsSulfonylureas, %195 (13.1)67 (18.3)49 (13.1)51 (13.7)28 (7.4)<0.001Glinides, %95 (6.4)29 (7.9)28 (7.5)16 (4.3)22 (5.8)0.164Metformin, %575 (38.6)155 (42.3)160 (42.8)141 (37.9)119 (31.6)0.005Thiazolidinediones, %32 (2.1)9 (2.5)12 (3.2)7 (1.9)4 (1.1)0.220α‐glucosidase inhibitors, %504 (33.8)161 (44.0)140 (37.4)116 (31.2)87 (23.1)<0.001DPP‐4 inhibitors, %192 (12.9)55 (15.0)57 (15.2)45 (12.1)35 (9.3)0.048GLP‐1 receptor agonists, %97 (6.5)23 (6.3)16 (4.3)34 (9.1)24 (6.4)0.062SGLT‐2 inhibitors, %167 (11.2)47 (12.8)42 (11.2)45 (12.1)33 (8.8)0.313Insulin, %513 (34.5)157 (42.9)146 (39.0)114 (30.6)96 (25.5)<0.001Blood pressure, mmHgSystolic136.59 ± 18.07137.14 ± 19.32137.13 ± 17.55136.04 ± 18.17136.04 ± 17.190.708Diastolic83.11 ± 11.9281.02 ± 11.4782.23 ± 11.7083.66 ± 11.3385.53 ± 12.70<0.001TC, mmol/L4.98 ± 1.574.71 ± 1.324.84 ± 1.245.19 ± 1.725.18 ± 1.84<0.001TG, mmol/L1.75 (1.27, 2.54)1.62 (1.18, 2.26)1.78 (1.27, 2.40)1.83 (1.32, 2.72)1.83 (1.29, 3.00)0.001HDL‐C, mmol/L1.08 ± 0.261.06 ± 0.281.06 ± 0.251.09 ± 0.261.10 ± 0.260.065LDL‐C, mmol/L3.01 ± 0.982.81 ± 1.022.95 ± 0.923.10 ± 0.953.15 ± 1.00<0.001FBG, mmol/L7.77 ± 2.897.49 ± 2.877.66 ± 2.807.85 ± 3.068.08 ± 2.790.038HbA1c, %8.68 ± 2.218.71 ± 2.348.72 ± 2.228.62 ± 2.178.67 ± 2.110.939Uric acid, μmol/L342.38 ± 104.00343.80 ± 108.65345.13 ± 101.88342.14 ± 104.15338.43 ± 101.460.833eGFR, mL/(min*1.73 m2)105.82 ± 24.1595.11 ± 24.69100.31 ± 24.53110.31 ± 20.88117.66 ± 19.58<0.001ACR, mg/g15.00 (7.30, 39.65)20.20 (9.78, 90.17)15.02 (7.83, 47.38)12.85 (7.00, 28.53)13.30 (6.40, 29.55)<0.001ACR, albumin to creatinine ratio; BMI, body mass index; CVD, cardiovascular disease; DHEA, dehydroepiandrosterone; DKD, diabetic kidney disease; DPP‐4, dipeptidyl peptidase‐4; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; FBG, fasting blood glucose; GLP‐1, glucagon‐like peptide‐1; HbA1c, glycosylated hemoglobin; HDL‐C, high density lipoprotein cholesterol; LDL‐C, low density lipoprotein cholesterol; SGLT‐2, sodium‐glucose cotransporter‐2; TC, total cholesterol; TG, triglycerides.Table 2 provides the clinical characteristics of the study population based on the diabetic retinopathy status. Overall, 346 of 1,519 patients (22.8%) had diabetic retinopathy. Those with diabetic retinopathy had a longer duration of diabetes mellitus, more instances of diabetic kidney disease and hypertension, and higher SBP, DBP, ACR, HDL‐C, FBG, and HbA1c, but lower BMI and eGFR values than those without diabetic retinopathy (all P < 0.05). Furthermore, patients with diabetic retinopathy were more likely to use metformin, α‐glucosidase inhibitors, and insulin than those without diabetic retinopathy (all P < 0.05). Finally, the proportion of medical insurance type significantly differed between patients with and without diabetic retinopathy (P = 0.025).Table 2Characteristics of patients categorized by the presence of diabetic retinopathyVariablesNon‐DRDR P Participants, %1,173 (77.2)346 (22.8)–Age, years55.25 ± 14.5356.77 ± 12.860.062Male sex, %648 (55.2)178 (51.4)0.213BMI, kg/m2 27.42 ± 5.6226.22 ± 4.75<0.001Current smoking, %343 (29.3)89 (25.9)0.214Current drinking, %325 (27.8)82 (23.8)0.147Insurance type, %Urban workers989 (84.3)273 (78.9)0.025Non‐working urban residents124 (10.6)55 (15.9)Self‐pay60 (5.1)18 (5.2)Duration of type 2 diabetes, year6.00 (0.50, 13.00)10.00 (4.00, 19.00)<0.001DKD, %286 (26.4)161 (50.5)<0.001CVD, %251 (21.4)83 (24.0)0.310Hypertension, %687 (58.6)224 (64.7)0.039Dyslipidemia, %877 (75.5)245 (71.8)0.176Use of hypotensive medications, %579 (49.5)180 (52.6)0.306Use of lipid‐lowering medications, %301 (25.7)88 (25.6)0.957Glucose‐lowering medicationsSulfonylureas, %140 (12.2)55 (16.2)0.052Glinides, %73 (6.3)22 (6.5)0.925Metformin, %423 (36.8)152 (44.8)0.007Thiazolidinediones, %26 (2.3)6 (1.8)0.584α‐Glucosidase inhibitors, %357 (31.0)147 (43.4)<0.001DPP‐4 inhibitors, %138 (12.0)54 (15.9)0.058GLP‐1 receptor agonists, %76 (6.6)21 (6.2)0.786SGLT‐2 inhibitors, %129 (11.2)38 (11.2)0.997Insulin, %336 (29.2)177 (52.2)<0.001Blood pressure, mmHgSystolic135.63 ± 17.26139.84 ± 20.260.001Diastolic82.74 ± 11.4584.36 ± 13.320.041TC, mmol/L4.95 ± 1.605.07 ± 1.430.221TG, mmol/L1.76 (1.27, 2.56)1.69 (1.25, 2.48)0.141HDL‐C, mmol/L1.06 ± 0.261.12 ± 0.27<0.001LDL‐C, mmol/L2.99 ± 0.983.07 ± 1.000.189FBG, mmol/L7.66 ± 2.768.15 ± 3.230.012HbA1c, %8.57 ± 2.209.07 ± 2.21<0.001Uric acid, μmol/L343.12 ± 103.97339.92 ± 104.220.618eGFR, mL/(min*1.73 m2)107.30 ± 23.19100.82 ± 26.56<0.001ACR, mg/g13.50 (6.99, 29.28)28.05 (9.43, 170.58)<0.001ACR, albumin to creatinine ratio; BMI, body mass index; CVD, cardiovascular disease; DKD, diabetic kidney disease; DPP‐4, dipeptidyl peptidase‐4; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; FBG, fasting blood glucose; GLP‐1, glucagon‐like peptide‐1; HbA1c, glycosylated hemoglobin; HDL‐C, high density lipoprotein cholesterol; LDL‐C, low density lipoprotein cholesterol; SGLT‐2, sodium‐glucose cotransporter‐2; TC, total cholesterol; TG, triglycerides. DHEA associations with diabetic retinopathyTable 3 presents the odds ratios (ORs) for the association of DHEA with diabetic retinopathy in three models. The diabetic retinopathy odds decreased significantly as the DHEA level increased incrementally in model 1 (unadjusted; OR [quartile 4 compared with quartile 1]: 0.46; 95% confidence interval [CI]: 0.33–0.65; P < 0.001 for the trend). Furthermore, a low serum DHEA level remained statistically associated with diabetic retinopathy after adjusting for age, insurance type, BMI, duration of diabetes mellitus, SBP, DBP, diabetic kidney disease, HDL‐C, FBG, HbA1c, and the use of metformin, α‐glucosidase inhibitors, and insulin in model 3 (OR: 0.51, 95% CI: 0.32–0.81; P = 0.012 for the trend). Moreover, when DHEA was log‐transformed with the base natural constant and analyzed as a continuous variable, a significant association between DHEA and diabetic retinopathy risk remained after adjusting for the abovementioned variables (OR: 0.71; 95% CI: 0.54–0.94; P = 0.015).Table 3Odds ratios of diabetic retinopathy by different status of DHEANo. of participantsNo. of casesModel 1Model 2Model 3OR (95% CI) P valueOR (95% CI) P valueOR (95% CI) P valueQuartile 1380113Reference–Reference–Reference–Quartile 2380830.66 (0.48, 0.92)0.0130.68 (0.49, 0.95)0.0220.56 (0.37, 0.85)0.006Quartile 3379880.72 (0.52, 0.99)0.0420.74 (0.52, 1.03)0.0760.70 (0.46, 1.07)0.102Quartile 4380620.46 (0.33, 0.65)<0.0010.47 (0.32, 0.69)<0.0010.51 (0.32, 0.81)0.005 P for trend<0.0010.0010.012As a continuous variable † 1,5193460.66 (0.54, 0.81)<0.0010.67 (0.53, 0.83)<0.0010.71 (0.54, 0.94)0.015 † DHEA was log‐transformed with base natural constant.Model 1: unadjusted. Model 2: adjusts for age and insurance type. Model 3: model 2 + BMI, duration of diabetes mellitus, SBP, DBP, DKD, HDL‐C, FBG, HbA1c, and use of metformin, α‐glucosidase inhibitors, and insulin.BMI, body mass index; CI, confidence interval; DBP, diastolic blood pressure; DHEA, dehydroepiandrosterone; DKD, diabetic kidney disease; DR, diabetic retinopathy; FBG, fasting blood glucose; HbA1c, glycosylated hemoglobin; HDL‐C, high density lipoprotein cholesterol; OR, odds ratio; SBP, systolic blood pressure.Figure 2 describes the overall dose–response association of DHEA with diabetic retinopathy in the restricted cubic spline. After adjusting for covariates, the odds of diabetic retinopathy decreased linearly with increasing DHEA concentrations (P‐overall = 0.044; P‐nonlinear = 0.364).Figure 2The overall dose–response association of dehydroepiandrosterone (DHEA) with diabetic retinopathy (DR) shown by the restricted cubic spline. The line indicated the adjusted ORs and 95% CI is shown by shaded areas. DHEA was log‐transformed with base natural constant. Adjusted for age, insurance type, BMI, duration of diabetes, SBP, DBP, DKD, HDL‐C, FBG, HbA1c, and use of metformin, α‐glucosidase inhibitors and insulin.Subgroup analyses of the DHEA and DR relationshipFigure 3 illustrates the association between DHEA and diabetic retinopathy in the subgroup analyses based on age, sex, obesity, hypertension, dyslipidemia, and the HbA1c level. The effect of DHEA on diabetic retinopathy risk was stable in all subgroups, and no interactions between DHEA and the subgroup variables were statistically significant (all P for interaction >0.05).Figure 3Subgroup analyses of the association of DHEA with diabetic retinopathy. DHEA was log‐transformed with base natural constant. Adjusted for age, insurance type, BMI, duration of diabetes mellitus, SBP, DBP, DKD, HDL‐C, FBG, HbA1c, and use of metformin, α‐glucosidase inhibitors and insulin.DISCUSSIONThis cross‐sectional study assessed the association of DHEA levels with diabetic retinopathy risk in patients with type 2 diabetes mellitus. To our knowledge, we are the first to report a significant association between low serum DHEA levels and DR in type 2 diabetes mellitus patients after adjusting for traditional risk factors. Moreover, the effect of DHEA on diabetic retinopathy was stable in the age, sex, obesity, hypertension, dyslipidemia, and HbA1c subgroups.Previous studies have evaluated associations of DHEA with macrovascular and microvascular diseases. In men, a low DHEA concentration was associated with an increased incidence of macrovascular diseases. A prospective study of the general population demonstrated that a low serum DHEA level was associated with a 5 year risk of developing coronary heart disease in men aged 69–81 years 8 . The Massachusetts Male Aging Study also reported a significant association between low serum DHEA levels and the development of ischemic heart disease 22 . Moreover, low DHEA levels correlated with cardiovascular disease, including coronary heart disease, myocardial infarction, and stroke, in participants with type 2 diabetes mellitus from 10 communities in Shanghai, China 9 . Similarly, our previous study identified a relationship between low DHEA levels and coronary heart disease in men with type 2 diabetes mellitus 15 . Conversely, a significant relationship was not identified between DHEA and the incidence of cardiovascular disease in studies of women in the general population or those with type 2 diabetes mellitus 9 , 15 , 23 . Sex‐specific differences regarding these associations remain unclear and require further investigation.Previous studies have described the relationship between DHEA and microvascular disease. The population‐based Rotterdam study showed that DHEA was not related to microvascular injury in men or women, evaluated by arteriolar and venular calibers of the retina 24 . However, a study of postmenopausal women with type 2 diabetes mellitus from Shanghai, China, reported that high DHEA levels were associated with diabetic kidney disease 9 . In contrast, a rat mesangial cell‐based study showed that DHEA had a protective effect against hyperglycemia‐induced lipid peroxidation and cell growth inhibition 25 . Our previous study also reported that low serum DHEA levels correlated with diabetic kidney disease in men with type 2 diabetes mellitus 10 . In addition, DHEA was shown to reverse bovine retinal capillary pericyte loss caused by glucose toxicity 12 . Similarly, the current study identified a strong negative correlation between the serum DHEA level and diabetic retinopathy in participants with type 2 diabetes mellitus. We presume that differences in the participants, outcomes, and adjusted risk factors partially explain these contrasting results.Presently, diabetic retinopathy is considered an inflammatory neurovascular complication of diabetes mellitus and is characterized by retinal capillary occlusion, vasculature leakage, retinal ischemia and damage, angiogenesis, and neovascularization 26 . Inflammation plays an important role in the pathogenesis of diabetic retinopathy 27 , and increases in endothelial cell adhesion molecules, such as VCAM‐1 and E‐selectin, cause an accumulation of leukocytes in retinal capillaries 28 , 29 , 30 , 31 . Leukocyte‐endothelium adhesion leads to endothelial cell apoptosis and the breakdown of the blood–retinal barrier 32 , thereby causing a low‐grade inflammatory state in the retina. In addition, the upregulated inflammatory cytokine expression in the serum and ocular samples of diabetic patients, including TNF‐α, IL‐6, IL‐8, and soluble VCAM‐1, have been associated with the severity of diabetic retinopathy 33 , 34 , 35 .Furthermore, DHEA inhibits inflammation and regulates immune responses. For example, in a lipopolysaccharide (LPS)‐induced lung inflammation model, DHEA restrained acute neutrophil recruitment by upregulating developmental endothelial locus 1 expression, which is decreased in an inflammatory state 36 . In mice with colitis, DHEA attenuates intestinal inflammatory injury through GPR30‐mediated Nrf2 activation and NLRP3 inflammasome inhibition 37 . Moreover, DHEA relieves Escherichia coli O157:H7‐induced inflammation in mice and LPS‐induced inflammation in RAW264.7 macrophages by blocking the activation of AKT, MAPK, and NF‐κB signaling pathways and increasing the activation of Nrf2, which is associated with autophagy 38 , 39 . Given that diabetic retinopathy is also an inflammatory complication of diabetes mellitus, we hypothesized that the anti‐inflammatory effects of DHEA partly explain the inverse relationship between DHEA and diabetic retinopathy.Neurodegeneration is an important part in the pathogenesis of diabetic retinopathy 40 . Neurodegeneration, including reactive gliosis, decreased retinal neuronal function, and neuronal apoptosis, is now regarded as an early event in the progression of diabetic retinopathy 41 , 42 . Diabetes mellitus‐induced neurodegeneration occurs before visible microangiopathy in diabetic rats and humans 43 , 44 . Moreover, DHEA has neuroprotective effects on stroke and traumatic brain and spinal injuries 45 , 46 , 47 . Furthermore, DHEA inhibits microglial inflammation by activating the TrkA‐Akt1/2‐CREB‐Jmjd3 pathway in subarachnoid hemorrhage and neuroinflammation models 48 , 49 . Additionally, DHEA prevents the apoptotic loss of neurons through its interaction with nerve growth factor 50 , and intravitreal DHEA injections reduces retinal damage caused by the excitatory amino acid, AMPA, in adult Sprague–Dawley rats 51 . Furthermore, BNN27, a novel C17‐spiroepoxide derivative of DHEA, reverses retinal injury in diabetic rats, targeting the neurodegenerative and inflammatory components of diabetic retinopathy 52 . Therefore, we speculate that the neuroprotective mechanisms of DHEA underlie the relationship between DHEA and diabetic retinopathy.Although animal experiments have presented favorable evidence regarding the effects of DHEA on inflammation and neurodegeneration, DHEA supplementation in humans has been controversial since these studies only included a small number of participants. For instance, a daily 50 mg dose of DHEA significantly increased insulin sensitivity 53 and decreased the total cholesterol and low‐density lipoprotein levels 54 , 55 in patients with hypoadrenalism and healthy postmenopausal women. Furthermore, 50 mg of DHEA per day for 6 months improved age‐related changes in fat mass, lean mass, and bone mineral density in older women and men 56 , 57 . However, a systematic review and meta‐analysis of randomized controlled trials indicated that DHEA administration reduced fasting plasma glucose levels but not insulin resistance 58 . In contrast, other studies have demonstrated that DHEA treatment does not affect insulin secretion 59 or lipid profiles 60 . DHEA supplementation did not affect cardiovascular parameters, arterial stiffness, or endothelial function in women with hypoadrenalism or hypopituitarism 61 , 62 . Thus, further clinical trials with larger sample sizes are needed to confirm the effect of DHEA on glucose metabolism and health.This study had several limitations. First, the causal association of DHEA with diabetic retinopathy could not be determined because of the study's cross‐sectional design. Second, we recruited hospitalized patients with type 2 diabetes mellitus; thus, these results should be confirmed before generalizing them to the general diabetic population. Third, we did not assess the association between low DHEA levels and the severity of diabetic retinopathy, especially sight‐threatening diabetic retinopathy because of the small number of patients with severe diabetic retinopathy. Finally, this study's sample size was relatively small, potentially limiting the power to test subgroup interactions.In conclusion, low serum DHEA levels were significantly associated with diabetic retinopathy in adult patients with type 2 diabetes mellitus in northern China, suggesting a potential role of DHEA in the pathogenesis of diabetic retinopathy. Further prospective studies are necessary to confirm these results and to identify the mechanisms underlying associations between DHEA and diabetic microvascular complications.DISCLOSUREThe authors declare no conflict of interest.Approval of the research protocol: The study protocol was approved by the institutional review board of Tianjin Medical University General Hospital (approval number: IRB2020‐YX‐027‐01).Informed consent: The requirement for informed consent was waived because the data were gathered from electronic medical records, and the participants’ identities were anonymized.Approval date of registry and the registration no. of the study: N/A.Animal studies: N/A.

PMC

Diabetes, Metabolic Syndrome and Obesity

PMC10821666

1-23-2024

10.2147/DMSO.S449374

Relationship Between Cardiometabolic Index and Insulin Resistance in Patients with Type 2 Diabetes

Wu Limin, Xu Jing

PurposeCardiometabolic index (CMI) has been suggested as innovative measures for assessing the cardiometabolic status. However, there is a lack of relevant studies on exploring the relationship between CMI and insulin resistance (IR). Consequently, this study aims to examine the relationship between CMI and IR in subjects with type 2 diabetes mellitus (T2DM).Patients and MethodsA cross-sectional study was performed on 2493 patients with T2DM (including 1505 males and 988 females). IR was measured through the homeostatic model assessment of insulin resistance (HOMA-IR), which was defined as HOMI-IR≥2.69. The relationship between CMI and IR was evaluated with Spearman's correlation, ROC analysis, multiple logistic regression, generalized smooth curve fitting and subgroup analysis.ResultsCMI was correlated with HOMA-IR in patients with T2DM (Spearman correlation coefficient = 0.391 in females and 0.346 in males, P<0.001). Through the multiple logistic regression analysis, CMI was significantly correlated with IR (OR=1.30, 95% CI=1.15–1.47 in males and OR=1.62, 95% CI=1.32–1.99 in females). In addition, a non-linear correlation between CMI and IR risk was identified. The AUC of CMI (AUC = 0.702 for males and 0.733 for females, all p < 0.01) was the largest compared with traditional indexes of adiposity and blood lipids. According to the subgroup analysis, the two had a more significantly positive correlation in females, the elderly and subjects with HbA1c < 7%.ConclusionIn patients with T2DM, elevated CMI is significantly correlated with IR, as a useful index of IR.

IntroductionGiven that IR is recognized as a prominent factor in various pathological conditions, including diabetes mellitus, atherosclerosis, hypertension and metabolic syndrome (MetS), it is imperative to accurately assess IR. The hyperinsulinemic-euglycemic clamp is considered as a gold standard for measuring IR in studies.1 However, its practicality is limited for routine clinical use due to issues of replicability, cost and accessibility.1–5 As a substitute, HOMA-IR is commonly employed in adults.6 Nevertheless, the calculation of HOMA-IR necessitates the measurement of fasting plasma insulin, which is not typically performed in clinical settings. Therefore, an accurate, simple and cost-effective diagnostic test is needed to predict IR.The correlation between obesity, dyslipidemia and IR has been extensively documented in the literature.7,8 Specifically, the TG/HDL-C ratio has emerged as a valuable index for predicting the susceptibility to NAFLD and effectively evaluating IR in NAFLD.9–11 The Waist-to-height ratio (WHtR) is a composite measure that incorporates both height and waist circumference (WC). Compared with the conventional single-body measurement index, WHtR demonstrates superior capability in identifying abdominal obesity, assessing cardiac metabolic risk, IR and various noncommunicable diseases.12–15 Recently, Wakabayashi et al introduced a novel index known as CMI.16 CMI is calculated by multiplying WHtR with the TG/HDL-C ratio, thereby integrating lipid and obesity parameters into a straightforward and reproducible marker for the effective detection of NAFLD and diabetes.16,17 Furthermore, a number of recent studies have demonstrated a strong relationship between CMI and various diseases affecting people’s health conditions, such as stroke, hypertension, kidney diseases and cardiovascular diseases.18–21 These findings suggest that CMI is of great values as an index for metabolic diseases. However, there is currently a lack of relevant studies on exploring potential links between CMI and IR.Therefore, this study aims to explore the relationship between IR and CMI in patients with T2DM through a comprehensive cross-sectional analysis and to determine whether CMI can be used as a novel and practical biomarker for diagnosing IR.Materials and MethodsStudy DesignIn this cross-sectional study, 2493 patients with T2DM admitted to the Department of Endocrinology of the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University between January 2020 to August 2022 were included. The study has received the approval from the hospital’s ethical review committee (Approval Number: LCKY2020-01), and written consent was obtained from all patients with T2DM following the Declaration of Helsinki. Inclusion criteria encompassed a T2DM diagnosis based on WHO criteria, age of 20 years or older, and the availability of complete clinical and biochemical parameters data. Exclusion criteria included were Patients with acute complications (diabetic ketoacidosis, diabetic hyperosmolar coma, or lactic acidosis); Patients with recent history of surgery, trauma, severe infection, immune diseases, and malignant tumors; Patients with T1DM, pregnancy, previous history of hyperthyroidism, hypothyroidism, cardiovascular diseases, kidney, liver and muscle diseases.Anthropometric MeasurementsThe data collected upon admission included a history of hypertension, duration of diabetes mellitus (DD), hypoglycemic drugs, smoking habits, alcohol intake and physical measurements such as blood pressure, height, weight and WC. Specifically, the definitions of hypertension, smoking and alcohol status have been previously described in this study.22 Obesity and overweight were defined as BMI ≥ 28 kg/m2 and 24kg/m2≤BMI <28 kg/m2, respectively.Biochemical MeasurementsBlood samples were collected in patients on an empty stomach and 2h after breakfast on the second day of admission. LDL-C, alanine aminotransferase (ALT), glutamyl transpeptidase (GGT), HbA1c, UA, 2-h PPG, TC, 2-hour postprandial C-peptide (2h PCP), fasting C-peptide (FCP), albumin, TG, creatinine, HDL-C, FPG and aspartate transaminase (AST) were determined as previously described.22 Total number of missing values was less than 2%. Multiple imputation was performed for missing values.Assessment of IRHOMA-IR formula was used to evaluate IR. HOMA-IR=1.5+FPG [mmol/L] × FCP [pmol/L]/2800.23 IR was defined as HOMI-IR≥2.69, based on an epidemiology survey conducted in China.24,25Cardiometabolic IndexIt was worth noting that CMI was considered a continuous variable, and its calculation involved the formula [WC (cm)/height (cm)]×[TG (mmol/L)/HDL-C (mmol/L)].16The non-insulin-based markers of IR were calculated based on previously reported formulas, as follows:26Visceral Adiposity Index (VAI) was calculated as follows:For males: VAI = WC/(39.68 + (1.88 × BMI)) × (TG/1.03) × (1.31/HDL-C);For females: VAI = WC/(36.58 + (1.89 × BMI)) × (TG/0.81) × (1.52/HDL-C).Dysfunctional Adiposity Index (DAI) was calculated as follows:For males: DAI = WC/(22.79 + (2.68 × BMI)) × (TG/1.37) × (1.19/HDL-C);For Females: DAI = WC/(24.02 + (2.37 × BMI)) × (TG/1.32) × (1.43/HDL-C).Statistical AnalysisDifferences in WhtR and CMI were observed between genders, leading to separate analyses of males and females. The normal distribution of the data were determined through Kolmogorov–Smirnov tests. For continuous variables, normally distributed data were expressed by means and standard deviations (SDs), while asymmetrically distributed data were expressed by medians (interquartile ranges, IQRs). To compare the two groups, the Mann–Whitney U-test or t-test was adopted for continuous variables, while chi-square test was employed for categorical variables. In addition, Spearman's correlation was utilized to explore the relationship between CMI and metabolic risk factors. Patients were divided into quartiles based on their CMI levels (≤0.49, 0.49–0.79, 0.79–1.27, ≥1.27 in females, ≤0.53, 0.53–0.86, 0.86–1.54, ≥1.54 in males), with the first quartile representing the lowest one (as reference group) and the fourth quartile representing the highest. A binary logistic regression model was employed to examine the relationship between CMI quartiles and IR. In Model 1, no covariate was adjusted. Model 2 was adjusted for BMI and age. Based on Model 2, DD, hypoglycemic drugs, SBP, DBP, HbA1c, serum creatinine, serum albumin, uric acid, ALT, AST, GGT, drinking and smoking were added to Model 3. Subgroup analysis was conducted to stratify the patients according to HbA1c, BMI, gender and age. In order to identify the potential non-linear relationship between CMI and IR probabilities, the generalized smooth curve fitting were adopted. The diagnostic efficacy of CMI in detecting IR was evaluated through the ROC curve analysis. The statistical analysis was conducted with EmpowerStats software and R, with the significance determined at P < 0.05.ResultsCharacteristics of ParticipantsAs depicted in Table 1, the prevalence of IR was found to be 18.1% of males and 21.1% of females. The HOMA-IR, proportion of subjects with hypertension and hypoglycemic drugs, weight, WC, BMI, WhtR, SBP, FPG, ALT, AST, GGT, creatinine, FCP, 2-h PCP, TG, uric acid and CMI levels was observed to be higher in patients with IR compared to those without IR of both genders (P<0.001). Female subjects with IR were found to be significantly older than the non-IR subjects (P<0.001). Additionally, IR subjects exhibited lower levels of LDL-C and HDL-C in comparison to non-IR subjects.Table 1Baseline Characteristics of the T2DM Patients Stratified by Insulin Resistance and GenderMaleP-valueFemaleP-valueIR positiveIR NegativeIR PositiveIR NegativeN2311274172816Age, years54.6±16.655.3±14.10.52666.6±13.861.3±13.5<0.001Duration of diabetes, year5.0±6.24.2±5.60.0408.8±7.08.5±7.10.117Hypertension, n (%)58.642.0<0.00167.648.3<0.001Height, cm169.8±6.3169.0±7.30.113155.9±6.0156.8±6.80.116Weight, kg76.9±15.369.6±11.8<0.00162.7±10.459.0±10.1<0.001Body mass index, Kg/m226.6±4.424.5±10.5<0.00125.7±3.624.1±7.30.006Waist circumference, cm92.7±12.788.2±22.20.00388.5±10.584.6±11.1<0.001Waist-to-height ratio0.55±0.070.52±0.130.0090.57±±0.070.54±0.08<0.001Systolic blood pressure, mmHg146.4±23.6139.1±26.7<0.001149.4±24.5141.5±27.80.001Diastolic blood pressure, mmHg87.4±7.284.2±25.00.05182.5±1.781.7±7.90.264Current smoking, %40.241.81.00001.30.601Current drinking, %11.310.20.7261.800.504Metformin, %53.537.4<0.00166.753.5<0.001α-glucosidase inhibitors, %36.126.1<0.00149.029.1<0.001Thiazolidinediones, %8.82.1<0.00111.85.8<0.001Hemoglobin A1c, mmol/L8.9±2.29.5±2.30.0018.6±1.79.5±2.3<0.001FPG, mmol/L8.1±2.36.3±1.7<0.0018.3±2.06.8±2.1<0.0012-h PPG, mmol/L16.0±3.816.4±3.80.22216.9±4.017.2±3.90.360FCP, ng/mL1.99±0.800.66±0.37<0.0011.94±0.730.65±0.37<0.0012-h PCP, ng/mL4.63±2.512.53±1.82<0.0014.68±2.542.42±1.80<0.001HOMA-IR3.32±0.692.00±0.29<0.0013.34±0.642.02±0.30<0.001Albumin, g/dl41.0±5.540.4±4.20.08141.4±6.239.8±3.6<0.001Creatinine, umol/L100.9±79.174.6±28.9<0.00173.1±40.657.2±32.8<0.001Uric acid, umol/L421.1±117.3355.9±98.5<0.001377.4±123.8300.4±89.3<0.001ALT, U/L38.8±45.730.7±39.00.00629.6±21.723.3±28.00.007AST, U/L31.9±40.725.1±19.6<0.00127.6±16.322.4±13.6<0.001GGT, U/L66.2±83.951.5±117.30.07650.1±60.129.6±49.4<0.001Total cholesterol, mmol/L4.60±1.494.56±1.360.6294.58±1.374.68±1.200.315Triglycerides, mmol/L2.88±3.241.98±1.98<0.0012.57±1.981.74±1.15<0.001HDL-cholesterol, mmol/L0.94±0.330.99±0.280.0191.00±0.291.14±0.31<0.001LDL-cholesterol, mmol/L2.65±1.032.84±1.080.0132.66±1.152.90±1.060.007CMI1.78±1.681.18±1.23<0.0011.63±1.420.93±0.76<0.001Notes: Values are mean±SD or number (%). P<0.05 was deemed significant (comparison between IR positive and IR negative).Abbreviations: FPG, fasting plasma glucose; HDL-c, High density lipoprotein cholesterol; ALT, alanine aminotransferase; GGT, glutamyl transpeptidase; AST, aspartate transaminase; LDL-c, Low density lipoprotein cholesterol; 2-h PPG, 2-h postprandial plasma glucose; 2h PCP, 2-hour postprandial C-peptide; FCP, fasting C-peptide; HOMA, homeostatic model assessment of insulin resistance; CMI, cardiometabolic index.Correlation Between CMI and Metabolic ParametersThe correlation between CMI and the metabolic parameters can be seen in Table 2 with Spearman correlation. It was observed that in males, there was a positive correlation between CMI and BMI (r=0.429), WC (r=0.388), DBP (r=0.166), HbA1c (r=0.094), TC (r=0.228), FPG (r=0.098), FCP (r=0.363) and HOMA-IR (r=0.346). In females, BMI (r=0.322), WC (r=0.344), SBP (r=0.076), DBP (r=0.141), TC (r=0.152), FPG (r=0.141), FCP (r=0.397) and HOMAI-IR (r=0.391) were correlated with CMI (all P<0.001) (Table 2).Table 2Spearmen’s Correlation of CMI Levels with Clinical and Biochemical ParametersVariableMaleFemalerPrpBMI0.429<0.0010.322<0.001WC0.388<0.0010.344<0.001SBP0.0330.2010.0760.017DBP0.166<0.0010.141<0.001HbA1c0.0940.0010.0240.503TC0.228<0.0010.152<0.001LDL-C−0.0020.9460.0380.231FPG0.098<0.0010.141<0.001FCP0.363<0.0010.397<0.001HOMA-IR0.346<0.0010.391<0.001Abbreviations: BMI, body weight index; WC, waist circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; HbA1c, glycosylated hemoglobin; TC, total cholesterol; LDL-C, low density lipoprotein cholesterol; FPG, fasting plasma glucose; FCP, fasting C-peptide; HOMA, homeostatic model assessment of insulin resistance; CMI, cardiometabolic index.Correlation Between CMI and IRTable 3 presents the results of binary logistic analysis examining the relationship between CMI and IR in patients with T2DM. The analysis was adjusted for BMI, age (Model 2), DD, hypoglycemic drugs, SBP, DBP, HbA1c, serum creatinine, serum albumin, uric acid, ALT, AST, GGT, drinking and smoking (Model 3), higher CMI quartiles was correlated with an increased risk of IR (P<0.001). Moreover, a positive correlation was also observed in the non-linear relationship between CMI and IR assessed by smooth curve fittings (Figure 1).Table 3Association of the Insulin Resistance with CMI QuartilesCrude ModelModel IModel IIOR (95% CI)POR (95% CI)POR (95% CI)PMale Q1RefRefRef Q21.70 (0.98–2.96)0.0601.67 (0.96–2.91)0.0681.65(0.89–3.07)0.112 Q32.75 (1.63–4.63)<0.0012.72 (1.61–4.58)<0.0012.97(1.68–5.25)<0.001 Q44.70 (2.91–7.59)<0.0014.67 (2.87–7.61)<0.0013.91(2.19–6.98)<0.001Female Q1RefRefRef Q22.14 (1.12–4.08)0.0211.99 (1.04–3.82)0.0391.16(0.59–2.26)0.668 Q34.02 (2.20–7.38)<0.0013.54 (1.92–6.54)<0.0012.56(1.40–4.68)0.002 Q46.87 (3.82–12.38)<0.0016.45 (3.55–11.71©)<0.0012.87 (1.49–5.52)0.002Notes: Crude model: adjusted for none. Model I: adjusted for age and BMI. Model II: adjusted for age, BMI, DD, hypoglycemic drugs, SBP, DBP, HbA1c, serum creatinine, serum albumin, uric acid, ALT, AST, GGT, drinking, smoking.Abbreviations: BMI, body mass index; DD, duration of diabetes mellitus; SBP, systolic blood pressure; DBP, diastolic blood pressure; HbA1c, glycosylated hemoglobin; ALT, alanine aminotransferase; GGT, glutamyl transpeptidase; AST, aspartate transaminase; CMI, cardiometabolic index. Figure 1The smooth curve fit for the association between CMI and prevalence of IR. Solid redline represents the smooth curve fit between variables. Blue bands represent the 95% of confidence interval from the fit. Adjusted for: age, BMI, DD, hypoglycemic drugs, SBP, DBP, HbA1c, serum creatinine, serum albumin, uric acid, ALT, AST, GGT, drinking and smoking.Subgroup Analysis to Assess the Relationship Between CMI and IRTo evaluate the impact of subgroups on the relationship between CMI and IR, subgroup analyses were conducted based on age, BMI, HbA1c and gender (Table 4). It was found that the p values for the subgroups were less than 0.005. CMI was independently correlated with IR, and this independently relationship was more obvious in female patients with T2DM, with age≥60 years old and HbA1c<7%. In addition, when the non-linear relationship was characterized by smooth curve fittings, the positive correlation between CMI and IR did survive in most groups (Figure 2).Table 4Association Between CMI and Insulin Resistance Stratified by Age, BMI and HypertensionOR (95% CI) p ValueP for InteractionStratified by gender0.029 Male1.30 (1.15–1.47), <0.001 Female1.62 (1.32–1.99), <0.001Stratified by age0.016 Age<60 years old1.23 (1.08, 1.41), 0.003 Age≥60 years old1.74 (1.43, 2.12), <0.001Stratified by BMI0.142 BMI<24kg/m21.65 (1.33, 2.03), <0.001 BMI≥24kg/m21.26 (1.11, 1.42), <0.001Stratified by HbA1c0.020 HbA1c<7%2.33 (1.43, 3.80), 0.001 HbA1c≥7%1.35 (1.21, 1.50), <0.001Notes: Gender, age, BMI, hypertension (not adjusted for in the subgroup analyses), DD, hypoglycemic drugs, serum creatinine, serum albumin, uric acid, ALT, AST, GGT, drinking, smoking were adjusted.Abbreviations: BMI, body mass index; DD, duration of diabetes mellitus; ALT, alanine aminotransferase; GGT, glutamyl transpeptidase; AST, aspartate transaminase; CMI, cardiometabolic index. Figure 2Subgroups analysis for the association between CMI and prevalence of IR by gender, age, BMI and HbA1c level.The Predictive Value of CMI for IRThe ROC of CMI, TG/HDL, WHtR, VAI, DAI, BMI, WC and uric acid to diagnose IR is shown in Figure 3. Table 5 shows that the AUC for CMI in the ROC analysis was 0.702 (95% CI: 0.665–0.738) in males, 0.733 (95% CI: 0.690–0.777) in females, which was considerably higher than that of TG/HDL, WHtR, VAI, DAI, BMI, WC and uric acid (P < 0.001), suggesting that CMI may be a better index for IR than traditional indexes of adiposity and blood lipids, although its diagnostic accuracy is still somewhat limited.Table 5The Results of ROC Analysis of CMI, TG/HDL, WHtR, VAI, DAI, BMI, WC and Uric Acid for the Diagnosis of IRNutritional IndicesCut-offSensitivity (%)Specificity (%)AUC95% CIMale CMI0.7777.553.30.7020.665–0.738 TG/HDL1.4976.648.40.6520.615–0.688 WHtR0.5542.476.60.6120.571–0.653 VAI1.9377.448.00.6640.615–0.689 DAI1.3076.548.30.6590.610–0.684 BMI24.668.057.70.6580.620–0.697 WC98.869.388.00.6160.574–0.658 UA368.567.761.10.6740.634–0.714Female CMI0.8974.861.90.7330.690–0.777 TG/HDL1.6366.362.10.6880.645–0.731 WHtR0.5279.740.40.6120.568–0.656 VAI3.0968.661.80.6890.642–0.729 DAI1.7968.661.20.6850.638–0.725 BMI23.773.350.60.6380.595–0.681 WC81.879.140.70.5990.555–0.643 UA347.555.274.70.6890.642–0.737Abbreviations: CMI, cardiometabolic index; TG/HDL, triglycerides/ high density lipoprotein cholesterol; WhtR, waist-to-height ratio; VAI, visceral Adiposity Index; DAI, dysfunctional Adiposity Index; BMI, body mass index; WC, waist circumference; UA, uric acid. Figure 3Receiver operating characteristic curves of TG/HDL, WHtR, VAI, DAI, BMI, WC and uric acid to identify IR.DiscussionIn this study, compelling evidence were presented supporting a positive correlation between CMI and an increase in HOMA-IR and the risk of IR among a large cohort of patients with T2DM. This relationship remains consistent across various demographic factors, including age, BMI, gender and HbA1c. Furthermore, the findings reveal a non-linear relationship between CMI and the risk of IR.The analysis on the ROC indicates that CMI outperforms TG/HDL, WHtR, VAI, DAI, BMI, WC and uric acid in detecting IR, suggesting that CMI serves as a highly specific and sensitive marker for IR. CMI represents a novel clinical marker that combines the measurements of TG/HDL-C ratio and WHtR. Wakabayashi et al discovered it for the first time in 2015, which proved its important role in evaluating DM.16 Subsequent investigations have expanded upon this finding, revealing a strong correlation between CMI and conditions such as cardiovascular disease, hypertension, kidney disease and stroke,18–21 which indicates its potential as a metabolic disease index. Nevertheless, there is currently a dearth of data regarding the relationship between CMI and IR. In this sense, this study has successfully confirmed the relationship between CMI and IR in patients with T2DM, utilizing a substantial sample size for the first time.The distribution of body fat accumulation plays a significant role in the development of DM and progression of IR, as indicated by previous studies.27,28 Previous studies have established a robust correlation between various conventional obesity indexes, such as BMI and WC, and IR. Moreover, the WHtR and WC have been proposed as superior measures to BMI in identifying cardiovascular risk factors, including IR and T2DM.13,14 The TG/HDL-C ratio is a reliable and simple measurement of IR that has been extensively studied in relation to T2DM.13,29,30 In summary, when combined with WHtR and TG/HDL-C, it is believed that CMI can provide a comprehensive assessment of obesity and dyslipidemia, thus making it a practical tool for identifying IR. As shown in Figure 3, the results showed that CMI had the largest AUC compared with TG/HDL, WHtR, BMI and WC, indicating its superior performance in detecting IR.Previous studies have also highlighted the relationship between CMI and various metabolic-inflammatory diseases. For instance, a study conducted by Ichiro Wakabayashi et al examined a cohort of 10,196 subjects undergoing health check-ups and found a positive correlation between elevated CMI values and hyperglycemia and risk of diabetes.16 Zou et al discovered that CMI could effectively predict NAFLD in general population in Japanese.31 Luo et al found that high CMI values were positively associated with incident cardiovascular disease in patients with obstructive sleep apnea and hypertension.32 Sun et al investigated 11,956 rural residents in China and found that CMI is independently and positively associated with the risk of ischemic stroke.20 Another study by Alleva demonstrated a significant positive correlation between CMI and metabolic syndrome in females suffering from obesity.33 The findings align with these previous studies, as a correlation between CMI and various factors including BMI, WC, DBP, TC and FPG was observed in patients with T2DM, albeit the correlation is not strong. These results highlight the potential value of CMI in future clinical applications and warrant further promotion.Age, HbA1c and BMI were significantly correlated with HOMA-IR. Therefore, further subgroup analysis showed a significant interaction between HbA1c levels, age and gender between CMI and IR risk. The study revealed that the correlations were more pronounced in female participants, those who were older, and those with HbA1c levels below 7%. Importantly, for the above population, IR is often neglected. Consequently, CMI should be recognized as a crucial determinant for identifying IR, particularly in this population.IR was associated with long term damage to organs, especially eyes, kidney, nerves and the heart in patients with T2DM.34 CMI usage is simple and low cost, which has a strong correlation with IR and has some predictive power. This allows clinicians to find IR in a timely manner in clinical work to delay or even prevent the development of diabetes mellitus. It will improve the T2DM patient’s life and life treatment and save economic costs.The specific mechanism through which CMI contributes to IR remains unclear. The observed relationship between abnormal lipid metabolism and subjects with assessed CMI may provide an explanation for these findings. In the case of obese subjects with a high WhtR, an excess of free fatty acids can impede insulin’s function in glucose metabolism, thereby leading to the development of IR.35 Additionally, subjects with abdominal obesity may experience a decrease in binding affinity and reduction in the quantity of insulin receptors on target tissues, resulting in a diminished capacity to respond to glucose.36,37 Moreover, an elevated triglyceride (TG) status plays a role in the development of IR similar to that of abdominal obesity. Additionally, reduced levels of HDL-C may adversely affect the functioning of beta cells, and lead to decreased insulin output and sensitivity.37,38 In summary, there exists a “vicious cycle” between IR and high CMI.The advantage of this study is that we have well characterized the subjects on a large population basis and tested whether there are differences in CMI and IR among different populations through subgroup analysis, thereby enhancing the dependability of the findings. Nevertheless, certain limitations should be acknowledged. Firstly, the establishment of a causal relationship between CMI and IR is precluded by the utilization of cross-sectional research design. Secondly, the adoption of the HOMA-IR was proposed as an evaluation tool. However, it is important to note that HOMA-IR has been associated with FPG, which exhibits a strong correlation with hepatic IR, but not to muscle IR.39 Additional research is necessary to investigate the relationship between CMI and IR with the gold standard hyperinsulinemic-euglycemic clamp. Moreover, it is important to note that the research population was limited to subjects with T2DM. Consequently, a prospective cohort study involving a larger and more diverse population, including subjects without DM, is needed to validate and promote the current findings, and this correlation with IR that could support further evidence for the treatment of diabetes in primary prevention.ConclusionIn summary, the extensive cross-sectional study demonstrates that CMI serves as a novel and useful biomarker for biochemical and anthropometric parameters, exhibiting an independent and positive correlation with IR, and appears to have higher IR AUC values than traditional indexes of adiposity and blood lipids.

PMC

Indian Journal of Occupational and Environmental Medicine

PMC10257243

Jan-01-2023

10.4103/ijoem.ijoem_163_22

Tobacco use and Oral Premalignant Lesions among Auto-Rickshaw Drivers in Belagavi, North Karnataka

Patil Amaresh P., Yogeshkumar S.

Background:Auto-rickshaw drivers (ARDs) are under constant physical and mental pressure due to illiteracy, poverty, lack of awareness about hazards of addictions, and other factors that lead to various habits majority being tobacco use. Studies have found that tobacco use is very prevalent among ARDs in comparison to general population. Tobacco use is commonly associated with cancers. Oral Pre-Malignant Lesions (OPMLs) are the strongest risk factor for majority of oral cancers. We studied the prevalence of OPML among ARDs of Belagavi and their association with tobacco use.Methods:It was a cross-sectional study conducted among 600 regular ARDs of Belagavi City during January to December 2016. We selected two ARDs that were the last in line from 300 major auto-rickshaw stands. We adapted the questionnaire from Global Adult Tobacco Survey questionnaire. After getting informed consent, we collected the data by personal interview and performed an oral visual examination for OPML for all the study participants. Data were analyzed using SPSS software. Institutional Ethics Committee approved the study.Results:Prevalence of tobacco was 62.17%. One-third of participants (30.17%) had OPMLs. Leukoplakia (62.43%) was the most common lesion. OPMLs were significantly associated with tobacco use and duration of tobacco use.Conclusions:About 30% of ARDs had an OPML. Chewing tobacco, gutkha, lime with tobacco, and cigarette were significantly associated with OPML.

INTRODUCTIONThree-wheeled motor vehicles, commonly called auto-rickshaws, are the cornerstone of urban mobility. They continue to be the most prevalent mode of transportation in Tier II and III cities, where metros and application-based cab aggregators have not yet made their mark. Auto-rickshaw drivers (ARDs) work for prolonged hours and constitute an essential part of the urban informal sector in India. The constant physical and psychological stress caused by irregular shifts, fluctuations in fuel prices, long waiting hours, peer pressure, illiteracy, poverty, lack of knowledge about the hazards of tobacco, and other socioeconomic factors lead to various habits majority being tobacco use.Because of job-related sedentariness and idle breaks between drives, ARDs are more likely to use tobacco. A few studies have shown that tobacco use is highly prevalent ranging from around 64% to 84% among ARDs, compared to 28.6% among Indian adults aged 15 years and more.Oral premalignant lesions (OPMLs) are relatively common, occurring in about 2.5% of the general population and are an important target for cancer prevention. It is possible to detect them visually and they are readily amenable to screening and diagnosis. However, there is sufficient evidence to indicate that tobacco (smoking, smokeless tobacco, inhaled tobacco, and tobacco substitutes like pan masala or betel nut quid) triggers the cascade of premalignancy responsible for most oral lesions, including oral cancers. Here, we tried to determine the prevalence of tobacco use and OPML and the association between tobacco use and OPML among ARDs of Belagavi.MATERIALS AND METHODSStudy designIt was an observational, cross-sectional study conducted from January to December 2016 on registered ARDs in Belagavi city. Based on the estimated prevalence of tobacco use of 84% and an absolute error of 3%, a sample size of 597 was calculated and rounded to 600.We included those participants who were registered, regular ARDs aged 18 years or more and residents of Belagavi for at least one year and excluded those who drove part-time.Sampling procedureAs per the information from the regional transport office, the city had around 300 major auto-rickshaw stands. Following convenient sampling method, we visited all the 300 stands and among each stand, we purposively selected two ARDs who were last in the queue. In this way, the participants could be interviewed and examined for the study, which could not have happened if we selected someone else from the queue, as they would have had to discontinue the study if their auto-rickshaw was hired meanwhile.Study toolsWe collected the data using a questionnaire adapted from the Global Adult Tobacco Survey (GATS) and tailored to the study requirements. Investigator was trained by a preventive oncologist with an expertise in identifying OPML. Training spanned over a week and was imparted using pictorial representation and audio visual aids. Investigator performed an oral visual examination using a torch and disposable wooden sticks.Data collectionFollowing a written informed consent, we interviewed and examined the participants in the auto-rickshaw stands or any convenient place nearby. We gave free will to participants to discontinue the study at any time if they had privacy concerns, confidential concerns, or concerns related to their work. We ensured that the participants had no hindrance in their work.Ethical considerationsThe Institutional Ethics Committee of J. N. Medical College of KLE University, Belagavi approved the study with the approval letter numbered MDC/DOME/379 dated November 19, 2015. We counseled tobacco users to quit after the study concluded and offered help to those willing to quit either with counseling or through tobacco cessation clinics. Those unaware of or misinformed about tobacco hazards received the correct information through interpersonal communication.Data analysisWe analyzed the data through descriptive statistics and a Chi-squared test using the SPSS software. P <.05 was accepted as the statistical significance value.RESULTSThe mean age of the participants was 39.71 ± 11.07 years [Table 1]. The mean duration in the present occupation was 15.80 years ± 10.11 years and participants worked over a length of 9.43 ± 1.82 hours per day. Majority of them (89.83%) were married, 371 (61.83%) stayed in nuclear families, and 346 (57.67%) resided in pucca houses. Among the participants, the prevalence of current use of tobacco was 62.17% . Cigarettes (87.05%) were the most commonly smoked form, while gutkha (54.93%) was the most common smokeless form. Most users had initiated their habit before 20 years of age (46.38%). The mean age at initiation was 23.22 ± 8 years for smoke form and 23.36 ± 7.99 years for smokeless form. The mean duration of use was 15.31 ± 10.29 years.Table 1Sociodemographic and occupational profile of participants (n=600)Characteristicsn (%)Age (years) < 30159 (26.5) 31-40175 (29.17) 41-50164 (27.33) 51-6090 6012 Religion Hindu143 (23.83) Muslim457 (76.17)Educational qualification Illiterate49 (8.17) Primary School141 (23.5) High School327 (54.5) Pre university college59 (9.83) Diploma/Graduate24 Socioeconomic status (Modified BG Prasad SES Scale) Class I17 (2.83) Class II65 (10.83) Class III196 (32.67) Class IV265 (44.17) Class V57 (9.5)Number of years in the present occupation < 10235 (39.17) 11-20189 (31.5) 20176 (29.33)Length of working hours on an usual day 6-8 hours51 (8.5) 8-10 hours218 (36.33) 10-12 hours191 (31.83) ≥ 12 hours140 (23.34)Usual number of night shifts per week 0582 ≥ 118 Among the participants, 181 (30.17%) had one or the other OPML [Figure 1]. Leukoplakia was the most common OPML found in 113 (18.83%) participants, followed by oral sub mucus fibrosis in 84 (14%) participants [Figure 2]. Buccal mucosa was the most common site with OPML in the oral cavity observed among 146 (24.33%) participants, followed by vestibule in 35 (5.83%) participants.Figure 1Presence of Oral Premalignant lesions (n = 600)Figure 2Type of Oral Premalignant lesions (n = 600)OPMLs were significantly associated with tobacco use, smokeless form, and smoke form. Among smokeless forms, chewing tobacco, gutkha, and lime with tobacco were significantly associated, whereas cigarettes were the only smoking form significantly associated with OPML [Table 2]. OPMLs were significantly associated with the duration of tobacco use, duration of smokeless tobacco use, and smoke form of tobacco [Table 3].Table 2Association between tobacco use and oral premalignant lesions among study participants (n=600)CharacteristicsOral Premalignant LesionsTotal χ 2 PresentAbsentAssociation of OPML with tobacco Tobacco user161 (43.16)212 (56.86)373 χ2=79.054, df=1, P<0.00001* Non user20 (08.81)207 (91.19)227 Total181 (30.17)419 (69.83)600 Association of OPML with smokeless tobacco Smokeless user123 (43.31)161 (56.69)284 χ2=44.217, df=1, P<0.00001* Non user58 (18.35)258 (81.65)316 Total181 (30.17)419 (69.83)600 Association of OPML with smoking form of tobacco Smokers55 (39.57)84 (60.43)139 χ2=7.591, df=1, P=0.0058* Non smokers126 (27.33)335 (72.67)461 Total181 (30.17)419 (69.83)600 Association of OPML with combined use of tobacco Combined users17 (34.00)33 (66.00)50 χ2=0.38, df=1, P=0.5376* Non users164 (29.82)386 (70.18)550 Total181 (30.17)419 (69.83)600 *Chi-squared test, P <0.05 is significantTable 3Association between duration of tobacco use and oral premalignant lesions among study participants (n=373)CharacteristicsOral Premalignant LesionsTotal χ 2 PresentAbsentAssociation of OPML with duration of tobacco use 20 years33 (35.48)60 (64.52)93 Total123 (43.31)161 (56.69)284 Association of OPML with duration of smoking 20 years02 (09.42)19 (90.48)21 Total55 (39.57)84 (60.43)139 *Chi-squared test, P <0.05 is significantDISCUSSIONARDs spend most of their day dealing with pollution, noise, continuous stress, accelerations and decelerations, lateral swaying, and whole-body vibrations when moving. Moreover, poor lifestyle practices such as irregular meals, poor quality toilets, absence of restrooms, inadequate water supply, poor posture while driving, and stressful driving conditions contribute to their health problems. Such working conditions may be associated with various health issues.We conducted the study to find the prevalence of OPML among ARDs of Belagavi and the association of OPML with tobacco use. All the 600 study participants were male, consistent with other studies. Most of the participants in this study had a high school education (54.50%). The educational level of participants was higher when compared with other similar studies. We can attribute the increase in education to better literacy levels in south India.Mean years in present occupation was 15.80 ± 10.11 years, whereas it was 17.70 ± 7.62 years in the study done in Nagpur. The mean length of working hours per day was 9.43 ± 1.82 hours, while it was 11.52 ± 2.29 hours in the study done in Nagpur.The prevalence of tobacco use in the present study was 62.17%. The study among ARDs in Gwalior showed the prevalence to be 84.26%, 64.44% among ARDs in Bareilly, Uttar Pradesh, and 69% among ARDs in South Delhi. We can attribute the difference in prevalence with the Gwalior study to the fact that the participants in the present study had higher education and the Gwalior study found a negative association between educational level and tobacco use. These studies reveal a comparatively high prevalence among ARDs compared to Indian adults aged 15 years and more, which was just 28.6%, as per GATS 16-17. Also, the study found a much higher prevalence compared to Belgaum city, where it was 29.41%.We can attribute this comparatively higher prevalence among ARDs to various occupational factors coupled with low levels of education, poverty, lack of knowledge about hazards, other socioeconomic factors, and many unknown factors.The most frequently reported form of tobacco was the smokeless form. Similar results are found in multiple studies done among similar subgroups and agree with the GATS survey done among adult Indian males. This is also particularly true in ARDs, as their hands are preoccupied with driving and smoking would be cumbersome while driving, whereas chewing can be continued even while driving.About 30% of ARDs had OPML, consistent with a study conducted in Mumbai among taxi drivers, which found 35% had precancerous oral conditions. However, a study conducted in rural areas of Belgaum found that only 12.4% had oral lesions. Prevalence varies based on epidemiological factors such as gender, age, and risk factors.In the present study, around 62.43% of those with OPML had leukoplakia, 46.41% had oral sub mucus fibrosis, only 1.65% had erythroplakia, 3.87% had leukoeryhtroplakia, 11.05% had nonhealing ulcer in the oral cavity, and 7.73% had melanoplakia similar to another study.In the study, tobacco use, smokeless tobacco use, and smoke form were significantly associated with OPML, which was insignificant in the combined use of tobacco. Numerous medical literature and studies have cited tobacco as one of the most frequently reported etiological factors for OPML. A review of Indian literature found a strong association between tobacco chewing and different OPML such as leukoplakia and erythroplakia. Cigarette smoking is a significant risk factor for OPML, mainly leukoplakia.The duration of any form of tobacco use was significantly associated with OPML. However, no specific trend was observed. A retrospective study done in India concluded that an increase in the duration of tobacco consumption increases the risk of developing oral cancer. Another study done in rural Maharashtra highlighted that the prevalence of leukoplakia increased with the increase in the duration of the chewing form of tobacco. As per a study done in Taiwan, the annual incidence rate of leukoplakia was higher with high consumption and long duration of smoking.LIMITATIONS AND RECOMMENDATIONSBesides social desirability bias, we might have missed ARDs plying exclusively at night. We could not do regression analysis that would have predicted true association between duration of tobacco use and OPML. We propose establishing a workplace tobacco cessation model for this group and targeting tobacco cessation activities for young ARDs to prevent future addiction and health hazards. To intervene at the right time, we have to continuously follow-up and treat ARDs with OPML. There is a need for strict implementation of legislations governing tobacco use.Financial support and sponsorshipNil.Conflicts of interestThere are no conflicts of interest.

PMC

Asian Biomedicine: Research, Reviews and News

37551369

PMC10388770

10.2478/abm-2021-0015

Placental α-microglobulin-1 in cervicovaginal fluid and cervical length to predict preterm birth by Thai women with symptoms of labor

Chawanpaiboon Saifon, Titapant Vitaya, Pooliam Julaporn

AbstractBackgroundPresence of placental α microglobulin-1 (PAMG-1) in cervicovaginal fluid is a bedside test to predict preterm delivery.ObjectiveTo determine whether the accuracy of a positive PAMG-1 test result to predict preterm birth within 7 days and 14 days in our hospital setting can be improved by adding cervical length.MethodsWe recruited 180 pregnant women who attended the labor ward of Siriraj Hospital, Thailand, from 2016 to 2018 for this prospective observational study of diagnostic accuracy. We used data from 161 women who met inclusion criteria including symptoms of preterm labor between 200/7 and 366/7 weeks’ gestation without ruptured membranes and with cervical dilatation <3 cm and effacement <80%. Presence of PAMG-1 in cervicovaginal fluid was tested using a PartoSure kit, cervical length was measured by transvaginal ultrasound, and the time to spontaneous delivery was calculated.ResultsPregnant women with labor pain who had cervical length <30 mm (45/161; 28%) went into delivery within 7 days, and women with a cervical length <15 mm (11/14; 79%) went into delivery within 7 days. When the PAMG-1 test result was positive and cervical length was ≤15 mm, the positive predictive value (PPV) was 83%; and when cervical length was ≤30 mm the PPV was 69%. The optimal cut off from receiver operating characteristic curve analysis showed that a cervical length <25 mm and PAMG-1 positive result has a PPV of 80% to predict preterm birth within 7 days and 90% within 14 days. The area under the curve (95% confidence interval) for a positive PAMG-1 result and cervical length ≤25 mm to predict preterm birth <7 days was 0.61 (0.50, 0.73) and <14 days was 0.60 (0.49, 0.70).ConclusionsCervical length ranging 15–30 mm combined with a positive PAMG-1 test result has a high accuracy to predict imminent spontaneous delivery within 7 days by women with preterm labor and cervical dilatation <3 cm in clinical practice.

Preterm or premature birth remains a major problem worldwide, and the trend of preterm birth has increased in many developed countries at least up to 2007 . The World Health Organization (WHO) defines preterm birth as all births before 37 completed weeks of gestation . Short and long-term complications of prematurity are reported . The statistical unit of the Department of Obstetrics and Gynecology at Siriraj Hospital has reported that the annual incidence of preterm births steadily increased from 9.4% in 2004 to 13.7% in 2010 . The 2015 preterm birth rate for the USA (based on obstetric estimate of gestational age) was 9.63% . Spontaneous preterm birth in the UK occurs in 7%–12% of pregnancies before 37 weeks’ gestation and in about 4% of pregnancies before 34 weeks’ gestation . In most developed countries, 60%–80% of neonatal mortality and 75% morbidity are from preterm birth . There is support for steroid therapy to reduce respiratory morbidity [7, 8, 9] and tocolytic agents to inhibit uterine contraction and postpone delivery . Corticosteroid treatment shows maximum benefit from 24 h after administration . However, preterm birth is difficult to diagnose and this results in overtreatment of threatened preterm labor, which was modeled to have a high cost of 4,653 EUR per case in 2013 (USD 6,179; Federal Reserve Foreign Exchange Rate G.5A), at least in The Netherlands . Several agents with different modes of action have been used to inhibit uterine contractility with a maximum time of 136 days in Germany . In 50%–80% of pregnant women, preterm labor does not lead to preterm birth within 7 days and 50%–70% of women in this group who received a placebo gave birth close to term . The sign of preterm labor alone has a false positive rate >50% , which may lead to unnecessary hospital admission for preterm labor management.Methods to predict spontaneous preterm birth include cervical examination for dilatation and length [14, 15, 16], and testing cervicovaginal fluid for fetal fibronectin [6, 10], prolactin , and phosphorylated insulin-like growth factor binding protein 1 (phIGFBP-1), also known as placental α microglobulin-1 (PAMG-1) [18, 19], are effective in excluding spontaneous preterm delivery within 7–14 days. An ideal predictor would be a test that identifies who would deliver within 48 h to 7 days with the purpose of guiding the rational use of antenatal corticosteroids and tocolytic agents, decreasing unnecessary admissions, and correcting triage of patients that need in utero transfer [14, 15].PAMG-1 was initially referred to as a specific α-1 globulin of the placenta [20, 21], then as phIGFBP-1 , and was first isolated from human amniotic fluid as reported by Petrunin et al. in 1976 (cited by reference 1 in ). PAMG-1 is found in the amniotic fluid, blood, and vaginal discharge of pregnant women [18, 23]. The concentration of PAMG-1 is several thousand-fold higher in amniotic fluid than it is in cervicovaginal secretions . Therefore, PAMG-1 is an important biomarker for premature rupture of the fetal membrane (PROM). Moreover, during uterine contractions, PAMG-1 passes through chorioamniotic pores in fetal membranes, and through microperforations from degradation of the extracellular matrix of fetal membranes, possibly as a result of the inflammatory processes of labor or infection [18, 22].The predictive accuracy of PAMG-1 is similar to that of qualitative fetal fibronectin [21, 24, 25]. However, to our knowledge, only one study has shown PAMG-1 to have a higher positive predictive value (PPV) than fetal fibronectin for predicting preterm labor . A systematic review and meta-analysis showed that PAMG-1 is highly predictive of preterm birth within 7–14 days of preterm labor .Cervical length measurement in symptomatic women can be used to predict preterm labor to shorten hospital stay without compromising patient care . Cervical length measurement, fetal fibronectin, and uterine contraction monitoring during pregnancy have been proposed to predict preterm delivery, but their practical beneficial remains uncertain [16, 26]. A cervical length >30 mm or a negative fibronectin obtained from a patient with possible preterm labor can avoid overdiagnosis and unnecessary treatment . However, a woman with a cervical length 2,000 beds in Bangkok, Thailand. All participants provided their written informed consent before entering the study.The included patients were those with a singleton pregnancy who had symptomatic painful uterine contraction every 4 times in 20 min or 8 times in 60 min with a cervical opening 30 mm, or a negative PAMG-1 test result, was not considered be at risk of imminent spontaneous delivery within 2 days, 7 days, or 14 days, and by contrast, those patients with a cervical length of ≤30 mm, or a positive PAMG-1 test result, were considered to be at risk of imminent spontaneous delivery within 7 days or 14 days.Statistical analysisWe estimated the sample size for adequate sensitivity and specificity with a 95% confidence interval (CI) and 15% error of detected sensitivity using in house software from Mahidol University, Bangkok, Thailand. All other statistical analyses were completed using PASW Statistics for Windows (version 18.0; SPSS). Data are expressed as mean ± standard deviation (SD), median (range) for continuous variables, and frequency (percentages) for categorical variables. The diagnostic accuracy of PAMG-1, cervical length, and a combination of the 2 indicators for predicting preterm birth was assessed using receiver operating characteristic (ROC) curves and determining the area under the curve (AUC). The sensitivity, specificity, PPV, NPV, and accuracy with 95% CI were calculated.ResultsFrom the total of 180 pregnant patients recruited, 19 were excluded from the study because of indicated deliveries (10 patients), incomplete data for cervical assessment (5 patients), and loss to follow-up (4 patients). Therefore, we included 161 patients in the study (Figure 1). All 161 patients underwent a PAMG-1 test and cervical length measurement. Their mean age was 28.9 years with an average body weight of 65 kg. Most of the patients were primigravid. Gestational age was calculated from ultrasound in about 43%. Some 145 (91%) patients went into spontaneous vaginal delivery; other deliveries were assisted or by cesarean section (Table 1).Figure 1Flow of participants.Table 1Patient demographic characteristics Demographic data n = 161 Maternal age (years)28.9 ± 6.9Body weight (kg)65.0 ± 12.5Height (cm)157.7 ± 6.2BMI (kg/m2)26.2 ± 4.8Income (Thai baht per month)*20,000 (0–70,000) Occupation Government19 (12%) Housewife35 (22%) Labor86 (53%) Merchant11 (7%) Own business4 (3%) Student6 (4%) Gravida1 (1, 6) Parity0 (0, 5) Abortion0 (0, 3) Gestational age at first visit (weeks)10 (4, 34) Gestational age at admission (weeks)33.2 (21.1, 36.3) Number of antenatal care10 (0, 12)Calculate gestational age by source LMP34 (21%) LMP/ultrasound58 (36%) Ultrasound69 (42%)Maternal medical illness None144 (89%) Gestational diabetes mellitus2 (1%) Hypertension3 (2%) Pulmonary disease5 (3%) Thyroid disease7 (4%) Iron deficiency13 (8%)Hemoglobinopathy None121 (75%) α-Thalassemia trait8 (5%) β-Thalassemia trait1 (1%) Hemoglobin E trait28 (17%) Homozygous hemoglobin E3 (2%) VDRL reactive†1 (1%) Anti-HIV positive‡2 (1%) HBS Ag positive§25 (16%)Maternal complication None137 (85%) Gestational diabetes mellitus11 (7%) Gestational hypertension5 (3%) Mild preeclampsia2 (1%) Severe preeclampsia6 (4%)Mode of delivery Cesarean section8 (5%) Spontaneous vaginal delivery147 (91%) Vacuum extraction6 (4%)Data are presented as n (%), mean ± SD, and median (range).*U.S. Federal Reserve Foreign Exchange Rate G.5A. 1 USD = 35.26 , 33.91 , 32.30 . Participants recruited March 2016 to September 2018.†VDRL reactive means positive screening test for syphilis.‡HBS Ag positive means positive screening test for antibodies to hepatitis B surface antigen.§Anti-HIV positive means positive screening test for antibodies to HIV. BMI, body mass index; HBS Ag, hepatitis B surface antigen; HIV, human immunodeficiency virus; LMP, last menstrual period; SD, standard deviation; VDRL, venereal disease research laboratory test.Some patients may have more than one type of thalassemia.Of the 161 symptomatic pregnant women with cervical length ≤30 mm, 34 (21%) went into spontaneous delivery within 7 days and 11 (7%) went into spontaneous delivery within 14 days (Table 2). Of the 14 symptomatic pregnant women with a cervical length ≤15 mm, 11 (79%) went into spontaneous delivery within 7 days (Table 3).Table 2Symptomatic patients with cervical length <30 mm and delivery within 7 days and 14 days All 161 patients included (symptomatic with cervical length <30 mm) Delivery <14 days45 (28%)Delivery <7 days34 (21%)Delivery 7–14 days11 (7%)Delivery ≥14 days116 (72%)Total161 (100%)Table 3Symptomatic patients with various cervical lengths and delivery within 7 days and 14 days All symptomatic patients with cervical length <30 mm Delivery <7 days n (%) Delivery <14 days n (%) Cervical length 30 mm (n = 97)9 16 When PAMG-1 testing was combined with cervical length measurement <30 mm, the NPV was 83% to predict preterm birth within 7 days, and 77% to predict preterm birth within 14 days (Table 4). When PAMG-1 testing and cervical length measurement ≤15 mm was used, the PPV was 83% (Table 5). The optimal cut-off from ROC curve analysis shows that a cervical length <25 mm and a PAMG-1 positive result has a PPV of 80% to predict preterm birth within 7 days and 90% to predict preterm birth within 14 days (Tables 6–8; Figures 2 and 3).Table 4Performance of a positive PAMG-1 test result and cervical length <30 mm in symptomatic patients to predict preterm birth within 7 days and 14 days Indicator Preterm birth <7 days Preterm birth <14 days Sensitivity (%) Specificity (%) PPV (%) NPV (%) Sensitivity Specificity PPV NPV PAMG-110/34 112/127 10/25 112/136 12/45 103/116 12/25 103/136 95% CI15, 4881, 9321, 6175, 8815, 4282, 9428, 6968, 83Cervical length 9955, 9869, 83PAMG-1 or cervical length <30 mm26/34 77/127 26/76 77/85 30/45 70/116 30/76 70/85 95% CI59, 8952, 6923, 4682, 9651, 8050, 6928, 5173, 90CI, confidence interval; NPV, negative predictive value; PAMG-1, positive placental α microglobulin-1 test result; PPV, positive predictive value.Table 5Performance of a positive PAMG-1 test result and cervical length <15 mm in symptomatic patients to predict preterm birth within 7 days and 14 days Indicator Preterm birth <7 days Preterm birth <14 days Sensitivity (%) Specificity (%) PPV (%) NPV (%) Sensitivity (%) Specificity (%) PPV (%) NPV (%) PAMG-110/34 112/127 10/25 112/136 12/45 103/116 12/25 103/136 95% CI15, 4881, 9321, 6175, 8815, 4282, 9427, 6968, 83Cervical length 99)5/6 126/155 5/45 115/116 (>99)5/6 115/155 95% CI5, 3196, >9936, >9974, 874, 2495, >9936, >9967, 81PAMG-1 or cervical length17/34 108/127 17/36 108/125 19/45 99/116 19/36 99/125 <15 mm 95% CI32, 6878, 9130, 6579, 9228, 5878, 9136, 7071, 86CI, confidence interval; NPV, negative predictive value; PAMG-1, positive placental α microglobulin-1 test result; PPV, positive predictive value.Table 6ROC curve analysis of a positive PAMG-1 test result and cervical length to predict preterm birth within 7 days and 14 days Indicator Preterm birth <7 days Preterm birth <14 days Sensitivity (%) Specificity (%) PPV (%) NPV (%) Sensitivity (%) Specificity (%) PPV (%) NPV (%) PAMG-110/34 112/127 10/25 112/136 12/45 103/116 12/25 103/136 95% CI15, 4881, 9321, 6175, 8815, 4282, 9428, 6968, 83Cervical length 99)9/10 115/151 95% CI11, 4194, >9944, 9876, 8810, 3595, >9956, >9969, 83PAMG-1 or cervical length <25 mm25/34 94/127 25/58 94/103 27/45 85/116 27/58 85/103 95% CI56, 8766, 8130, 5784, 9644, 7464, 8133, 6074, 89CI, confidence interval; NPV, negative predictive value; PAMG-1, positive placental α microglobulin-1 test result; PPV, positive predictive value; ROC, receiver operating characteristic.Table 7ROC curve analysis of cervical length (<25 mm) to predict preterm birth within 7 days and 14 days Measurement Preterm birth <7 days Value (95% CI) Preterm birth <14 days Value (95% CI) Area under curve0.78 (0.68, 0.88)0.73 (0.64, 0.82)Sensitivity (%)68 (50, 83)53 (38, 68)Specificity (%)84 (77, 90)84 (76, 90)PPV (%)54 (38, 69)56 (40, 71)NPV (%)91 (84, 95)82 (74, 89)Accuracy (%)81 (74, 87)75 (68, 82)CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value; ROC, receiver operating characteristic.Table 8AUC (95% CI) for PAMG-1, cervical length <25 mm, PAMG-1 and cervical length <25 mm, and PAMG-1 or cervical length <25 mm Indicator Preterm birth <7 days AUC (95% CI) Preterm birth <14 days AUC (95% CI) PAMG-10.59 (0.47, 0.70)0.58 (0.48, 0.68)Cervical length 30 mm was combined with a positive PAMG-1 result, 9/97 patients (9%) delivered within 7 days. However, patients with cervical length measurements between these two cutoffs had varying rates of delivery within 7 days. In our present study, 14/50 (28%) patients with cervical length 15–30 mm delivered within 7 days of testing.The present study suggests that a PAMG-1 test result is useful when combined with a cervical length of 15–30 mm. The ROC curve analysis found that an optimal cut off of cervical length <25 mm with a positive PAMG-1 result has a PPV of 80% to predict preterm birth within 7 days, and 90% within 14 days.A limitation of the study is that patients with any bleeding per vagina, rupture of membranes, or who had undergone a vaginal examination had to be excluded. Therefore, the target population is not fully representative and the test can only be used in a limited population excluding those with the conditions described. Another limitation is that we did not meet our expected sample size target of 163 patients because of an unexpectedly high loss to follow up. Nevertheless, the sample size of 161 patients obtained did not unduly affect the results of the present study.It is already known that a short cervix or cervical length <15 mm is a good predictor of imminent spontaneous preterm delivery within 7 days. When cervical length measurement 15–30 mm was used as a cutoff, it was the least accurate predictor of imminent spontaneous preterm delivery within 7 days. This study adds that a cervical length 15–30 mm combined with a positive PAMG-1 test result will provide a higher predictor of imminent spontaneous preterm delivery within 7 days than either method alone, and should be considered for clinical practice. Our present findings support those of Nikolova et al. and Bolotskikh and Borisova , who reported predictive results for a cervical length 15–35 mm combined with a positive PAMG-1 test result.ConclusionCervical length 15–30 mm combined with testing cervicovaginal fluid for PAMG-1 has a high accuracy to predict imminent spontaneous delivery within 7 days by pregnant women with preterm labor and cervical dilatation <3 cm in our clinical practice setting.

PMC

The Veterinary Quarterly

37489957

PMC10388792

10.1080/01652176.2023.2241551

Effects of exercise on urinary biochemical parameters and proteins in a group of well-trained military working dogs

Spinella Giuseppe, Valentini Simona, Matarazzo Micheletino, Tidu Lorenzo, Ferlizza Enea, Isani Gloria, Andreani Giulia

AbstractExercise-induced proteinuria has been widely investigated in humans, also in relation to intensity and duration of activity. Instead, there are only limited publications regarding urinary biochemical parameters and urinary proteins before and after physical activity in dogs. This paper aimed to investigate the effects of exercise on urinary biochemistry and proteins in military dogs. Twenty-four dogs were enrolled in this study. All the dogs were clinically sound, and they were examined before and after activity. Pulse rates (PR) and respiratory rate (RR) were monitored. Urine was sampled before and after a training session of search activity. Standard urinalysis was carried out, urine total proteins and creatinine were measured and the urinary protein:creatinine ratio was calculated; finally, the urinary proteins were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Clinical examination before and after activity did not reveal any pathological finding. After activity, the PR was slightly increased, while the RR was notably increased (p < 0.05). Total proteins, albumin, and their ratio with creatinine were significantly higher after exercise when considering all the dogs included or only the females while, when considering only the males no significant difference was detected. The clinical relevance of this study was related to the possibility of using urine as a non-invasive sample for monitoring health status after training activity and exercise in dogs. An increase in microalbuminuria after search activity, measured using SDS-PAGE could be considered an early biomarker of renal function during training sessions.

IntroductionMilitary working dogs (MWDs) play a fundamental role together with the army in managing humanitarian missions on both national and international territories. Their function is well recognized by intergovernmental organizations, such as the United Nations (UN) and the North Atlantic Treaty Organization (NATO), both of which have the aim of maintaining international peace and security. Training carried out correctly can lead to highly specialized fitness in these dogs, resulting in the prevention of professional traumatic injuries (Spinella et al. 2022).Human sport medicine recommends that adults should engage in moderately-intense cardiorespiratory exercise for 150 min per week (30 min sessions 5 days per week) in order to have a proper level of physical wellness (Hesketh et al. 2020). Adult dogs should also undergo similar activities; however, while conditioning, it is of utmost importance to prevent work overload and muscle fatigue.Changes in physiological, hematological and metabolic parameters during exercise contribute to the internal load, also defined as ‘the relative biological stressors imposed on the athlete during training or competition’ (Bourdon et al. 2017). The internal load depends on several factors, including the typology of the exercise and the extent of the canine training as well as on environmental factors (Cerqueira et al. 2018; Spinella et al. 2021). Depending on the type of stimulus, regular exercise leads to changes in the proteome and metabolome in an extremely complicated network of signalling and metabolic pathways (McGlory et al. 2017).To monitor the correct execution of the training, clinical and hematological examinations should be carried out frequently. However, hematological monitoring requires repeated blood samplings and could potentially stress dogs. To avoid the invasiveness of sequential blood samplings, urine could be considered an interesting alternative for investigating the health status and pre- and post-exercise changes in athletic and working dogs.Urine represents an ideal biological sample as it can easily be obtained with non-invasive procedures and repeated after a short time. In veterinary medicine, the application of proteomics to urine samples has been reported in the literature, for the most related to companion animals (Ferlizza et al. 2020; Miller 2020). Urinary protein profiles represent an interesting opportunity for monitor pathophysiological adaptations of kidney function or the possible site of renal damage. For example, decreased urinary uromodulin is related to tubular dysfunction in dogs and cats (Ferlizza et al. 2015, De Loor et al. 2013) while an abundance of proteins with a high and intermediate molecular mass (MM) is indicative of glomerular proteinuria (Hokamp et al. 2018).The aim of this prospective study was to investigate the effects of exercise on urinary biochemical parameters and urinary proteins separated using SDS-PAGE electrophoresis in well trained MWDs.Materials and methodsA standing agreement between the Italian Army and the Department of Veterinary Medical Sciences of Bologna University for the use of their data and for the development of the present study was stipulated (f.n. M_D SSMD REG2020 0051733 − 27/03/2020 SMD –IGESAN). The study was carried out according to European Union Directive 2010/63/EU and was approved by the Animal Welfare Committee of the University of Bologna (Project ID 914).Twenty-four trained military working dogs (MWDs) belonging to the Italian Army were enrolled in this study (Table 1): 23 German Shepherd dogs and one Belgian Shepherd Malinois. The inclusion of the one dog of a different breed is not expected to influence the results. Fifteen dogs were female (14 intact females and one spayed) and 9 were male (6 intact, one neutered and 2 monorchid). The dogs, which were selected among those available at the military centre (not on mission), were those capable of carrying out search activities. All the dogs had a level of training established by a military veterinarian and appropriate for search activity. The dogs were fed with premium (Protein: 26% – Fat content: 17% – Crude ash: 6.4% – Crude fibers: 1.4%) or super-premium (Protein: 32% – Fat content: 30% – Crude ash: 7.8% – Crude fibers: 1.8%) commercial food. The dogs were randomly divided into two groups according to typology of activity: ten dogs performed only a standard 20-minute training of search activity (Group A) and 14 dogs additionally exercised 10 min on a treadmill after the search activity (Group B). The activity on the treadmill was performed at trot with a mean velocity of 12 km/h. Mean environmental temperature and relative humidity were recorded: the mean environmental air temperatures ranged from 26.5 °C on the 1st day to 23.5 °C on the 2nd and the 3rd days of the study, and the relative humidity from 71% on the1st day to 75% on the 2nd and 3rd days of the study.Table 1.Age, weight, breed, and type of activity of the dogs enrolled in the study. All the dogs enrolledFemales (intact/ spayed)Males (intact/neutered/monorchid)Specimens2414/16/1/2Age (months)32 ± 18 33 ± 22 30 ± 10 Weight (Kg)30 ± 4.5 (29.5)27.4 ± 2.6 34.4 ± 1.9 Breed23 GS; 1 BSM15 GS8 GS; 1 BSMSearch activity (Group A)1055Search activity and treadmill (Group B)14104The data are reported either referring to all the dogs enrolled in the study, or divided into males and females. The data for age and weight are reported as mean ± SD with the median in parentheses.GS: German Shepherd; BSM: Belgian Shepherd MalinoisAll the dogs were routinely monitored by military veterinary personnel throughout their activity of MWDs. However, before each work session, all the dogs underwent complete signalment and physical examination to ensure their current healthy status. None of the dogs had received any medication with steroids or non-steroidal anti-inflammatory drugs (NSAIDs) within 30 days before the study. All the dogs that participated in the study were normothermic before performing the exercise. Pulse rate (PR), respiratory rate (RR) and rectal body temperature (BT) were recorded before the activity; the PR and RR were also evaluated after the activity. The PR was detected by palpation of the femoral artery, and the RR was measured by thoracic visual observation. In both the male and the female dogs, ten mL of mid-stream urine were sampled during spontaneous voiding into sterile urine cups before the daily feeding and watering activities, and with the animals at rest (T0). Within 30 min after the search activity a second sample of urine was obtained using the same procedure (T1); this second sample represented the first urination after the exercise. Moreover, in 12 dogs, the lactate concentration was also randomly evaluated using a rapid lactometer (Roche Diagnostics spa, Germany) to monitor the potential changes induced by exercise.UrinalysisAll the urine samples were kept refrigerated (+4 °C) and were processed on a routine basis within 2 h after collection. In particular, the urinalysis consisted of a macroscopic examination evaluating the color and turbidity. Urine specific gravity (USG) was measured using a manual refractometer (Giorgio Bormac, 41012 Modena, Italy), the chemical evaluation was carried out using a semi-quantitative dipstick test (Combur10Test, Roche Diagnostic, Mannheim, Germany). After centrifugation at 1500 g for 10 min, urine sediment at T0 was observed under both high (400x) and low microscopic fields (100x). Urine supernatants were divided into aliquots and stored in part at −20 °C for a maximum of 7 days for total protein and creatinine determination, and in part at − 80 °C for the subsequent proteomic analysis.Urine protein to creatinine ratioUrine total proteins (uTP) and creatinine (uCr) were measured using commercial kits (Urinary/CSF Protein, OSR6170, and Creatinine OSR6178, Olympus/Beckman Coulter, Atlanta, GE, USA) on an automated chemistry analyzer (AU 480, Olympus/Beckman Coulter, Atlanta, GE, USA). The urine protein:creatinine ratio (UPC) was calculated using the following formula: UPC = uTP (mg/dL)/uCr (mg/dL).One-D-ElectrophoresisAfter thawing and centrifugation at 3000 × g for 10 min, the urinary proteins in the supernatants were separated using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system (NuPAGE, Thermo Fisher Scientific, Waltham, MA, USA) as previously described (Ferlizza et al. 2015); SDS-PAGE is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins which will undergo electrophoresis and then separates them based on their molecular mass. Briefly, three µg of protein were loaded on 4-12% polyacrylamide gel in MOPS buffer with SDS (Thermo Fisher Scientific, Waltham, MA, USA). If the uTP concentration was lower than 0.100 µg/µL, urine was concentrated using spin columns with a molecular mass cut-off of 3 kDa (Vivaspin 500, Sartorius, Goettingen, Germany), following the manufacturer’s instructions. Each gel was also loaded with standard proteins of known molecular mass (Precision Plus Protein Standard, Biorad, Hercules, CA, USA). The gels were stained with Coomassie brilliant blue (PageBlu protein staining solution; Thermo Fisher Scientific, Waltham, MA, USA). After staining, each gel was digitalized (ChemidocMP, BioRad, Hercules, CA, USA), and pherograms were obtained using commercial software (ImageLab, BioRad, Hercules, California, USA). The bands at 100, 67 and 18 kDa were identified on the basis of the molecular mass as previously reported by Ferlizza et al. . The quantification of bands at 100 and 67 kDa was carried out using an internal standard of quantity as described by Ferlizza et al. .Statistical analysisThe data regarding the blood and urine chemistry were analyzed using statistical software (R version 3.4.4). Normal distribution was tested graphically and using the Shapiro-Wilk normality test, the data were expressed as mean ± standard deviation (SD) or standard error (SE) with the median in parentheses. The variables between the T0 and T1 samples were compared using the Wilcoxon or the t Student test depending on their distribution, assuming p < .05 as a significant probability.ResultsTwenty-three German Shepherd dogs and one Belgian Shepherd Malinois were enrolled (Table 1) in this study. Clinical examination before activity started at 8.30 am of each day with an interval of 10 min for each dog. All the dogs enrolled were found to be completely sound after a physical examination carried out by two licensed veterinarians. The clinical values for BTs, RRs, PRs and blood lactate are reported in Table 2.Table 2.Clinical data, serum and urinary analytes in specimens grouped as all dogs and as females and males (n = 24; except for blood lactate, n = 12) at rest (T0) and within 30 min after the search activity (T1). All the dogs enrolledFemaleMale T0T1T0T1T0T1RR77 ± 35136 ± 3575 ± 31138 ± 3982 ± 43133 ± 29PR81 ± 1585 ± 1882 ± 1882 ± 1880 ± 1790 ± 17Blood lactate2.45 ± 0.61 (2.4)2.04 ± 0.52 (1.8)2.68 ± 0.53 (2.4)2.0 ± 0.65 (1.7)2.22 ± 0.65 (2.1)2.08 ± 0.43 (1.8) Urine biochemistry from the dipstick analysis pH6.4 ± 0.6 (6.0)7.1 ± 0.9 (7.0)6.4 ± 0.6 (6.0)6.8 ± 0.9 (6.5)6.3 ± 0.7 (6.0)7.5 ± 0.7 (7.0)USG1051 ± 11 1044 ± 16 1051 ± 14 1044 ± 17 1050 ± 7 1043 ± 16 Pro mg/dL25 ± 10 28 ± 7 22 ± 12 27 ± 9 30 ± 0 30 ± 0 UBG µmol/L2 ± 6 neg2 ± 6 neg2 ± 5 negBil mg/dLnegnegnegnegnegnegGlu mmol/LnegnegnegnegnegnegKet mmol/LnegnegnegnegnegnegEry (+/number of specimens)++/2++/2++/2++/2negnegLeu (+/number of specimens)+/3+/5negneg+/3+/5The The data for RRs and PRs are reported as mean ± SD. The data for blood lactate and urine biochemistry from the dipstick analysis are reported as mean ± standard deviation (SD) with the median in parentheses. In the same row and for each group, the same superscript number indicates a significant difference between T0 and T1 (p 60 kDa, such as albumin (Sentürk et al. 2007). By contrast, during intense exercise glomerular and tubular (mixed) proteinuria occurs due to excessive filtration or the low reabsorption of albumin and other low-molecular mass proteins by the tubule (Lippi et al. 2012). Various mechanisms have been reported to explain transient post-exercise proteinuria, namely decreased circulation in the renal districts which results in inflammation (Lippi et al. 2012), hypoxia (Joyce et al. 2020) and the increased production of reactive oxygen and nitrogen species (Sentürk et al. 2007). Hemodynamic changes in kidney vessels produce an increase in glomerular membrane permeability and the passage into the ultra-filtrate of macromolecules, such as albumin (Sentürk et al. 2007). The concentration of albumin in urine also depends on tubular function since this protein is also reabsorbed mainly in the proximal tubule by an endocytic mechanism (Christensen et al. 2012).In dogs, microalbuminuria is defined as a concentration of albumin in the urine of >1 mg/dL which is below the limit of detection of semi-quantitative screening tests (30 mg/dl), while an albumin concentration of >30 mg/dL is defined as overt albuminuria (Whittemoreet al. 2006). In this study, the mean albumin values measured in dogs before activity were slightly above (in the female group and that of both sexes) or under (in the male group) the cut-off of 1 mg/dL while after exercise, they did not exceed the cut-off of 30 mg/dL, despite a significant increase in the female group and that of both sexes.Albuminuria can also be expressed as the urinary albumin:creatinine ratio (uAC). In veterinary medicine, uAC values 0.3 are indicative of overt albuminuria (Pasławska et al. 2020; Falus et al. 2022). Gary et al. found a microalbuminuria prevalence of 15% in healthy dogs at T0 and reported that mild exercise on a treadmill for 20 min did not affect urinary albumin concentration. In the present study, the prevalence of microalbuminuria at T0 was 25%, regardless of sex; however, after exercise, the prevalence doubled, indicating that search activity alone or associated with a treadmill led to an increase in post-exercise albuminuria. However, the finding that the mean concentration of albumin after exercise did not exceed the upper limits of microalbuminuria (0.3) gave the Authors the confidence that the training of military dogs and the intensity of exercise were correctly set.Using SDS-PAGE, it was also possible to measure the urinary uromodulin concentration, obtaining a mean value similar to that reported in healthy dogs by Ferlizza et al. . Uromodulin is a glycoprotein abundantly present in the urine of healthy dogs. Previous research involving dogs showed that serum and urinary uromodulin concentration could be correlated with the glomerular filtration rate and that its decrease was considered to be a promising and early biomarker of renal/tubular dysfunction (Ferlizza et al. 2020; Seo et al. 2022). The concentration of uromodulin in the urine of the dogs analyzed in the present study did not present significant changes after physical activity, suggesting the absence of renal/tubular impairment (Table 3).Finally, the wide variability of urinary total protein and albumin concentration determined after physical activity was in accordance with the data reported by other authors (Pasławska et al. 2020). In addition to interindividual physiological variability, the development of post-exercise proteinuria could also indicate underlying medical conditions, such as early renal disease or diabetes mellitus, requiring additional investigation. Therefore, the monitoring of proteinuria by SDS-PAGE at regular intervals in canine athletes could be useful in identifying subjects with a higher risk of developing renal failure, as it has been suggested in human medicine (Lippi et al. 2012).ConclusionsOverall, the results of the present study were in accordance with those of other authors; however, also revealed interesting and remarkable findings. First, the method of separating and quantifying urinary proteins using SDS-PAGE electrophoresis was sensitive enough to find even a slight increase in microalbuminuria after search activity in well trained dogs. Second, albuminuria could be considered an early and non-invasive biomarker for monitoring renal function during training sessions. Finally, the physiological presence of arginine esterase in the urine of intact males could influence UPC, and uromodulin and albumin concentrations and mask the post-exercise proteinuria.One limitation of the study was the presence of intact and neutered specimens in the male group; additional research is needed to evaluate the effect of castration on the urinary proteome in male dogs. An additional limitation of the study was the limited number of specimens in the male group. Furthermore, additional studies, also carried out with metabolomic techniques, are needed to investigate the presence of other possible urinary biomarkers indicative of the impairment of renal and muscular function after physical activity.Supplementary MaterialSupplemental MaterialClick here for additional data file.

PMC

Journal of Advanced Research

36736695

PMC10703728

2-02-2023

10.1016/j.jare.2023.01.021

North and East African mitochondrial genetic variation needs further characterization towards precision medicine

Fähnrich Anke, Stephan Isabel, Hirose Misa, Haarich Franziska, Awadelkareem Mosab Ali, Ibrahim Saleh, Busch Hauke, Wohlers Inken

Graphical abstract Highlights•Novel sequencing data from 159 Sudanese human mitochondrial genomes.•Combined with 11 published North and East African data sets yields 641 MT genomes.•Analyzed with world-wide sequencing data from 1000 Genomes and the HGDP.•Haplogroup characterization for North and East Africa.•Fifteen potential novel haplogroups.•Differences in haplogroups L0a1 and L2a1. IntroductionMitochondria are maternally inherited cell organelles with their own genome, and perform various functions in eukaryotic cells such as energy production and cellular homeostasis. Due to their inheritance and manifold biological roles in health and disease, mitochondrial genetics serves a dual purpose of tracing the history as well as disease susceptibility of human populations across the globe. This work requires a comprehensive catalogue of commonly observed genetic variations in the mitochondrial DNAs for all regions throughout the world. So far, however, certain regions, such as North and East Africa have been understudied.ObjectivesTo address this shortcoming, we have created the most comprehensive quality-controlled North and East African mitochondrial data set to date and use it for characterizing mitochondrial genetic variation in this region.MethodsWe compiled 11 published cohorts with novel data for mitochondrial genomes from 159 Sudanese individuals. We combined these 641 mitochondrial sequences with sequences from the 1000 Genomes (n = 2504) and the Human Genome Diversity Project (n = 828) and used the tool haplocheck for extensive quality control and detection of in-sample contamination, as well as Nanopore long read sequencing for haplogroup validation of 18 samples.ResultsUsing a subset of high-coverage mitochondrial sequences, we predict 15 potentially novel haplogroups in North and East African subjects and observe likely phylogenetic deviations from the established PhyloTree reference for haplogroups L0a1 and L2a1.ConclusionOur findings demonstrate common hitherto unexplored variants in mitochondrial genomes of North and East Africa that lead to novel phylogenetic relationships between haplogroups present in these regions. These observations call for further in-depth population genetic studies in that region to enable the prospective use of mitochondrial genetic variation for precision medicine.

IntroductionMitochondria are energy-producing, double-membrane-bound organelles that contain their own genome and that are central to many processes in eukaryotic cells . The mitochondrial (MT) genome is circular and typically 16,569 bases long in humans . Mitochondrial dysfunction is often linked to inefficient oxidative phosphorylation (OXPHOS) capacity with compromised ATP and increased mitochondrial reactive oxygen species (ROS) production that change cellular function and signaling . Dysfunction accompanies various pathologic conditions, such as metabolic diseases , neurodegenerative disorders , and autoimmune conditions as well as cancer and is also linked to aging . Its root cause might stem from variations of the MT genome, which has thus moved into the center of research as a determining factor for complex diseases and precision medicine .Since mitochondria are exclusively maternally inherited in humans , the mitochondrial DNA (mtDNA) phylogeny represents a global maternal genealogy, which has been summarized in an established and widely applied reference phylogenetic tree called PhyloTree , . Its current version 17 is based on more than 24,000 mitochondrial sequences and constitutes more than 5,400 haplogroups, i.e., recurrent genetic variant profiles common in human mitochondrial DNA .Recent studies assessed mitochondrial haplogroups in worldwide human diversity reference data sets from the 1000 Genome Project (1000G; n = 2504) , , the Human Genome Diversity Project (HGDP; n = 623 in ; n = 102 in ) and very recently gnomAD . Such mitochondrial phylogenetic studies have traced modern human settlement of the world and support the hypothesis of Africa as origin of modern humans . The “Out-of-Africa” theory proposes the so-called “mitochondrial Eve” , which represents the mitochondrial Reconstructed Sapiens Reference Sequence (RSRS) mtDNA as the phylogenetic root .Previous studies on mtDNA genetic variation in North and East African individuals largely focused on identifying and characterizing phylogenetic clades with respect to time of origin and geographic dispersal , , , , , , . However, with advances in next-generation sequencing technologies and decreasing sequencing costs , the wide-spread use of mitochondrial genetic variation for medical purposes becomes feasible. Many applications in this context can be considered as precision medicine , i.e. they provide personalized diagnostics or treatment.They fall into two areas: primary mitochondrial diseases and common diseases . Primary mitochondrial diseases are caused by a genetic defect that impairs MT function, resulting into a range of severe symptoms. They affect one of 4,300 people . To clinically diagnose these genetic diseases, the underlying causal variant or variants need to be identified on an individual basis, a process in which variants that occur in healthy individuals are ruled out. Thus, reference data that are representative of human genetic mitochondrial variation will improve diagnosis of primary mitochondrial diseases.The second area of application are common, polygenic diseases. Differences in mitochondrial DNA sequences have been associated with a range of them, e.g. type 2 diabetes and multiple sclerosis or Parkinson’s disease . Considering the central biological role of mitochondria, mtDNA variation is particularly promising for disease stratification, identification of disease modifiers and inclusion into personalized risk assessment. MtDNA for precision medicine in common diseases has thus been considered a comparably novel field with large potential . Genetics-based precision medicine for common diseases currently utilizes genetic variants genome-wide, e.g. via genome-wide association studies (GWAS), polygenic scores or in the context of machine learning . The latter can be considered in method development stage, and because mtDNA variants need special attention (e.g. due to haploid variants and hetroplasmy), they are, due to technical reasons, typically not considered in genome-wide approaches. Eventually, doing so has potential for clinical intervention down the line, since mitochondria are central to main biological processes of the cell. However, whether this can be exploited therapeutically needs to be seen once respective fields develop.Owing to these recent developments, we here view mitochondrial genetic variation of North and East Africa from the point of its application in primary mitochondrial disease diagnostics and prospective precision medicine.In this work, we address the state of mtDNA reference data from the regions of North Africa and East Africa. As of now, there are limited genetic data available from this world region, as it is not included in large genome projects such as 1000G , gnomAD or Topmed . An exception is Egypt, for which an initial reference data set has been compiled and efforts are ongoing to construct a much larger Egyptian genome reference towards genomics-based precision medicine .To address this reference data limitation, we compiled and subsequently quality controlled data from overall 844 mitochondrial genomes (cf. Suppl. Table 1). These derive from a newly sequenced Sudanese cohort (n = 159), an Egyptian cohort with targeted mitochondrial sequencing (n = 217) and an Egyptian cohort having whole genome sequencing data (n = 110). Both Egyptian cohorts have previously been investigated for haplogroup distribution , and are here combined with mitochondrial sequencing data of ancient Egyptian mummies (n = 90) as well as mtDNA sequences from Genbank from individuals with origins in North or East African countries (n = 268).In addition to providing additional mitochondrial genomes from this understudied region, we reanalyzed all available next-generation sequencing data using a novel tool, called haplocheck , to identify potential sample contamination and phantom variants . Haplocheck is a recent tool which allows to detect sample contaminations from next-generation sequencing data by assessing if multiallelic variant calls can be broken down into a mixture of two haplogroups, one denoted as “major”, the other as “minor”. The presence of two haplogroups indicates that genetic material of two individuals was jointly sequenced and thus DNA from the individual relating to the major haplogroup contaminated with DNA from an individual carrying the minor haplogroup. Our combined and new quality-controlled data results in 146 exceptionally high-quality and 641 high quality mitochondrial genome sequences after quality control that we used to phylogenetically re-characterize genetic MT variation in North and East Africa with respect to worldwide mtDNA diversity. We find that the haplogroups of the region cover nearly all worldwide clades and identified 15 potential novel subhaplogroups, among them two that descend from L0a1a1 and and two that are related to L2a1, for which sequences from North and East Africa suggest a different phylogenetic relationship as described by the reference PhyloTree.Results & discussionNorth and East African mitochondrial data collection and filteringTo comprehensively cover the phylogenetic tree of the North and East African population, we sequenced DNA from a cohort of 159 Sudanese individuals which we combined with mitochondrial sequencing data from 217 Egyptians. An additional 110 MT genomes were extracted from whole genome sequencing data of Egyptians from Pagani et al. and Wohlers et al. . We further included mapped mitochondrial sequencing data from 90 ancient Egyptian mummies from Schuenemann et al. ; for three of these mummies, WGS data were available (cf. Supplementary Table 1 for details).As controls, we used two worldwide whole-genome references. These were 1000G with 2,504 samples and HGDP with 828 samples from which we generated mitochondrial DNA sequences. Further, we obtained quality-controlled and aligned Genbank sequences from countries in North and East Africa from the supplement information of McInerny et al. , which we manually traced to their eight respective publications provided in Suppl. Table 1. Sampling location or ethnicity of donors as well as the number of sequences are provided in Fig. 1.Fig. 1Mitochondrial DNA data compiled from North and East Africa. Blue color denotes cohorts referred to as North African, green color cohorts refer to Northeast African and yellow color cohorts refer to East African. Cohorts analyzed from raw next-generation sequencing data are denoted by three numbers: The overall number of samples; the number of samples remaining after lenient QC filtering; and the number of samples remaining after strict QC filtering. Locations with only one number refer to Genbank MT sequences obtained via McInerny et al. . Whenever Northeast Africa is not shown separately, its cohorts are included in North Africa and East Africa.Five of eight studies relating to Genbank entries included only specific haplogroups. These are two studies of haplogroup U6 (Maca-Meyer et al. , n = 11; Olivieri et al. , n = 28) in Berbers from Morocco, two studies of haplogroup L3 in Morocco (Harich et al. , n = 8) and Ethiopia (Soares et al. , n = 70), and one study of both M1 and U6 in Moroccans (Pennarun et al. , n = 56). We also included 18 Genbank MT sequences from ancient Moroccans (Fregel et al. ). Genbank-related studies that did not focus on a specific haplogroup covered 35 Egyptian (Kujanová et al. ) and 42 Tunisian mtDNA sequences (Costa et al. ).To exclude study-specific batch effects and sequencing errors, we applied a two-step quality control (QC) filter on all next-generation sequencing-based North and East African data sets. This was important to correctly assess haplogroup distributions and identify putatively new haplogroups. For the filter step, we kept all samples from Genbank as well as those (i) having a haplocheck-reported major haplogroup equal to a minor haplogroup, and (ii) having contamination assessed and not detected (i.e. “NO”). An overall of 641 samples from four next-generation sequencing-based data for North and East African samples and from eight Genbank studies passed this lenient filter (cf. Suppl. Table 2 and Suppl. Table 3 for aligned sequences). For the second, strict QC filtering step, we excluded Genbank sequences, as contamination checks for them were infeasible, and additionally required a minimum average sequencing coverage greater than 1000x, which is considerably higher than the minimum 600x coverage required by haplocheck to detect a 1% or greater contamination . There are 146 samples passing this filter (cf. Suppl. Table 4). The number of total and filtered samples in each QC step are provided in Fig. 1 for every cohort included. For mitochondrial sequences obtained from Genbank, only the overall number is provided.Haplogroup-based contamination assessment for diverse sequencing settingsWe assessed potential contamination and related sequencing and variant calling metrics for all six sequencing-based data sets, including the worldwide references 1000G and HGDP (cf. Supplementary Tables 5–13 and 14–17 for details). Haplogroups assigned to 543 HGDP samples by Lippold et al. using targeted mitochondrial sequencing, were largely consistent with the haplogroups assigned in this work (47% identical haplogroup; 86% a subclade of the previously assigned haplogroup; cf. Suppl. Table 18). Average coverage of the 1000G and HGDP data sets is high with a median greater than 10,000x, while the other four cohorts have a significantly lower average coverage of 840x (Egyptian MT sequencing), 670x (Egyptian WGS), 163x (ancient Egyptian mummies) and 992x for the Sudanese MT sequencing cohorts, respectively (see Fig. 2 b).Fig. 2Characterization of the six sequencing-based data sets using numbers reported by haplocheck. (a) Histogram of number of samples with haplocheck contamination status not available (“NA”) (gray), not contaminated (“NO”) (blue) and predicted contaminated (“YES”) (red); (b) Box plots for sequencing coverage; (c) Box plots for haplogroup quality of those samples not predicted to be contaminated; (d) For those samples predicted to be contaminated, box plots of the distance between major and minor haplogroup, i.e. the number of phylogenetic nodes between them within the phylogenetic tree; (e) Box plots of the contamination levels for samples predicted as contaminated. Box plots display median and lower/upper quartiles; whiskers denote the most extreme data point no more than 1.5 times the interquartile range; outliers are data points extending beyond whiskers.Contamination is identified using the computational tool haplocheck, which identifies polymorphic, i.e. multiallelic sites within samples. It attempts to decompose the variants into a so-called major and another, minor, haplogroup. If such decomposition is possible and coherent, then a mix of a sample of the major haplogroup with another sample of the minor haplogroup is predicted and the percentage of minor haplogroup allele frequency reported as contamination level. Interestingly, we found a large degree of heterogeneity in contamination calls among the studies. Few of the WGS-based 1000G samples are predicted to be contaminated, yet more than 30% of the WGS-based HGDP samples were reported to be contaminated (Fig. 2a), albeit at a very low contamination level of 2% (median 1.2%, Fig. 2e). Comparable, contamination at a low level with a minor haplogroup that has a high phylogenetic distance from the major haplogroup is predicted for WGS samples of Wohlers et al. and Pagani et al. Possibly, these contamination calls are caused by NUclear MiTochondrial DNA sequences (NUMTs) causing mapping artefacts, although a core genome coverage of 30x, a typically large haplogroup quality (Fig. 2c) and a large phylogenetic distance between major and minor haplogroup detectable based on unusual mutation patterns (Fig. 2d) indicate that this is rather not the case. Low-level contamination reports are possibly an artefact caused by erroneous multiallelic variant calls, e.g. due to low sequencing base accuracy, low or uneven coverage or NUclear MiTochondrial DNA sequences (NUMTS). The number of multiallic variant calls varies largely between samples and systematically between data sets (cf. next section) although a low number of multiallelic sites representing true heteroplasmy is expected. Low-level contamination reports may thus be artefacts and no true contaminations. Particularly for WGS data sets, incorrect multiallelic variant calls may also be caused by NUclear MiTochondrial DNA sequences (NUMTs), which occur in nearly every individual and are typically small (20%) are observed for one HGDP sample and five samples from the Sudan cohort (cf. Fig. 2e) and may indicate partial swaps of sample material, e.g. during pipetting. Indeed, haplocheck reports that two Sudanese samples of haplogroups K1a and M1a1 may have been mixed at similar ratio. For the ancient Egyptian mummy data set, haplocheck repeatedly identifies minor haplogroups related to H2a2a, which we consider an artifact caused by a lack of sequencing coverage that causes the revised Reference Sequence (rCRS) reference bases to be called, which refer to haplogroup H2a2a1. This is supported by the very low coverage for this cohort.Haplogroup validation with Nanopore long-read sequencingWe performed Nanopore long-read sequencing to validate haplogroups obtained from whole-genome sequencing data as well as from targeted mitochondrial sequencing data. We used 10 Egyptian samples that were whole genome-sequenced at 30x (covering haplogroups of H, L, M, and T). For all of them, the major haplogroup was confirmed (Suppl. Table 19). Further, we performed Nanopore sequencing for eight Sudanese samples for which mitochondrial sequencing data were available. The major haplogroup for all of them was confirmed, with the exception of L2a1 + 143, which according to Nanopore sequencing was assigned to the closely related haplogroup L2a1′2′3′4, with no contamination predicted (Suppl. Table 19). Manual inspection shows that all four variants that distinguish L2a1′2′3′4 from L2a1 + 143 were clearly detectable from Nanopore sequencing, but had a large fraction of reads carrying the reference allele (chrM:143 16%; chrM:12693 16%; chrM:15784 24%; chrM:16309 7%). Interestingly, we observe a large number of predicted contaminations (n = 13/18; 72%) with subhaplogroups of H2a2a, which, again, indicates a reference bias caused by a sequencing or variant calling artefact. Nanopore sequencing has a comparably low base accuracy and base calling errors affecting the reference base may be preferentially reported by MT DNA callers which have been designed for short-read data that has higher base accuracy. As technologies, protocols and tools for long-read data develop quickly, constant benchmarking is needed and has recently shown the validity for Nanopore-based somatic MT variant calling .Multiallelic variant calls and heteroplasmyBesides homoplastic variants, which are monoallelic, mtDNA variants can be heteroplasmic, i.e. mtDNA with different sequences and thus more than one allele is present in a sample or tissue. Heteroplasmy typically affects one or few individual sites in the mtDNA. At low levels of heteroplasmy, i.e., if only a small subset of mtDNAs carry a different allele, it is difficult to distinguish true heteroplasmic variants from multiallelic variant calls caused by artefacts. Highly accurate detection of heteroplasmic variants, especially at low heteroplasmy levels, was not the scope of our study and needs dedicated sequencing and analysis. For example, heteroplasmic variant detection depends on sequencing coverage and thus coverages larger than the ones of the data sets of this study are typically used. Nevertheless, we investigated the overall number of monoallelic and multiallelic variant calls generated under haplocheck’s default settings to characterize the next-generation sequencing data sets (see Suppl. Tables 6, 8, 10, 12, 15 and 17). Since a contamination, which is considered a mix of mtDNAs from two haplogroups, results in multiallelic variant calls that do not represent heteroplasmy, we exclude all samples which haplocheck predicted to be contaminated. For those samples not predicted to be contaminated (and thus not affected by artificial multiallelic calls), the results are displayed in Fig. 3(a) and (b).Fig. 3Boxplots of number of monallelic and multiallelic variant calls with respect to rCRS for all six next-generation sequencing-based data sets. Box plots display median and lower/upper quartiles; whiskers denote the most extreme data point no more than 1.5 times the interquartile range; outliers are data points extending beyond whiskers.The number of monoallelic variant calls reflects the haplogroups present in the respective data set, because the larger the haplogroup’s phylogenetic difference from the reference sequence rCRS haplogroup H2a2a1, the more variants will be called with respect to rCRS. As such, data sets containing for example many African haplogroups L will have more samples with many monoallelic, i.e., homoplasmic variant calls. This also explains the comparably large number of monoallelic variant calls in the Sudanese data set. However, numbers of heteroplasmic variants, represented by multiallelic variant calls are expected to be comparable between data sets and independent of the haplogroup content of the respective data set. This is however not the case and differences in the distributions of multiallelic variant calls can be observed between data sets. These differences indicate technical artefacts such as NUMTS, uneven or low coverage and base errors. Distributions of number of multiallelic variant calls are comparable for all whole-genome sequencing data sets, i.e. 1000G, HGDP and Pagani et al. with a median number of multiallelic variant calls of 12, 14 and 12, respectively. Laricchia et al. recently report for the gnomAD WGS data set of 56,434 individuals that most multiallelic variant calls are caused by NUMTS. This may also be the case here, because the Sudan mitochondrial amplicon sequencing data set has a much lower number of multiallelic variant calls (median 4), likely representing an upper bound of true heteroplasmic variants expected in blood of healthy individuals. However, the Egyptian mitochondrial amplicon sequencing data set has most multiallelic variant calls (median of 45). Finally, the Egyptian mummy data set has the widest range. Large numbers of multiallelic variant calls in mitochondrial sequencing observed here are caused by low and uneven coverage in conjunction with base errors. These base errors are possibly also reflecting input DNA quality, which decreases with time and environmental exposure. An example for this is the ancient DNA from Egyptian mummies, for which multiallelic variant calls also reflect mtDNA damage caused by the mummification process. Thus, for accurate heteroplasmic variant identification down to low heteroplasmy levels, deep sequencing at an even coverage will be needed.Distribution of Western Asian and Northeast African mitochondrial haplogroup diversityThe sequential accumulation of polymorphisms in the mtDNA sequence over time across generations has led to the emergence of geographically isolable variation profiles and haplogroups. As a result, geographic regions have characteristic distributions of mtDNA haplogroups. The analysis of 9,264 worldwide sequences (see Methods) yielded haplogroup distributions for distinct geographical regions as shown in Fig. 4a.Fig. 4Worldwide haplogroup distributions by geographic region using top-level alphabetic clade denominators as well as first level subclades of haplogroup L. Please note that for some haplogroups geographic incidence of individual subclades can differ substantially (e.g. different B subclades occurring in America and Asia). (a) Haplogroup level. (b) Haplogroups have been assigned to continents of highest prevalence, representing their hypothetical regions of origin. We decided on the following assignment: Africa: L haplogroups; Asia: E, F, G, M, Y, Z; Middle East: N, R, U, I, W; Europe: H, J, K, T, V; America: A, C, D, X; Oceania: B, P, Q. Please note that tracing geographic origins of haplogroups is complex, e.g. due to autochthonous subclades, and the haplogroup levels and groupings we chose here are not representing all these relations, e.g. for haplogroup B.As described previously, multiple haplogroups are predominant in different continental region. Here, we decided on the following assignment: Africa: L haplogroups; Asia: E, F, G, M, Y, Z; Middle East: N, R, U, I, W; Europe: H, J, K, T, V; Americas: A, C, D, X; Oceania: B, P, Q. Of note, we are aware that haplogroups overlap between continents, e.g., especially in Europe and the Middle East. Further, some haplogroups have autochthonous subclades, e.g. U6 in North Africa. The grouping above therefore does not entail exclusivity with respect to the listed haplogroups. Based on our assignment, we determined the relative proportion of haplogroups associated with different continents for each clustered group (see Fig. 4b). Regions in Asia, North America, Northeast Africa, and the Middle East are particularly diverse. Historically, this can be attributed to events such as colonization and slave trade in the Americas as well as early human migration in the Middle East . In addition to its unique transcontinental location between Africa and Asia, Northeast Africa is adjacent to the center of modern human settlement in the world and has been affected by complex events of prehistoric back migrations .The relative frequency of mitochondrial haplogroups of the 641 QC filtered North and East African samples are depicted in Fig. 5a and the individual haplogroup assignment per sample is given in Supplementary Table 2.Fig. 5Histograms of haplogroup frequencies. Blue dashed line boxes indicate predominantly North African and yellow dashed line boxes predominantly East African haplogroups within this region. (a) For all n = 641 North, Northeast and East African samples passing the lenient QC filter and including Genbank sequences. This includes also Genbank sequences of studies addressing particular haplogroups (e.g. L3, M1 and U6) and thus is not a geographically representative distribution. (b) Tunisian haplogroup numbers out of overall 42. (c) Egyptian haplogroup numbers out of overall 297. (d) Sudanese haplogroup numbers out of overall 113. (e) Haplogroup numbers in ancient Egyptian mummies. (f) Haplogroup numbers in ancient Moroccan individuals.Country-specific haplogroup frequencies for Tunisia, Egypt, Sudan, ancient Morocco and ancient Egypt are shown in Fig. 5b-f. Geographic neighborhoods are clearly reflected in the haplogroup distributions and indicate a geographic gradient from North Africa via Northeast Africa towards East Africa. This can be noted particularly for haplogroups H and U, that are most prevalent in Tunisia, less abundant in Egypt, and rare in Sudan. Conversely, L3 and L0 are prevalent in Sudan, and occur to a lesser extent in Egypt but are rarely observed in Tunisia. Interestingly, L2 is commonly observed in both Tunisia and Sudan, but rarer in Egypt. Overall, most haplogroup clades are observed in the Egyptian cohort (compiled from three data sets). This finding might be explained by the larger sample sizes and compilation of various data sets available for Egypt, compared to only one data set each for Tunisia and Sudan. Yet, more haplogroup clades reach high fractions compared to Tunisia and Sudan, supporting the hypothesis that Egypt is the region of highest mitochondrial DNA diversity within North- and East Africa.Northeast African mitochondrial lineages cover nearly all major worldwide cladesIn order to elucidate the haplogroup abundance in North and East Africa relative to the rest of the world, we computed a phylogenetic tree from the 641 sequences in combination with the 1000G and HGDP reference data. The resulting trees in Fig. 6, Fig. 7 demonstrate that nearly all worldwide mitochondrial clades are represented in North and East Africa and that, apart from the haplogroup M that is mainly represented by M1, a variety of subclades occurs. M1 is a North and East African subclade of M, that otherwise only occurs outside of Africa . It is absent from the 1000G and present in HGDP data only once.Fig. 6Phylogenetic tree of 641 North and East African mitochondrial sequences together with sequences from 1000G as worldwide reference data sets. This tree was computed by a Neighbor Joining-based tree and is colored according to haplogroup assigned by haplocheck (left) and colored according to data source, with North, Northeast and East African mitochondrial sequences compiled in this study shown in red and reference data shown in gray (right).Fig. 7Phylogenetic tree of 641 North and East African mitochondrial sequences together with sequences from HGDP as worldwide reference data sets. This tree was computed by a Neighbor Joining-based tree and is colored according to haplogroup assigned by haplocheck (left) and colored according to data source, with North, Northeast and East African mitochondrial sequences compiled in this study shown in red and reference data shown in gray (right).The worldwide haplogroups not observed in North and East Africa were A, B, D, E, F, G, P, Y and Z, which are predominantly North Asian, Northeast Asian, East Asian, Southeast Asian, American or Oceanian, respectively.The predominantly European haplogroup H is more common in Tunisia and Egypt and less common in Sudan. The same applies to the haplogroups I, J, K, N and U.Haplogroup I is a distant sister clade of N1a1b, as described earlier . Haplogroup J is also observed in both ancient data sets. North African sequences belong to J1 as well as J2 and are with respect to RSRS more deep-rooting than 1000G and HGDP sequences.Haplogroup K represents a subgroup of U8, as expected . In the North and East African data, only K1 was observed, whereas 1000G and HGDP samples also covered K2.African haplogroups L0, L1, L2 and L3 show a high abundance in Egypt, Sudan and Tunisia with the exceptions of L0 in Tunisia and L1 in Sudan being absent and rare, respectively. L3 is the founder of haplogroups N and M, which have given rise to all worldwide haplogroups outside of Africa, and thus L3 has been studied extensively and exclusively , . Haplogroup L3 is observed in all data sets, but predominantly in Sudan with a share of 40%. This is in line with previous studies that suggest East Africa as the origin of L3 .Besides pan-African haplogroups, we observe haplogroups L4, L5 and L6. All these haplogroups have previously been observed mainly in East Africa , . Due to their low prevalence being restricted to this region, they have not been studied by explicitly sequencing mtDNA sequences of these haplogroups.All but one sample relating to North and East African M haplogroups belong to the M1 clade, which is the only M subclade in Africa . The data sets compiled here cover a large diversity of M1. Haplogroup M1 has neither been observed in the comparably small Tunisian nor in the ancient Moroccan data set, but was present in the ancient Egyptian mummy data.Sequences belonging to the N subhaplogroups populated subclades that were located at different positions in the phylogenetic trees. All of them occured in the same phylogenetic branch, the same holds true for mtDNAs belonging to haplogroup R. In the R subclade, we only observe haplogroups R0a (n = 17) in North and East Africa, which previously has been described to be worldwide most frequent in the Middle East and North and East Africa , and encounter haplogroup R among the filtered sequences of an ancient Egyptian mummy once.Nearly all T subclades are detected in North or East Africa, and their proportion is elevated in both Moroccan and Egyptian ancient data sets. Haplogroup T, as well as its sister clade J, has previously been considered of Near Eastern origin , .North and East African haplogroup U mtDNAs cover all subclades (U1 to U8) except the rare U9 clade. Haplogroup U6 has been studied in a targeted way , , previously. Together with M1, it is the only clade of a worldwide haplogroup that is autochthonous in Africa, i.e., predominantly occurring there. Corresponding U6 sequences from and integrated into our data compilation as well as Egyptian and worldwide reference data support this notion.Finally, haplogroup X is present in data from all North and East African regions, including data from three ancient individuals. The North and East African data set contains 12 mtDNA sequences belonging to the phylogenetically old subclade X1, which is largely restricted to North Africa . The coalescence of X in the phylogenetic tree with 1000G and with HGDP, which both do not contain X1 mtDNA sequences, differs between both references and from the PhyloTree reference, because there are no or few reference mtDNA sequences from relevant haplogroups in the 1000G and HGDP data. In order to accurately resolve relationships between haplogroup X and other clades, more mtDNA sequences will be needed.Overall, many different mitochondrial haplogroups are present in North and East Africa; the largest variety is observed in Northeast Africa. A geographic location bordering different continents combined with prehistoric human migrations and a complex history shaped genetic variation observed in the region. One example is migration along the Nile river and competition between upper and lower Nile populations, likely contributing to differences in MT haplogroup frequencies we observe between Sudan and Egypt (e.g. L0 and L3). Another example are prehistoric migrations from Southwest Asia, contributing to a gradient from Northeast Africa to both North and East Africa with respect to haplogroups that have highest prevalence in Europe and Asia (e.g. haplogroup T). Beside prehistory, also throughout history, populations from many geographic regions likely influenced North and East African genetics via rule and trade, e.g. the Near East, Southern Europe, Central-, Western and East Africa.Clades occurring predominantly in North and East Africa are often poorly characterizedWe next investigated North and East African haplogroup profiles within the geographic context using selected 1000G and HGDP populations from neighboring regions (Fig. 8). A corresponding histogram is shown in Supplementary Fig. 1.Fig. 8Haplogroup frequencies of selected 1000G and HGDP populations together with the Sudanese and Egyptian haplogroups of this work (highlighted in box). The populations are colored according to geographic region. Shown in the histogram are clades that are particularly relevant and/or prevalent in North or East Africa and discussed in this work., e.g. L0a1a1 and L2a1. Note that a sequence is counted only once, i.e., a sequence of haplogroup M1 is not counted towards haplogroup M.This diagram shows that both Egyptian and Sudanese mitochondrial profiles are intermediate between the profiles of populations assessed by these public references, constituting a genetic continuum between them. For example, 1000G project data covers African, European, South Asian, East Asian and Admixed American populations, none of which represents mitochondrial haplogroup occurrences observed in Sudan or Egypt according to data generated and compiled in this work (cf. Fig. 8). Middle Eastern and North African populations of the HGDP reference show a combination of predominantly European, African and Asian haplogroups comparable to what is seen in Sudan and Egypt. However, HGDP population sample sizes are low and thus corresponding haplogroup distributions may not be wholly representative.Further, some haplogroups from the North and East African region are absent from both major global references 1000G and HGDP. This applies especially to specific haplogroups that exclusively or predominantly occur in North and/or East Africa, e.g., subclades of L0a1a1, L3, L4, L6, M1, U6 and X1. Some of them, with a particular interest to worldwide human prehistoric migration routes, have explicitly been addressed by studies and selectively been sequenced before (e.g., L3, M1 and U6, which are included in our study). However, local clades that are not or not yet linked to archaeological questions are more poorly characterized, e.g., L0a1a1.Finally, there is little information on rare haplogroups such as L4 and L6. Some of them differ at many mtDNA positions from all other, well characterized haplogroups. Since variant alleles may convey different mitochondrial function and disease susceptibility, especially rare haplogroups with large phylogenetic distance to common haplogroups will need better characterization in the context of mitochondrial genetics-based precision medicine.Variation suggesting novel haplogroupsTo obtain a data set of exceptional high-quality, we next selected 146 samples with high sequencing coverage and without predicted contamination (provided in Suppl. Table 4). The phylogenetic tree of these sequences with corresponding haplogroups is depicted in Fig. 9.Fig. 9Phylogenetic tree of the 146 exceptional high-quality sequences used for detection of novel haplogroups. This tree was computed by a Neighbor Joining-based tree. The circular tree depicts the topological information, while branch lengths are not preserved. An alternative visualization preserving branch lengths is shown in Supplementary Fig. 2.In the search for novel and previously undefined subclades, we investigated this data set using the phantom mutation module of the online haplogrep2 tool. In this module, sequences are checked for recurrent variants that are not haplogroup-defining and that have a ‘Soares score’ of less than or equal to 2, i.e., having been detected no more than twice in a large collection of mitochondrial sequences (according to Table S3 of Soares et al. ). We checked for such variants in samples of the same haplogroup to ensure that they were likely specific to haplogroup carriers and defined a novel, previously undefined subclade. A summary is provided in Table 1.Table 1Haplogroups having potentially novel and previously undescribed sub-haplogroups. The table columns denote, in this order, haplogroup, recurrent novel variants, the number of samples after strict filtering (# HQ; out of n = 146) and after lenient filtering (#LQ; out of n = 641–146 = 495) and the Soares score , which represents the previous occurrence of the variant in worldwide data sets.HaplogroupSubhaplogroup variant# HQ# LQSoares scoreHV1b + 1528640T; 4590G210; 0I567C300I567C; 4772C230; 2L0a1a18869G211L0a1a18017T390L2a1c5186G; 9116C202; 2L2a1 + 1437897A; 398C; 9389G201; 2; 2L2e113356C200L3d5a15457A; 14410A210; 1L3e4a470G; 9455G220; 1L3e5711C220L3f1a112441C; 736T; 328G;15151G; 13967T; 14554G330; 0; 0; 1; 2; 2M1a216182G; 2858G; 15401G; 1282A201; 1; 1; 1R0a2c5981C210V9126C210For ten putatively novel haplogroups, we find further support among samples with lower sequencing coverage. In seven cases, we found one novel, recurrent variant in at least two individuals of the same haplogroup, for example variant 8017 T in 12 carriers of L0a1a1. In eight cases, more than one recurrent variant was detected within carriers of the same haplogroup, e.g. six carriers of L3f1a1 share six novel variants (12441C; 736 T; 328G;15151G; 13967 T; 14554G). Thus, 14 of 30 potential novel haplogroup variants have a Soares score of 0 (47%), 9 have a score of 1 (30%) and the remaining 7 have a score of 2 (23%). The haplogroups of which potential novel subhaplogroups have been identified are either lineages that are largely restricted to North and/or East Africa, e.g., L2a1, M1, and different L3 clades (d, e and f), or are potential local novel subclades diverging from basic haplogroups, e.g., I and V.Variation suggesting revision of phylogenetic relationshipsFor the 146 exceptional high-quality sequences, we examined the phylogenetic tree according to the PhyloTree reference to search for potential new haplogroups using a function from the web-based haplogrep2 tool (see Supplementary Fig. 3). We manually investigated all variants present in the mtDNA sequence as well as all variants expected for the haplogroup assigned, but found to be missing in the sequence. We found two clades, L0a1a1 and L2a1, which were better represented, when revising or extending the phylogenetic tree provided by PhyloTree (Fig. 10).Fig. 10Phylogenetic tree according to the PhyloTree reference for those samples that were assigned to subclades (a) L0a1 and (b) L2a1. We highlight with magenta, gray and turquoise boxes those variants that indicate that a different phylogenetic tree may better explain the samples from North and East Africa, see legend in Figure a). Variants denoted for the leaves of the tree, i.e. samples, denote variant differences w.r.t. the haplogroup assigned. Shared back mutations (magenta) denote that parental haplotypes may be missing in PhyloTree. Mutations repeatedly observed in a subtree (gray) suggest that child haplogroups are missing. Variants that occur in multiple samples and differ from variants defining a parental haplogroup (turquoise) suggest that different variant combinations and haplogroup specifications may better explain North and East African mtDNA sequences.There are eight samples assigned to L0a1 subclades. In most of them, the haplogroup-defining variant 95C is not observed, which is already noted within the PhyloTree reference. Further, three samples show a back mutation at position 185. Such a back mutation is expected for haplogroup L0a2 according to PhyloTree, meaning the samples we sequenced may link clades L0a1 and L0a2. In one sample, an expected mutation at position 200 was not observed, showing the possibility that 95C and 185 together with 310, 8017, 12,738 and possibly 16,519 define a hitherto undescribed subclade of L0a1.The second branch of interest is L2a1. Out of 19 samples to which sub-haplogroups of L2a1 have been assigned, most showed extensive, recurrent deviations from PhyloTree (Fig. 10 b). For example, resolving the haplogroup variants 16,189 and 16,192 within the PhyloTree reference seems not representative for North and East African samples, although their location in the hypervariable region may also have resulted in the observed inconsistencies. Further, variants 3705C, 13752, 7897, 9389, 5186 and 9116, which are recurrent in the L2a1 clade, are not represented in sequences appearing in PhyloTree, yet. However, if included, they would necessitate this.ConclusionWe investigated whether current global mitochondrial genetic data adequately represents the genetic mitochondrial variation of the North and East African regions. Genetic data from this region have been sparse until now, despite an apparent interest due to autochthonous ancestry components being identified , and despite the importance of the region as origin or transit of modern humans outside of Africa. Besides an in-depth analysis of mitochondrial sequence data underlying Egyptian haplogroup distributions reported previously , we have generated novel sequence data of mitochondrial genomes from Sudan, another region with few genetic data available. We characterized North and East African mitochondrial DNA variation by combining these data with publicly available sequence data as well as with mtDNA sequences from Genbank relating to 10 other studies. This work has resulted, to the best of our knowledge, in the largest mitochondrial data collection from this region to date.With a stringent quality control via the novel tool haplocheck, we related North and East African phylogenetics to the worldwide context by restricting the analysis to mitochondrial sequences unaffected by potential contamination or other shortcomings such as insufficient sequencing coverage. An additional filter for a minimal 1000x average sequencing coverage results in exceptionally high-quality mitochondrial sequences, which makes us very confident that novel mitochondrial variations, novel haplogroups and observed PhyloTree inconsistencies are indeed caused by a hitherto insufficient genetic representation of this geographic region. Thus this work complements recent efforts to improve mitochondrial reference data , .We find that North and East African mitochondrial genomes are phylogenetically diverse and cover many of the major haplogroups seen in global populations. Likewise, specific subclades are prevalent in the region, supporting that North and East Africa, and particularly Northeast Africa harbors ancestry components typically attributed to Africa, Europe and Asia , all of which are likely present in the region since prehistoric times . Although our study focused on assessing the state of mtDNA reference data for precision medicine, the combined data allowed to rediscover geographic differences in genetic ancestry that many earlier studies related to prehistorical and historical conditions and events , e.g. migration between Egypt and Sudan along the Nile river . Further, several North or East African clade representatives highlighted by our work and not studied in-depth so far may still add further information to worldwide maternal human genetic history (e.g., the haplogroups L4, L6, N, I and X).Mitochondrial DNA has previously been used extensively to study population history, also for the region of North and East Africa. The focus of our study, however, is the prospective use of mtDNA variants as genetic reference data in the context of precision medicine. Thus, we would like to note that genetic differences increase gradually with geographic differences , defining a continuous genetic spectrum. Accordingly, for medical purposes, reference data covering this spectrum is important.In conclusion, the initial North and East African mitochondrial reference data set compiled as part of our work calls for further extension in order to support genetics-based precision medicine.Material and methodsHaplogroup data compilation for worldwide mitochondrial haplogroup distributionBased on a comprehensive literature research, n = 7414 publicly available mitochondrial mtDNA sequences with available haplogroup and geographic information in the form of the country of origin were collected. Additionally, data from two reference data sets, the 1000 Genomes data set (n = 2504) and the Human Genome Diversity Project (n = 828) were added, resulting in a total number of n = 10,746 entries. As quality assurance, for all database entries haplogroups were reassigned using for mitochondrial sequences haplogrep2 and for next-generation sequencing-based cohorts haplocheck . Subsequently, entries were filtered such that only those remained that were representative for the population of a region. Towards this, studies analyzing only specific haplogroups as well as data from archaeological studies was removed. Eventually, 9,264 entries were retained for further analysis.Objective geographic grouping for worldwide mitochondrial haplogroup distributionsFor determining the relative amount of haplogroups per geographic region, we grouped the database entries into clusters of minimal geographic distance and having a size of at least 100 entries. At the beginning, only countries with less than 100 entries were considered, i.e., those that could not form a group on their own. Countries with an initial number less than 100 were assigned to group 1, all other countries to group 2. In order to classify a country from group 1, an attempt was made to combine it in a first step with other countries or already connected groups of countries in group 1. A continent-dependent maximum distance was set as a termination criterion. For Europe, this was set to 1000 km, for Africa to 1750 km, for the Middle East to 1600 km, for Asia to 1900 km, for the Americas to 2600 km and for Oceania to 1600 km. This distance was determined as a function of the average area of a country on the continent and the existing sample density. If after this first step, the sample number of a country or a group was still less than 100, the countries with an initial sample number greater than or equal to 100 were included as possible connection partners. In this second step, the country or group to be classified was in any case merged with the country from group 1 or group 2 with minimal distance.Mitochondrial DNA short-read sequencingSudanese samples were acquired under approval of the Central Institution Review Board, Al-Neelain University, Sudan, IRB Serial Number NU-IRB-17-7-7-91. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Genomic DNA samples were processed for library preparation, using the Human mtDNA Genome protocol for Illumina Sequencing Platform ( as described previously , for Egyptian samples and 63 Sudanese samples. In brief, a primer set [MTL-F1 (AAAGCACATACCAAGGCCAC) and MTL-R1 (TTGGCTCTCCTTGCAAAGTT); MTL-F2 (TATCCGCCATCCCATACATT) and MTL-R2 (AATGTTGAGCCGTAGATGCC)] was used to amplify the mtDNA by long-range PCR (LR-PCR). Library preparation of the LR-PCR products was performed using a Nextera XT DNA Library Preparation Kit (Illumina Inc., CA, USA). For 96 Sudanese samples, we applied a slightly modified protocol to improve the evenness of the coverage. More specifically, the LR-PCR was conducted using another primer set [hMT-1_F (AAATCTTACCCCGCCTGTTT) and hMT-1_R (AATTAGGCTGTGGGTGGTTG); hMT-2_F (GCCATACTAGTCTTTGCCGC) and hMT-2_R (GGCAGGTCAATTTCACTGGT)] and the library preparation of the LR-PCR products of hMT1 and hMT2 primers was conducted using DNA Prep Tagmentation (Illumina Inc.). This approach allows the fragment size of the tagmented LR-PCR products to be evenly 300 bp, as the transposome reaction occurs every 300 bp on the beads, in contrast to the Nextera XT DNA library preparation Kit, by which transposome reaction occurs randomly, thus, results in various size of the tagmented LR-PCR products (i.e., larger fragments were removed by beads purification steps). The libraries prepared by both methods were further purified, concentration quantified using Qubit Fluorometer (Thermo Fisher GmbH, Dreieich, Germany) and the library size was determined by 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA, USA). The final library was sequenced on the Illumina MiSeq sequencing platform, using v2 chemistry (2 × 150 bp paired-end reads) (Illumina Inc.).Mitochondrial DNA long-read sequencingGenomic DNA (gDNA) from peripheral blood for the long-read sequencing was prepared using Qiagen MagAttract HMW DNA kit (Qiagen, Hilden, Germany). Two GridION sequencing runs were conducted using one GridION Flow Cell version R10.3 (FLO-MIN111; Oxford Nanopore Technologies, Oxford, UK). Full-length mitochondrial DNA (16,595 bp) was amplified from gDNA by long-range PCR using a set of primers tagged with universal sequences (show in Italic): [US-hmt_F-2120 (TTTCTGTTGGTGCTGATATTGCGGACACTAGGAAAAAACCTTGTAGAGAGAG and US-hmt_R-2110 (ACTTGCCTGTCGCTCTATCTTCAAAGAGCTGTTCCTCTTTGGACTAACA)]. Fifty µl PCR reaction was prepared with 200 ng template gDNA, 50 nM of each forward and reverse primers, 25 µl LongAmp Hot Start Taq 2́ Master Mix (New England BioLabs, Frankfurt am Main, Germany). The PCR program was 94 °C for 1 min; 30 cycles of 98 °C for 10 s, and 68 °C for 16 min; 72 °C for 10 min; hold at 4 °C. The PCR products were purified using GeneJET PCR purification Kit (Thermo Fisher Scientific GmbH, Dreireich, Germany). The purified PCR product were barcoded by PCR using the primer sets, which were the same sequences as Nanopore PCR Barcoding Expansion 1–96 (EXP-PBC096; Oxford Nanopore Technologies, UK), but were commercially synthesized (Biomers.net, Germany). The barcoding PCR condition was 95 °C for 3 min; 15 cycles of 95 °C for 15 sec, 62 °C for 15 sec, and 65 °C for 16 min; 65 °C for 16 min; hold at 4 °C.The barcoded PCR products were further prepared for the DNA library using the Ligation Sequencing Kit (SQK-LSK110; Oxford Nanopore, UK) according to the manufacturer’s instructions. In brief, the end-repair, dA-tailing, sequencing adapter ligation, and final purification of DNA libraries were performed according to the manufacturer’s instructions using NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (New England BioLabs GmbH, Germany). The GridION Flow Cell was primed and the DNA library was loaded according to the manufacturer’s instructions. Both sequencing durations were approximately 24 h.Analysis of long-read MT sequencing dataBasecalling was performed with guppy version 6.0.1 + 652ffd1 and flowcell option FLO-MIN111, sequencing kit option SQK-LSK110 and barcode kit option EXP-PBC096, which defaults to guppy config file dna_r10.3_450bps_hac.cfg and model file template_r10.3_450bps_hac.jsn, i.e. using high accuracy base calling. Reads with a Q-score larger 10 were considered pass reads and kept for further analysis. PycoQC was run for quality control and mapping performed with minimap2 against GRCh38, which includes rCRS. Haplogroups were assigned based on this BAM file using the haplocheck tool within mitoVerse ( and Egyptian mitochondrial sequencing characteristics and QCRunning FastQC on all Sudanese and Egyptian mitochondrial sequencing FASTQ files resulted in no systematic, data quality compromising warnings or errors. Sudanese sequencing data has an average length of 140 bases, Egyptian sequencing comprises samples sequenced with 150 and samples sequenced with 300 bp target read length, resulting in an average length of 205 bases across the cohort. Average bases mapped per sample are about 18 Mb for the Sudanese cohort and about 30 Mb for the Egyptian cohort, amounting to an approximate average coverage of more than 1000X. In the Sudanese cohort, five samples have less than 1000 reads and are considered dropouts. The Egyptian cohort does not contain samples with less than 1000 reads.Variant calling, haplogroup assignment and contamination detectionRaw reads from FASTQ files of Sudanese and Egyptian sequencing data were mapped to mitochondrial sequence MT of GRCh38, which resembles the historic revised Cambridge Reference Sequence (rCRS), a sequence of haplogroup H2a2a1. For samples with available BAM files, we used these directly. Our analysis is largely based on haplocheck (version 1.1.2) and its internal accompanying tools haplogrep2 and mutserve (version 1.3.4) as implemented via the mtDNA-server . Variant caller mutserv was run with default settings, which amounts to reporting heteroplasmic variants at a level larger than 1% (--level 0.01), calling deletions (--deletions), base alignment quality turned off (--noBaq), considering a mapping quality of 20 (--mapQ 20) and a base quality of 20 (--basQ).Phylogenetic analysesIn order to obtain a FASTA format mitochondrial sequence that relates to the haplogrep2 output reported by haplocheck, we perform variant calling with mutserv using the default parameters also used within haplocheck. Then we select from the VCF those variants for which the major base is the non-reference base. Using bcftools consensus, we then convert the VCF file into FASTA. Note that mutserv does not call insertions, only deletions which are denoted with a gap symbol (within FASTA, it is “-“), thus ensuring that the sequence length is always exactly the length of rCRS, i.e. 16,569 bases, and that sequences align correctly without re-alignment. The same holds for the Genbank files processed by McInerny et al. . Thus, we can simply combine FASTA files of cohorts by pasting them into one combined FASTA file. The combined FASTA file was then loaded into Jalview where a Neighbour Joining-based tree was computed and exported into Newick format. This tree was then visualized with ggtree using various graphical settings.Ethics statementThis study was approved by the Central Institution Review Board, Al-Neelain University, Sudan, IRB Serial Number NU-IRB-17–7-7–91. All subjects gave written informed consent in accordance with the Declaration of Helsinki.CRediT authorship contribution statementAnke Fähnrich: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – review & editing, Visualization. Isabel Stephan: Software, Formal analysis, Investigation, Data curation, Writing – review & editing, Visualization. Misa Hirose: Investigation, Resources, Data curation, Writing – review & editing. Franziska Haarich: Investigation, Resources, Data curation, Writing – review & editing. Mosab Ali Awadelkareem: Validation, Investigation, Resources, Data curation, Writing – review & editing. Saleh Ibrahim: Conceptualization, Validation, Investigation, Resources, Writing – review & editing, Project administration, Funding acquisition. Hauke Busch: Conceptualization, Validation, Investigation, Resources, Writing – review & editing, Project administration, Funding acquisition. Inken Wohlers: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization, Supervision, Project administration.Declaration of Competing InterestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Multiple Sclerosis Journal - Experimental, Translational and Clinical

PMC10387702

7-24-2023

10.1177/20552173231187810

The socioeconomic impact of disability progression in multiple sclerosis: A retrospective cohort study of the German NeuroTransData (NTD) registry

Dillon Paul, Heer Yanic, Karamasioti Eleni, Muros-Le Rouzic Erwan, Marcelli Guiseppe, Di Maio Danilo, Braune Stefan, Kobelt Gisela, Wasem Jürgen

BackgroundMultiple sclerosis (MS) is a progressively debilitating neurologic disease that poses significant costs to the healthcare system and workforce.ObjectiveTo evaluate the impact of MS disease progression on societal costs and quality of life (QoL) using data from the German NeuroTransData (NTD) MS registry.MethodsCross-sectional cohort study. The cost cohort included patients with MS disability assessed using Expanded Disability Status Scale (EDSS) in 2019 while the QoL cohort included patients assessed using EDSS and EuroQol-5 Dimension 5-Levels between 2009 and 2019. Direct and indirect medical, and non-medical resource use was quantified and costs derived from public sources.ResultsWithin the QoL cohort (n = 9821), QoL worsened with increasing EDSS. Within the cost cohort (n = 7286), increasing resource use with increasing EDSS was observed. Societal costs per patient, excluding or including disease-modifying therapies, increased from €5694 or €19,315 at EDSS 0 to 3.5 to €25,419 or €36,499 at EDSS 4 to 6.5, and €52,883 or €58,576 at EDSS 7 to 9.5. In multivariate modeling, each 0.5-step increase in EDSS was significantly associated with increasing costs, and worsening QoL.ConclusionThis study confirms the major socioeconomic burden associated with MS disability progression. From a socioeconomic perspective, delaying disability progression may benefit patients and society.

IntroductionMultiple sclerosis (MS) is a lifelong demyelinating disease of the central nervous system affecting approximately 2.8 million people worldwide in 2020, with a prevalence of 36 per 100,000 people globally, and 303 per 100,000 in Germany. 1 MS presents heterogeneously, and may include upper/lower extremity disabilities, emotional and cognitive disturbances, balance and coordination disruption, spasticity, abnormal speech, bladder and bowel problems, and fatigue.2,3 As MS progresses, myelin degradation and axonal loss lead to deterioration in nervous communication with the brain.2,4 Subsequently, patients experience progressive loss of motor and sensory functions.2,4 Although there is no cure for MS, symptoms may be managed with physiotherapy, social support, symptomatic medications, and disease-modifying therapies (DMTs).The socioeconomic burden of MS extends beyond healthcare costs. MS poses not only a significant burden to patients and their families but also to wider society.5,6 MS disability severity is typically measured using the Expanded Disability Status Scale (EDSS), which is commonly used in MS randomized clinical trials.7,8 With disability progression, as reflected in higher EDSS, quality of life (QoL) decreases while socioeconomic costs increase. 9 In patients with MS (PwMS), increased disability correlates with decreased workforce participation.9,10 Costs associated with short- to long-term work absences increase with MS progression.11,12 Productivity costs, especially due to reduced employment, are enhanced by the early age of diagnosis. 13 Direct non-medical costs including care provided by family and professionals play a large role with increasing disability.9,10,14,15Several survey-based studies have previously highlighted increasing socioeconomic costs and decreasing QoL associated with increased MS disability. 10 These studies have relied on patient recruitment and self-completion of questionnaires. In this cross-sectional, registry-based cohort study of PwMS in Germany, we evaluated the broad impact of disease progression on societal costs and patient QoL, using information routinely collected as part of the patient's standard of care within a registry.MethodsStudy design and populationThis cross-sectional cohort study of PwMS used routinely collected data from the NeuroTransData (NTD) MS registry. 16 All PwMS were eligible for inclusion. Two cohorts were evaluated. The QoL cohort included data from PwMS who visited NTD clinics between 2009 and 2019. Eligible visits included EDSS and EuroQol-5 Dimension 5-Levels (EQ-5D-5L) assessments on the same day (defined as the index date). The socioeconomic costs cohort (cost cohort) included data from PwMS with ≥1 visit to NTD clinics in 2019 during which EDSS was assessed (index date). This was the most recent year of full data availability at time of analysis and excluded the impact of COVID-19 restrictions on patient healthcare visits. Another visit 365 days prior to the index date with EDSS assessment was required to ensure adequate capture of patient characteristics and socioeconomic costs in that year.For both populations, patients may be eligible for the study at multiple time points, and for each patient, only the most recent eligible time point was included.Data source and settingData were retrieved from the NTD MS registry database provided by the NTD network, a Germany-wide network of neurologists and psychiatrists specialists founded in 2008. 16 We included patients from each of the 66 neurology and psychiatry clinics comprising the NTD network. The NTD MS registry database currently includes >22,000 PwMS with an average observation period of 5 years. The NTD MS registry routinely captures demographic, medical history, socioeconomic information, patient-reported outcomes, and clinical data during visits to NTD outpatient offices, which are performed on average of approximately 3.5 times per year. 17 A standardized dataset is collected, with the minimal dataset collected at each visit, while additional data are assessed depending on medical need. All data are pseudonymized and pooled to form the NTD MS registry database. 18 OutcomesThe following key variables were retrieved from the NTD registry: EDSS, EQ-5D-5L, healthcare resource utilization (HCRU), sociodemographics (age, sex, educational attainment, living status), and clinical history (disease duration, subtype, relapse history, progression event history, DMT history).To evaluate QoL, utilities were estimated from the EQ-5D-5L values using the German value set published in Ludwig et al., 2018. 19 The descriptive system comprises five dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, with each having five levels: no problems, slight problems, moderate problems, severe problems, and extreme problems.Societal costs were estimated from direct and indirect medical and non-medical resource use. HCRU categories were derived and compared based on prior studies 10 (Table 1). Records of healthcare visits, investigations, treatments, need for equipment, care, etc., from the index date visit and ≥1 other visit in the prior 12 months were used to describe HCRU and generate an annualized cost. If a record for a particular HCRU item was absent, the patient was considered not to use this resource. Costs associated with these variables were derived from prior studies that leveraged public sources (e.g., public information on average costs for inpatient stays, consultations, etc.),9,10 which were adjusted to 2019 values using the consumer price index. Where necessary, this information was supplemented with additional searches of public information to obtain sample costs, for example, pricing of assistive equipment from up to 10 product websites. The human-capital method using national German cost of labor was used to value productivity losses.Table 1.Healthcare resource utilization categories.CategoriesSubcategoriesRelevant NTD itemsDirect medicalInpatient careHospital visits, rehab visits Day admissionsHospital visits, rehab visits ConsultationsOccupational/speech/physio/psycho/acupuncture therapy/neurologist TestsCerebral MRI, spinal MRI, blood samples MedicationsWide range of medications, which are not DMTs including fampridine, cannabidiol/delta-9-tetrahydrocannabinol spray DMTsDMTsDirect non-medicalInvestmentsWheelchair, walker, stairlift, house modification, crutches, incontinence pads, diapers, bed pads, tub lift Community servicesCommunity care, care during day, domestic support Informal careCare by familyIndirectShort-term absenceSick days Long-term absence, invalidity, early retirementDisability pensions, other payouts for extended periods including “Uebergangsgeld” (interim pay) and “Krankengeld” (sick pay)We aligned our HCRU categories with those applied in Flachenecker et al. 10 DMT, disease-modifying therapy; HCRU, healthcare resource utilization; MRI, magnetic resonance imaging; NTD, NeuroTransData.Costs of care were estimated based on binary (yes/no) information indicating whether a certain kind of care is needed: daycare (care only provided during the day), care by family, short-term care (temporally restricted stay in an inpatient facility), care in an outpatient setting, and domestic aid (support by community services).As the number of hours of care is not captured, the average cost of informal care (care by family) per EDSS was imputed, based on prior studies. 10 Investment costs were assigned based on product lifetime with occasional maintenance; these costs and those based on product information were collected. For some products, costs and lifetime value are ill-defined, for example, “house modification” costs vary from €20,000 (complete adaption of a flat). We used the average costs spent per flat in Germany to make a flat barrier fee (excluding costs for tub lifts and stair escalators, as these are separate NTD items).Statistical analysisPatient characteristics for both cohorts were described and compared with the overall NTD population to ensure generalizability of findings.For QoL, average utilities per EDSS are reported. For this descriptive analysis, due to the small sample size at severe EDSS (EDSS 7–9), these patients were categorized to a single EDSS 7+ group. Subsequently, multivariable linear mixed regression modeling was performed to evaluate the association between EDSS and utility adjusted for confounders, including sociodemographics and MS disease characteristics.For HCRU, the proportion of PwMS with average usage of each HCRU item was reported per EDSS level. Similar to QoL analysis, severe MS patients were categorized to a single EDSS 7+ group. HCRU usage was then mapped to costs, and costs per category were reported according to three levels of disease severity (mild MS, EDSS 0–3.5; moderate MS, EDSS 4–6.5; severe MS, EDSS 7–9), and similar to prior studies.9,10 The prior study 9 relied on a patient-reported EDSS whereby EDSS category 0 to 3 would correspond to EDSS 0 to 3.5 in the official EDSS. Multivariate linear mixed regression modeling was performed to evaluate associations between cost and EDSS, adjusted for confounding. The NTD center was included as a random effect in both models. All analyses were performed using R Statistical Software (v4.1.2; R Core Team 2021) 20 and figures were produced using the package RStudio. 21 Ethical approval and patient consentThe data acquisition protocol was approved by the ethical committee of the Bavarian Medical Board (Bayerische Landesärztekammer; 14 June 2012, No. 11144) and reapproved by the ethical committee of the Medical Board North-Rhine (Ärztekammer Nordrhein, 25 April 2017, ID 2017071). Patient inclusion with informed consent is completed in the respective NTD practice as part of routine clinical care. Patients included in this analysis provided their informed consent (via tablets in NTD practices, electronic questionnaires, or via a patient portal) to the NTD registry. Patients explicitly agreed to any secondary use of their data.ResultsSample descriptionStudy population and patient demographicsThe final sample included 9821 and 7286 PwMS, for the QoL and cost cohorts, respectively (Figure 1). Patients were predominantly female (72%/72%), relapsing-remitting MS (RRMS) patients (85%/87%), treated with DMTs (71%/77%), with a mean EDSS of 2.7/2.6, and age of 46/47 years. Patient characteristics were comparable between the cohorts, and both were representative of the overall NTD population during 2019 (Figure 2), although some differences were noted. The cost cohort had a higher proportion of patients treated with DMTs (77%) than the QoL cohort (71%), which is likely reflective of the changing treatment landscape for the different eras from which patients were included in these cohorts (i.e., 2019 only versus 2009–2019).Figure 1.Population flowchart for the QoL and cost populations. Full covariate list: age, sex, living status, educational attainment, time since diagnosis, time since manifestation, MS subtype, time since last relapse, number of relapses in previous year, time since last confirmed disability progression, current DMT, and time since last DMT change. DMT, disease-modifying therapy; EDSS, Expanded Disability Status Scale; EQ-5D-5L, EuroQol-5 Dimension 5-Llevels; HCRU, healthcare resource utilization; MS, multiple sclerosis; NTD, NeuroTransData; QoL, quality of life.Figure 2.Study population demographics and MS disease characteristics. DMT, disease-modifying therapy; EDSS, Expanded Disability Status Scale; MS, multiple sclerosis; NTD, NeuroTransData; RRMS, relapsing-remitting multiple sclerosis; SD, standard deviation.Quality of lifeWith MS disability progression, QoL was observed to worsen with average utilities decreasing from 0.94 at EDSS 0 to 0.36 at EDSS 7+ (Figure 3). Compared with prior studies of PwMS in Germany, 10 we observed a sharper decrease in utility from moderate to severe EDSS levels (Figure 3). The multivariate linear regression model assessing the association of EDSS with utility identified statistically significant worsening of QoL with each 0.5-step increase in EDSS, adjusting for confounders (Figure 4A).Figure 3.Mean utility estimated with the EQ-5D-5L by EDSS level in the NTD registry QoL population (2009–2019) compared with Flachenecker et al. 10 NTD utility population (2009–2019) N = 9435, Flachenecker et al. 10 Patients with EDSS >7 are not included in this chart due to low numbers and wide error bars. Note the previous study relied on patient-reported EDSS, which has minor scoring differences at low EDSS compared with the official EDSS. EDSS, Expanded Disability Status Scale; EQ-5D-5L, EuroQol-5 Dimension 5-Levels; NTD, NeuroTransData; QoL, quality of life.Figure 4.Multivariate regression models of the association between EDSS and separately: (A) QoL (n = 9821) and (B) costs (n = 7286). QoL (measured using German value set); multivariate regression model was adjusted for age, living status, sex, time since last relapse, time since MS diagnosis, time since manifestation, time since last DMT change, number of relapses in previous year, time since last confirmed progression event, current DMT use, educational attainment, MS subtype, and included NTD center as a random effect. CI, confidence interval; DMT, disease-modifying therapy; EDSS, Expanded Disability Status Scale; MS, multiple sclerosis; NTD, NeuroTransData; QoL, quality of life.Direct non-medical healthcare resource utilizationInvestments were observed to increase with increasing EDSS score in the cost cohort, with a notable increase in use of aids and equipment from EDSS 4 onwards (Figure 5A). Overall, walking aids were adopted by 15.8% of the NTD cost cohort, 9.6% had wheelchairs, 4.8% had house modifications, and 3.3% had house lifts (Table 2). Community services and informal care were observed at lower EDSS scores, led by domestic support and family care, which became increasingly relevant at EDSS >5 (Figure 5B). Overall, 6.0% of patients required family care, and 18.7% of patients required domestic aid (Table 2).Figure 5.Percentage of patients per EDSS score within the NTD registry cost population : (A) requiring direct non-medical investments; (B) requiring direct medical care (including inpatient, outpatient, and short-term care) and direct non-medical care (including family, domestic, and community daycare); (C) incurring direct medical costs (including consultations, tests, and non-DMT medications [fampridine and cannabinoids]); (D) with indirect costs due to short-term and long-term leave. NTD cost population N = 7286. Note: for those of working age less than 65 years, n = 6838, and for those of working age and working full time, n = 3195. AU, sick day; DMT, disease-modifying therapy; EDSS, Expanded Disability Status Scale; MRI, magnetic resonance imaging; NTD, NeuroTransData; y, year.Table 2.Percentage of patients per EDSS category within the NTD registry cost population .Overall a n = 7286EDSS 0–3.5 n = 5230EDSS 4–6.5 n = 1750EDSS 7+ n = 306 Direct medical MS patients using outpatient nursing care126 (1.7)17 (0.3)59 (3.4)50 (16.3)MS patients using care during day17 (0.2)3 (0.1)8 (0.5)6 (2.0)MS patients using short-term care13 (0.2)2 (0.0)6 (0.3)5 (1.6)MS patients using inpatient care26 (0.4)5 (0.1)13 (0.7)8 (2.6)MS patients with spinal MRI in last 12 months1335 (18.3)1029 (19.7)284 (16.2)22 (7.2)MS patients with cranial MRI in last 12 months3055 (41.9)2391 (45.7)614 (35.1)50 (16.3)MS patients with blood test in last 12 months1908 (26.2)1426 (27.3)446 (25.5)36 (11.8)MS patients with speech therapist visit in last 12 months68 (0.9)24 (0.5)27 (1.5)17 (5.6)MS patients with occupational therapist visit in last 12 months350 (4.8)108 (2.1)179 (10.2)63 (20.6)MS patients with psychotherapist visit in last 12 months258 (3.5)178 (3.4)70 (4.0)10 (3.3)MS patients with physiotherapist visit in last 12 months2182 (30.0)978 (18.7)986 (56.3)218 (71.2)MS patients with acupuncturist visit in last 12 months35 (0.5)18 (0.3)13 (0.7)4 (1.3)MS patients with cortisone therapy in last 12 months797 (10.9)562 (10.8)210 (12.0)25 (8.2)MS patients with fampridine therapy in last 12 months637 (8.7)92 (1.8)440 (25.1)105 (34.3) Direct non-medical MS patients using crutches906 (12.4)123 (2.4)660 (37.7)123 (40.2)MS patients using wheelchair635 (9.6)18 (0.4)381 (24.9)236 (86.5)MS patients using walker707 (9.7)56 (1.1)509 (29.1)142 (46.4)MS patients using stairlift237 (3.3)83 (1.6)105 (6.0)49 (16.0)MS patients using house modifications353 (4.8)52 (1.0)195 (11.1)106 (34.6)MS patients using catheter105 (1.4)12 (0.2)53 (3.0)40 (13.1)MS patients using incontinence pads726 (10.0)284 (5.4)354 (20.2)88 (28.8)MS patients using diapers152 (2.1)20 (0.4)85 (4.9)47 (15.4)MS patients using bed pads190 (2.6)39 (0.8)83 (4.7)68 (22.2)MS patients using tube lift87 (1.2)5 (0.1)48 (2.7)34 (11.1)MS patients using walking aids (crutches or ambulators)1150 (15.8)128 (2.5)774 (44.2)248 (81.1)MS patients using domestic aid1364 (18.7)614 (11.7)594 (33.9)156 (51.0)MS patients using care by family439 (6.0)62 (1.2)258 (14.7)119 (38.9) Indirect % of working age (below 65 years) b 6838 (93.9)5086 (97.3)1514 (86.5)238 (77.8)MS patients on full disability pension (of below 65 years)1261 (18.4)447 (8.8)665 (43.9)149 (62.6)MS patients working full time (of below 65 years)3195 (46.7)2821 (55.5)348 (23.0)26 (10.9)MS patients on disability pension (partial or full) of below 65 years1383 (20.2)508 (10.0)723 (47.8)152 (63.9)MS patients with sick days last 12 months (of full-time working population)333 (10.4)288 (10.2)43 (12.4)2 (7.7)EDSS, Expanded Disability Status Scale; MRI, magnetic resonance imaging; MS, multiple sclerosis; NTD, NeuroTransData. a % calculated on n of those of working age. b % calculated on n of those working full-time.Direct medical resource useDuring the 12-month period, we observed an overall rate of 0.36% of patients requiring inpatient care, while overall outpatient nursing care was 1.73%, which increased with EDSS (Figure 5B; Table 2). All patients had ≥1 neurologist consultation in the 12 months prior to index date, whereas <5% of patients had speech therapist, occupational therapist, and psychotherapist consultations. For physiotherapy, 30% had at least one consultation, which increased with EDSS (Figure 5C). Usage of medical investigations in the 12 months prior to index date was low; however, magnetic resonance imaging (MRI) and blood tests are not fully captured within the NTD database (Figure 5C).Indirect resource use and productivity lossesOverall, 93.9% (n = 6838) of the cost cohort were of working age (i.e., <65 years). Full-time employment amongst those of working age in the cost cohort was 46.7% (n = 3195), which decreased with increasing EDSS score (Figure 5D). Of the full-time employed population, 10.4% (n = 333) reported ≥1 day of sick leave in the 12 months preceding the index date. However, sick days are likely underreported in the NTD database, as non-MS sick days are not reported. Amongst those of working age, 20.2% (n = 1383) were receiving full or partial invalidity pensions, which increased from 10.0% at EDSS 0 to 3.5 to 63.9% at EDSS 7+ (Figure 5D).Socioeconomic costsSocietal costs per patient per year in 2019, excluding or including DMTs, increased from €5694 or €19,315 at mild EDSS, to €25,419 or €36,499 and €52,883 or €58,576 at moderate and severe EDSS levels, respectively (Table 3). The cost of DMTs per EDSS category were €13,621, €11,080, and €5693 for mild, moderate, and severe EDSS levels, respectively (Table 3). The multivariate linear regression model assessing the association of EDSS with overall costs, excluding DMTs, identified statistically significant increases in costs with each 0.5-step increase in EDSS, adjusting for confounders (Figure 4B). Similar results were observed in sensitivity analyses using overall costs including DMTs (Supplemental Figure S1).Table 3.Mean healthcare resource utilization costs per EDSS category using the NTD registry cost population .NTD (unit costs 2019). Average per patient/year (€) N = 7286CategoriesSubcategoriesEDSS 0–3.5 n = 4855EDSS 4–6.5 n = 2125EDSS 7–9.5 n = 306Direct medicalInpatient care/day admissions2507501418 Consultations48411851595 Test917131 Medications22915963043Direct non-medicalInvestments544601349 Community services305201015,087 Informal care5012147824IndirectShort-term absence21229763 Long-term absence, invalidity, early retirement401917,83622,474DMT costs 13,62111,0805693Total excluding DMT costs 569425,41952,883Total including DMT costs 19,31536,49958,576DMT, disease-modifying therapy; EDSS, Expanded Disability Status Scale; NTD, NeuroTransData.DiscussionThis retrospective real-world study of PwMS from the German NTD MS registry confirms EDSS-related trends associated with decreasing QoL and increasing socioeconomic costs associated with MS disease progression reported previously.9,10,22–26 Specifically, with each 0.5-step increase in EDSS, we observed a significant decrease in utility (6% on average based on EQ-5D-5L), and significant increase in annual socioeconomic costs (€3643 on average), becoming more pronounced at higher EDSS levels. Compared with current literature, 10 we observed a steeper decrease in QoL at moderate to severe EDSS levels, while for HCRU, at mild EDSS, absolute direct and indirect medical costs, and non-medical costs observed were lower, but increased at a steeper rate with disease progression.The greatest cost drivers evolved from DMTs for mild MS (70.5% of total costs) to productivity losses in moderate and severe disease (48.9% and 38.4%, respectively; DMT costs fell to 30.4% and 9.7% of total costs), findings in line with previous studies. 10 We observed how the EDSS-related increasing use of crutches (EDSS >2), walkers (EDSS >3), and wheelchairs (EDSS >3) reflect the motor-driven EDSS step definitions (in rare cases at low EDSS levels, the usage of such aids was observed due to temporary worsening of walking disability due to relapse activity). The use of incontinence pads increases from EDSS 0 linearly throughout all stages, as do diapers from EDSS >3. Modifications at home, stairlifts, and tub lifts become relevant at EDSS >5.Care needs increase linearly, with support within the family starting at EDSS 0, and care of patients within their families at EDSS >2. Comparing with direct medical care needs, outpatient nursing care becomes relevant at EDSS >4. Very few patients are in full inpatient care. This demonstrates that PwMS typically remain at home being supported and cared for by their families. Furthermore, the EDSS-related increasing use of physiotherapy (EDSS >0) and occupational therapy (EDSS >4) reflected the EDSS step definitions, while acupuncturist visits did not increase throughout all stages.Indirect costs associated with long-term workplace absences were also a large driver of EDSS-related cost increases. Full-time employment amongst those less than 65 years of age, decreased from 55% at low EDSS to 23% and 11% at moderate and severe EDSS levels, respectively. Similarly, those receiving full disability pensions increased from 10% at low EDSS, to 48% and 64% at moderate and severe EDSS levels, respectively.Compared with prior studies of PwMS in Germany, 10 we observed similar overall costs and trends with increasing EDSS (Supplemental Figure S2). Overall our findings mirror prior studies from Germany27–29 and other countries9,14,15 whereby the predominant societal cost at higher EDSS levels are the broader impacts resulting in indirect costs due to disability pensions and non-medical costs arising from community services and informal care. These findings highlight the potential value of halting or delaying disease progression through the early use of high-efficacy therapies.30,31There are strengths and limitations to our study. This study is the first to our knowledge to utilize routinely collected real-world data from outpatient neurology clinics to quantify MS disability progression-related trends in QoL and socioeconomic costs. The similarities with previous studies validate the use of routinely collected registry data within the NTD to quantify QoL and HCRU.9,10 However, a lower number of MS-related hospitalizations were observed, in part explained by a decline in the annualized relapse rate (ARR) during this study observation period. 17 In addition, the NTD has a relapse treatment protocol to prevent inpatient treatment and favors ambulatory treatment of relapses. Compared with other settings, this results in lower hospitalization rates. Since 2009, only 18% of NTD-managed PwMS had intravenous cortisone treatments administered in an inpatient setting. This may have resulted in the observation of lower MS-related hospitalization rates, although similar ARRs to prior studies. Further research would be required to quantify the impact on relapse outcome and cost of relapses resulting from this treatment protocol in an outpatient setting.Additional strengths in the current study include that EDSS was assessed by the patient's treating neurologist (a certified EDSS rater), while many prior studies relied on patients’ self-reported EDSS.9,10,32–34 Additionally, QoL was measured using the EQ-5D-5L rather than the 3L version relied on previously.9,10,24,35 Furthermore, the NTD population may be more representative of the RRMS distribution in PwMS in Germany, 36 when compared with a recent large-scale survey of PwMS in Germany 10 and prospective observational studies.28,29 We observed that the distribution of MS characteristics, particularly subtype, was more representative of MS population characteristics in comparison to prior survey-based studies, leading to further sample characteristic differences such as lower mean EDSS.10,28,29 Survey-based recruitment requiring self-participation may have excluded healthier PwMS at lower EDSS levels (e.g., recently diagnosed patients not currently enrolled into MS patient society groups), as well as sicker patients at higher EDSS who may decline to participate due to the severity of their disability. These sampling differences may have led to observations of lower costs at lower EDSS, and lower QoL at higher EDSS in the current study compared with the prior study. 10 However, overall, comparable trends of HCRU per EDSS were seen in both the current and prior study. 10 Similarly, prior prospective observational studies were restricted to RRMS patients treated with certain DMTs,28,29 while the current study included all MS patients regardless of treatment and subtype. Some limitations exist regarding the reporting of HCRU in the NTD registry, for example, visits for blood samples not involving a concurrent visit to the neurologist are not recorded in the database, which otherwise may be captured via surveys. MRI appointments at the early stage of MS diagnosis are potentially not fully captured which would have limited impact on the overall costs, however this may have a greater impact on costs at lower EDSS. Sick days are likely underreported in the NTD database. Furthermore, the recording of informal care usage was collected as “yes” or “no,” and costs that could not be accurately assigned as hours were not recorded. Similarly for investments, only the use of a particular item was known, hence the cost had to be assumed. Despite this, the NTD routinely captures a wide range of HCRU as part of standard practice, which are relevant and may be replicable over time. Future studies may consider further augmenting registry data with patient survey data to improve granularity of certain data elements. Bearing in mind the study limitations potentially leading to underestimate some costs, the burden of MS is likely higher than what is reported in our study.Within a clinically representative MS population, we identified worsening QoL and increasing societal costs as EDSS increases, in line with prior studies, but with a modestly greater decrease in QoL. Significant socioeconomic impacts including informal care requirements and disability pensions were also observed. These findings highlight the socioeconomic burden of MS disease progression and the potential value of halting or delaying disease progression through the early use of high-efficacy therapies. 37 Administering DMTs early in MS could lead to better socioeconomic outcomes related to employment, 38 reduce the need for informal care, and improve patient QoL. 39 From a socioeconomic perspective, delaying disability progression may benefit both patients and society.Supplemental Materialsj-docx-1-mso-10.1177_20552173231187810 - Supplemental material for The socioeconomic impact of disability progression in multiple sclerosis: A retrospective cohort study of the German NeuroTransData (NTD) registryClick here for additional data file.Supplemental material, sj-docx-1-mso-10.1177_20552173231187810 for The socioeconomic impact of disability progression in multiple sclerosis: A retrospective cohort study of the German NeuroTransData (NTD) registry by Paul Dillon, Yanic Heer, Eleni Karamasioti, Erwan Muros-Le Rouzic, Guiseppe Marcelli, Danilo Di Maio, Stefan Braune, Gisela Kobelt and Jürgen Wasem in Multiple Sclerosis Journal – Experimental, Translational and Clinical

PMC

Indian Dermatology Online Journal

PMC10718104

10-05-2023

10.4103/idoj.idoj_697_22

Schimmelpenning Syndrome: A Neuro-Oculo-Cutaneous Disorder

Gupta Apoorva, Goel Shitij, Thapar Rajeev K.

A 1.5-month-old baby girl with patchy hair loss over the scalp and yellow-to-black raised lesions over the face was brought in by the mother. The lesions were asymptomatic and constant in size since birth without any history of aggravation or regression. The baby was born following an uneventful full-term pregnancy with no perinatal insults. Cutaneous examination revealed six pale-to-hyperpigmented plaques over the left side of the scalp and face in a Blaschko-linear pattern. Ocular examination revealed a pedunculated mass and symblepharon at the left lateral canthus of the left eye and reacting pupils [Figure 1 and 2]. She had no abnormal neurological, cardiac, or per-abdomen clinical findings. She then presented 1.5 years later with delayed milestones, a developmental quotient of 27% and profound hearing loss in the left ear till 90 dB that was subsequently surfaced on conducting brainstem-evoked response audiometry (BERA). Schimmelpenning-Feuerstein-Mims syndrome is an epidermal nevus syndrome that characteristically presents with nevus sebaceous and cerebral, ocular, and skeletal defects, though other genitourinary, cardiovascular, endocrine, and dental abnormalities have been documented as well. Clinically, most epidermal nevi are present at birth. However, neurological symptoms may only develop over a period of time. There is currently no curative treatment for Schimmelpenning syndrome, but an inter-disciplinary approach coupled with careful evaluation of systems that have the potential to be involved is necessary. The skin lesion can be treated with emollients, topical salicylic acid, retinoids, vitamin D analog, laser, photodynamic therapy, shave dermabrasion, cryotherapy, or surgery for cosmetic reasons. Similarly, the treatment of ocular lesion can range from simple excision, amniotic membrane transplantation, lamellar keratoplasty, and penetrating keratoplasty to autologous limbal stem cell allograft along with eyelid reconstruction, tailored according to the residual eyelid defect.Figure 1A linear, sharply demarcated, yellowish plaque with multiple papillomatous and verrucous areas over the forehead, tip of the nose, philtrum, and the chest. Left bulbar conjunctiva shows a well-defined, circular, pedunculated mass of size 2 × 1 cm and symblepharon at the lateral canthusFigure 2Multiple sharply demarcated, pale-to-hyperpigmented papillomatous plaques present in a Blaschko-linear pattern on the left side of the face and scalp and periocular nodule on the left lateral canthusDeclaration of patient consentThe authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.Financial support and sponsorshipNil.Conflicts of interestThere are no conflicts of interest.

PMC

Radiology Case Reports

PMC10238602

5-26-2023

10.1016/j.radcr.2023.04.036

Type IIB urethral duplication in adult: A case report

Dewi Dian Komala, Gunawan Cristha Octaviani

Urethral duplication are rare anomalies of the lower urinary tract consisting of partial or complete development of an accessory urethra that common in males than female. The exact embryological mechanism for this condition remain unclear, it is thought to relate to a disruption of development of the lateral folds of Rathke during hindgut development. There are 3 types based on Effman's classification. Diagnosis of urethral duplication, a genital examination has to performed and confirmed by micturating cytourethrography and retrograde urethrography. The authors presented a case of 67-year-old man complaining of dysuria, who was diagnosed with urethral duplication Effman Classification type IIB accompanied by bulbar urethral stricture and diverticula and multiple bladder diverticula after performing retrograde urethrography and micturating cystourethrography. This is an extremely rare type of duplication of the urethra (type IIB) with late presentation. Further study may be required regarding the surgical management.

IntroductionUrethral duplication is rare anomalies of the lower urinary tract consisting of partial or complete development of an accessory urethra . It is more common in males, occurring usually in the sagittal plane. In females, the anomaly is rare and most often associated with bladder duplication . Urethral duplication are reported 300 case in the literature. Although the exact embryological mechanism for this condition remain unclear, it is thought to relate to a disruption of development of the lateral folds of Rathke during hindgut development . There is diversity of clinical manifestations, diagnosis is difficult as well as its classification. Patients can be either asymptomatic or symptomatic, most common clinical findings being incontinence, obstruction, recurrent urinary infection and double urinary stream . For the diagnosis of urethral duplication, a genital examination is performed and confirmed by micturating cystourethrography (MCUG) and retrograde urethrography. Urethral duplication has various types that bring a therapeutic challenge for urologic surgeon. Selection of surgical treatment depend on symptoms and the anatomy of urethra and the urinary bladder neck .Case presentationA 67-year-old man came to our institution with complaints of dysuria since about 1 week ago. The urine is yellow, not accompanied by blood and the patient does not have a fever. The patient had undergone urethral surgery because of previous urethral stricture followed by cystostomy procedure and urinary catheter placement since approximately 3 months ago. About 1 week ago the urinary catheter was removed and the patient complained of dysuria. The patient had a history of recurrent urinary tract infections approximately 1 year ago. The patient had no abnormal findings during the physical examination.Retrograde urethrography was performed, 50 cc of contrast is inserted through the external urethral orifice using Foley catheter. Contrast filling bulbar urethral, membranous urethral and prostatic urethral. There was a narrowing of the lumen in bulbar urethral accompanied by diverticula in bulbar urethral. Contrast appears to fill the bladder through the bifurcated urethra with a separate opening to the bladder.Then a voiding cystourethrography with water soluble contrast was carried out by infusion of ± 340 cc by drip into the urinary bladder through Foley catheter. On the appearance of the urinary bladder is fully filled and no vesicoureteric reflux is seen. There are multiple additional shadows, firm boundaries, regular edges, on the bladder wall. At the time of urination, the bladder neck appears open, the contrast exits through the urethra. Two separate proximal urethra were found that came out of the urinary bladder and merged distally, classified as type IIB according to Effman Classification. Fig. 1(A–C), Fig. 2(A–C).Fig. 1(A–C)Retrograde urethrogram (positive and negative fluoroscopic images). There was bulbar urethral stricture accompanied by bulbar urethral diverticle. Contrast appears to fill the bladder through the bifurcated urethra with a separate opening to the bladder.Fig 1Fig. 2(A–C)Voiding cystourethrogram (positive and negative fluoroscopic images). There are multiple diverticle on the bladder wall. Two separate proximal urethra were found that came out of the urinary bladder and merged distally.Fig 2The treatment planning for urethral duplication itself must be individualized for each patient depending on the type of deformity, the severity of the symptoms and other associated anomalies. In this case, after the patient was diagnosed with urethral duplication Effman Classification type IIB accompanied by stricture and bulbar urethral diverticula and multiple bladder diverticula, the patient was planned to undergo perineostomy which was indicated because of his urethral stricture.DiscussionUrethral duplication is a rare congenital abnormality. Embryogenesis is not well understood and various hypotheses exist, but no one can explain all type of presentation, the common pathological process is supposed to result from an abnormal relationship between the lateral folds of the genital tubercle and the central end of the cloacal membrane and the duplication commonly occurs in the sagittal plane with 1 urethra located centrally and the other dorsally . The frequency of this anomaly is mostly in males, and few cases in females . Being congenital, mostly the diagnosis was established in childhood or adolescence rarely in adult age. In this case, adult male patient was coming with dysuria. The urethral duplication has multiple presentation, with a lack of specificity going from the asymptomatic, dysuria, deformed penis, twin streams, urinary tract infection, symptoms of bladder outlet obstruction to various other signs like a renal failure as one of the worst . In this case, chief complain of patient is dysuria after catheter removal.Effmann et al, classified urethral duplication into 3 types. Type I: Blind-ending accessory urethra (incomplete urethral duplication) IA. Distal-duplicated urethras opening on the dorsal or ventral surface of the penis but not communicating with the urethra or bladder (the most common type) IB. Proximal-accessory urethra opening from the urethral channel but ending blindly in the periurethral tissues (rare). Type II: Completely patent accessory urethra. It is divided into 2 parts: A (2 meatuses) and B (1 meatus) IIA1:Two noncommunicating urethras arising independently from the bladder IIA2 : Second channel arising from the first and coursing independently into a second meatus (Y-type) IIB : Two urethras arising from the bladder or posterior urethra and uniting into a common channel distally. Type III: Accessory urethras arising from duplicated or septated bladders .Radiological investigation is mandatory to establish a diagnosis, identify the type of the anomaly and rule out associated with other anomalies . In this case, after the patient carried out a supporting examination such as voiding cystourethrography, it was found 2 proximal urethra that came out of the urinary bladder and merged in the distal body which was suggestive of urethral duplication based on Effman classification type IIB.The treatment of urethral duplication should be individualized for each patient according to the type of UD and the clinical presentation. Higher-grade types usually require complex multiple surgeries, while low-grade incomplete UD may remain untreated.ConclusionUrethral duplication are rare congenital urethral anomalies, and type IIB makes a rare subtype with additional late presentation. Further studies need to be carried out for the surgical management of urethral duplications, more specifically for IIB type.Patient consentWritten informed consent for publication of their case was obtained from our patient.