An additional cohort of mice received dexamethasone as a positive control for immunosuppression. Two and three doses of MSCs reduced lung inflammation, levels of interleukin (IL)‐4, IL‐13, and eotaxin; total leukocyte, CD4+ T‐cell, and eosinophil counts in bronchoalveolar lavage fluid; and total leukocyte counts in bone marrow, spleen, and mediastinal lymph nodes. Two and three doses of MSCs also reduced collagen fiber content and transforming growth factor‐β levels in lung tissue; however, the three‐dose regimen was more effective, and reduced these parameters to control levels, while also decreasing α‐actin content in lung tissue. Two and three doses of MSCs improved lung mechanics. Dexamethasone, two and three doses of MSCs similarly increased galectin levels, but only the three‐dose regimen increased CD39 levels in the thymus. Dexamethasone and the three‐dose, but not the two‐dose regimen, also increased levels of programmed death receptor‐1 and IL‐10, while reducing CD4+CD8low cell percentage in the thymus. In conclusion, multiple doses of MSCs reduced lung inflammation and remodeling while causing immunosuppression in HDM‐induced allergic asthma.In experimental house dust mite (HDM)‐induced allergic asthma, therapeutic administration of a single dose of adipose tissue‐derived mesenchymal stromal cells (MSCs) ameliorates lung inflammation but is unable to reverse remodeling. We hypothesized that multiple doses of MSCs might exert better therapeutic effects by reducing lung inflammation and remodeling but might also result in immunosuppressive effects in experimental asthma. HDM was administered intranasally in C57BL/6 mice. After the last HDM challenge, mice received two or three doses of MSCs  reduced lung inflammation and remodeling. However, three‐dose regimen was more effective at reducing remodeling parameters. Dexamethasone, two and three doses of MSCs similarly increased galectin levels, but only the three‐dose regimen increased CD39 levels in thymus. Dexamethasone and the three‐dose regimen, but not the two‐dose regimen, also increased levels of PD‐1 and IL‐10 in thymus. Although a single dose of mesenchymal stromal cells (MSCs) administered therapeutically was unable to ameliorate lung remodeling in house dust mite (HDM)‐induced allergic asthma, multiple doses of MSCs effectively reduced both lung inflammation and remodeling, while improving lung mechanics. Nevertheless, multiple doses of MSCs also resulted in immunosuppressive effects. This should be taken into account for future clinical trials in patients with severe asthma.1Asthma currently affects 1%‐18% of the population in different regions worldwide and represents a growing public health issue. It is a chronic inflammatory disease characterized by immune responses against allergens that result in lung remodeling, airway hyperresponsiveness, and airflow obstruction. Although many patients with asthma respond positively to corticosteroid therapy, with efficient relief of symptoms, it does not reverse established remodeling, and a cohort of patients still experiences poor control of symptoms and exacerbations.Aspergillus hyphal extract‐induced allergic asthma, a single dose of MSCs administered either locally or systemically resulted in reduction of both lung inflammation and remodeling.Mesenchymal stromal cell (MSC) therapy has demonstrated promising results in several lung disease models, but its impact on airway remodeling has been sometimes controversial.We hypothesized that, in experimental allergic asthma, multiple doses of MSCs might exert better therapeutic effects by reducing lung inflammation and remodeling but might also result in immunosuppressive effects. To address the therapeutic potential of a multiple‐dose MSC regimen, this study aimed to comparatively investigate the effects of two and three doses of human adipose tissue‐derived MSCs administered systemically in a model of HDM‐induced allergic asthma. Endpoints of interest included lung mechanics, histology, inflammation, and remodeling; protein levels of selected mediators; leukocyte counts in primary and secondary lymphoid organs; as well as expression of immunosuppression‐associated markers in the thymus.2Guide for the Care and Use of Laboratory Animals prepared by the U.S. National Academy of Sciences.This study was approved by the Ethics Committee of the Federal University of Rio de Janeiro Health Sciences Center . All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the 2.1pellet obtained was filtered through 10‐mm nylon membranes and centrifuged twice at 230g for 5 minutes. The pellet was then resuspended in Dulbecco's modified Eagle's medium (DMEM)  and cells were counted in a hemocytometer. Cells were cultured in DMEM low‐glucose medium containing 20% fetal bovine serum , 100 units/mL penicillin, and 100 μg/mL streptomycin antibiotic solution  at 37°C, in a 5% CO2 atmosphere. Nonadherent cells were removed 24 hours after the initial culture. After reaching 80% confluence, cells were detached with TrypLE (Life Technologies) and transferred to new flasks. At the third passage, cell viability, density, and final concentration  were determined by trypan blue exclusion in a hemocytometer. The resulting cells were used in experiments.MSCs were isolated and cultured as described elsewhere.2.2Dermatophagoides pteronyssinus; Greer Laboratories, Lenoir, North Carolina) diluted into 25 μL of PBS, three times a week, for three consecutive weeks, while control (CTRL) animals received 25 μL of sterile PBS under same conditions. One day after the last HDM challenge, the HDM group was further divided to randomly receive either two doses of MSCs (MSC‐2D) (105 AD‐MSC diluted in 50 μL of 0.9% NaCl per dose), three doses of MSCs (MSC‐3D), or three doses of saline (SAL) intravenously, via jugular injection under anesthesia (5% isoflurane). Each dose of MSCs or saline was administered once per day on three consecutive days. Seven days after the last HDM challenge, all animals were euthanized for data analysis  = 0.2 mL, and fraction of inspired oxygen (FIO2) = 0.21. The anterior chest wall was surgically removed and a positive end‐expiratory pressure of 2 cmH2O applied. Airflow and tracheal pressure (Ptr) were measured,L). Briefly, after end‐inspiratory occlusion, there is an initial rapid decline in PL  from the preocclusion value down to an inflection point (Pi), followed by a slow pressure decay until a plateau is reached. This plateau corresponds to the elastic recoil pressure of the lung (Pel). ΔP1,L selectively reflects the pressure used to overcome the airway resistance. Static lung elastance  was determined by dividing Pel by VT. Lung mechanics measurements were obtained 10 times in each animal.Seven days after the last HDM challenge, the animals were sedated , anesthetized , tracheotomized, paralyzed , and ventilated with a constant‐flow ventilator  set to RR = 100 bpm, tidal volume  was collected with a polyethylene cannula which was inserted into the trachea and through which a total volume of 1 mL of PBS containing 10 mM EDTA was instilled and subsequently aspirated. BALF was centrifuged at 300The left lung was removed, frozen immediately in liquid nitrogen, and stored at −80°C for molecular analysis, while the right lung was stored in 4% paraformaldehyde solution for histological analyses.Mediastinal lymph nodes, femoral bone marrow, spleen, and thymus were also removed and macerated with 1 mL PBS. Mediastinal lymph nodes and thymus were weighed before maceration. All samples were stored at 4°C.2.5For total cell counts, all samples were quantitated in a hemocytometer under light microscopy after dilution of the samples in Türk solution. Cell suspensions from BALF and thymus were blocked with anti‐CD16/32  and then stained with specific primary antibodies. T cells were characterized by using monoclonal anti‐mouse CD3 (Pe‐Cy5‐labeled), CD4 (FITC‐labeled), and CD8 (FITC‐labeled) antibodies, while eosinophils were characterized by staining with the anti‐mouse Siglec‐F antibody  in the polymorphonuclear‐gated populations. All data were acquired in a FACSCalibur flow cytometer  and analyzed using FlowJo X 10.0.7 software .2.6vs non‐stained tissue. To determine inflammation in the lung parenchyma, the slices were stained with hematoxylin and eosin. Inflammatory changes were graded according to a semiquantitative scoring system as absent (score 0), mild (score 1‐2), moderate (score 3), or severe (score 4) by two blinded researchers. The following scoring criteria were adopted: 0, no evidence of inflammation; 1, few inflammatory cells in the peribronchial area; 2, a ring of inflammatory cells (one cell layer deep); 3, a ring of inflammatory cells two to four cells deep; and 4, a ring of inflammatory cells more than four cells deep.Sections (4 μm thick) were cut, deparaffinized, and stained for histological analysis. For quantification of collagen fibers in alveolar septa, sections were stained with Sirius Red dissolved in saturated picric acid for subsequent analysis through polarized‐light optical microscopy , under ×400 magnification. The area occupied by fibers was determined by digital densitometric recognition  and divided by the area of alveolar septa. Results were expressed as the fractional area occupied by collagen fibers.2.7g for 5 minutes and 10 000g for 10 minutes), and the resulting supernatant assayed. Total protein content was measured by Bradford's reagent (Sigma‐Aldrich). Protein levels of IL‐4, IL‐13, eotaxin, IL‐10, transforming growth factor (TGF)‐β (lung tissue), and programmed cell death protein‐1 (PD‐1) and IL‐10 (thymus) were evaluated by ELISA  using matched antibodies in accordance with manufacturer instructions. The final concentration was normalized to total protein content and expressed as pg/mg.Lung tissue and thymus homogenates were used for mediator quantifications. Briefly, the right lung was isolated, homogenized in lysis buffer solution, centrifuged (6002.836B4) and expressed as the fold change relative to the CTRL group using the 2−ΔΔCt method, where ΔCt = Ct(target gene) − Ct(housekeeping gene).Thymic tissue was lysed for RNA extraction by the RNeasy Plus Mini Kit  in accordance with manufacturer instructions. The total RNA concentration was measured by spectrophotometry in a Nanodrop ND1000 system, and the first‐strand cDNA was synthesized from total RNA using the High‐Capacity cDNA Reverse Transcription Kit . Relative mRNA levels were measured with Bryt Green  using a Mastercycler ep realplex PCR system . All experiments were performed in triplicate. The relative level of each gene was normalized to the housekeeping gene acidic ribosomal phosphoprotein P0 , and statistical significance was established at P < .05.Differences among groups were assessed using one‐way ANOVA followed by Bonferroni's test or an unpaired Student's 33.1Protein levels of IL‐4, IL‐13, and eotaxin were higher in lung tissue homogenates of HDM‐SAL compared to CTRL mice. Both two and three doses of MSCs significantly reduced protein levels of these inflammatory mediators. No differences were observed in protein levels of IL‐10 among the experimental groups . Both two and three doses of MSCs reduced these parameters to CTRL‐comparable levels , spleen (P = .1318), or thymus (P = .7733), nor any other physiological alteration.HDM‐SAL animals demonstrated increased total leukocyte counts in bone marrow, spleen, mediastinal lymph nodes, and thymus compared to CTRL mice. Both the two‐dose and three‐dose MSC regimens reduced total leukocyte counts in bone marrow, spleen, and mediastinal lymph nodes, but not in the thymus Figure . To rule3.5To analyze whether multiple doses of MSCs may result in increased expression of immunosuppressive markers, a cohort of mice was treated with dexamethasone  after the last challenge with HDM, as a positive control for immunosuppression.+CD8+ cells, as well as a significant reduction in the percentage of CD4+CD8low and CD4+CD8− cells. The HDM‐MSC‐3D group also demonstrated an increased percentage of CD4+CD8+ cells and a reduction in the percentage of CD4+CD8low cells in comparison to HDM‐SAL mice. The HDM‐MSC‐2D group demonstrated a trend toward reduction of CD4+CD8low cell counts, but the difference was not statistically significant in comparison to the CTRL group (P = .0656)  3 times a week for 3 weeks and HDM group received 25 μg of HDM diluted in saline (25 μL). The HDM group was then treated with saline (50 μL) for 3 consecutive days after the last challenge, or 2 or 3 doses of 105 adipose tissue (AD) derived‐MSC diluted in saline (50 μL) for 2 or 3 consecutive days after the last challenge . All treatments were administered intravenously. Seven days after the last challenge, the animals were euthanized for data acquisition.Click here for additional data file.Supporting Information Figure S2 Representative histogram with the gating strategy used to quantify CD4+ T‐cells (top) and eosinophils (bottom) in bronchoalveolar lavage fluid.Click here for additional data file.Supporting Information Figure S3 Relative weight of mediastinal lymph nodes and thymus. Data are presented as means + SD of 6 animals/group. SAL: mice challenged with HDM and treated with saline. MSC‐2D and MSC‐3D: mice challenged with HDM and treated with 2 or 3 doses of AD‐MSCs, respectively. * Significantly different from CTRL (P < 0.05). # Significantly different from SAL (P < 0.05).Click here for additional data file.Supporting Information Figure S4 Dexamethasone therapy and three doses of MSCs modulated protein levels of programmed death receptor (PD)‐1, and interleukin (IL)‐10 in the thymic tissue of animals with HDM‐induced allergic asthma. CTRL, mice challenged with saline and treated with saline. HDM‐SAL, mice challenged with HDM and treated with saline. HDM‐DEXA, mice challenged with HDM and treated with dexamethasone. HDM‐MSC‐2D, mice challenged with HDM and treated with two doses of MSCs (105 cells per dose). HDM‐MSC‐3D, mice challenged with HDM and treated with three doses of MSCs. Data are presented as means ± SD of 6 animals/group. *Significantly different from CTRL (P < 0.05). #Significantly different from HDM‐SAL (P < 0.05). †Significantly different from HDM‐DEXA (P < 0.05). ‡Significantly different from HDM‐MSC‐2D (P < 0.05).Click here for additional data file.Table S1 Sequence of PCR primersClick here for additional data file.Table S2 Body weight of animals (g) before and after HDM‐induced allergic asthma and therapeutic protocolClick here for additional data file.Table S3 T‐cell subpopulations in the thymusClick here for additional data file.


Adequate dietary intake is important for promoting adaptation and prevention of musculoskeletal injury in response to large volumes of physical training such as Army Initial Entry Training (IET). The purpose of this study was to evaluate training volume and dietary intake and estimate energy balance in Army IET soldiers.Dietary intake was assessed by collecting diet logs for three meals on each of three, non-consecutive days during the first week of IET. Training volume was measured across 13 weeks of training using Actigraph wGT3X accelerometers. Training intensity was classified using Sasaki vector magnitude three cut points. Energy expenditure estimates were calculated during weeks two and three of training using the modified Harris-Benedict equation and by estimation of active energy expenditure using metabolic equivalents for each classification of physical activity. All data is presented as mean ± standard deviation.p < 0.001), whereby a 1 kg increase in body mass was associated with a 53 kcal energy deficit.A total of 111 male soldiers  completed diet logs and were monitored with Actigraphs. IET soldiers performed on average 273 ± 62 min low, 107 ± 42 min moderate, 26 ± 22 min vigorous, and 10 ± 21 min of very vigorous intensity physical activity daily across 13 weeks. The estimated total daily energy expenditure was on average 3238 ± 457 kcals/d during weeks two and three of IET. Compared to week one caloric intake, there was a caloric deficit of 595 ± 896 kcals/d on average during weeks two and three of IET. Regression analysis showed that body weight was a significant predictor for negative energy balance . This may directly affect soldier performance and injury frequency. IET soldiers undergo rigorous training, and these data may help direct future guidelines for adequate nourishment to optimize soldier health and performance.The online version of this article (10.1186/s12970-018-0262-7) contains supplementary material, which is available to authorized users. Initial Entry Training (IET) is a mentally and physically demanding military training program designed to transform civilians into soldiers. The transformation from civilian to soldier has become more challenging as the ability of IET soldiers to respond and adapt to IET has declined over recent decades. The American population has become less active due to changes in lifestyle factors such as increases in screen time in the place of physical activity, reductions in physically active jobs, and reduced active transportation  . ConsequSoldiers perform organized physical fitness training as well as occupational physical activity to improve physical fitness and learn soldiering skills during IET –10. OrgaRapid increases in training volume and intensity have been related to higher MSI rates . Few invDietary intake must match training volume to fuel physiological demands. Inadequate energy is detrimental to bone health , 15, immClearly understanding training volume and nutrition intakes of IET soldiers is critical in ensuring training success. However, few studies to date have examined these areas in the IET environment, and none have examined both factors together in IET soldiers. Thus, the primary aims of this study were to: 1) quantify total training volume; 2) determine dietary intake; and 3) estimate energy balance in soldiers participating in IET. Secondary aims were to determine how dietary intake of IET soldiers compares to current nutritional recommendations for active individuals  and to dThe Auburn University Institutional Review Board, Army Institutional Review Board, and the Director, Research & Analysis Directorate Army Center approved the study procedures. For inclusion, participants had to be at least 18 years of age, healthy with no apparent disease or MSI, and participating in IET. Interested potential participants from one training unit received verbal explanation of the study from the study team and provided written consent. One hundred eleven male IET soldiers  from one training unit of IET soldiers at Fort Benning, Georgia volunteered for this investigation.This study was 14 weeks in total duration. Diet analysis and body mass information was collected during the first week of IET. Physical activity data was collected daily beginning at week two and continued ended during week 14 of IET training.Anthropometric measures were conducted in a fasted state prior to morning physical training and breakfast on the morning of day three of IET. Urine specific gravity was evaluated the morning of testing using a handheld refractometer  to ensure participants were properly hydrated (USG below 1.03) . Height Dietary intake was recorded after each meal on three non-consecutive days  during the first full week of IET. Food menus from the cafeteria-style dining facility serving the IET soldiers were used to create meal specific diet logs  website . Food itParticipants were outfitted with Actigraph wGT3X monitors . Training volume and intensity were measured across weeks 2–14 and energy balance was estimated for weeks two and three of red phase during IET. Monitors were initialized using Actilife software version 13.1.1 . Each week of training a member of the research team met with 12 soldiers (3 per platoon) to instruct them to wear the monitors around the waist, on the right side of the body, at all times (awake and asleep), and to only remove for showering. At the end of each week the monitors were collected, and age, height, weight, ethnicity, and hand dominance were entered into the Actilife software for each subject. Sasaki Vector Magnitude 3 cut points were used were used to divide activity into three categories: moderate (2690–6166 cpm), vigorous (6167–9642 cpm) and very vigorous (> 9642 cpm) . A cut-pEnergy expenditure was estimated during weeks two and three of IET. Minutes per day were averaged for each classification of physical activity and then metabolic equivalents (MET) were assigned to each category of physical activity. The MET assignments are as follows: light = 2 METs , ModeratWe estimated resting energy expenditure (REE) using the Modified Harris-Benedict equation :\documenTotal energy expenditure was the sum of REE and AEE. Energy balance was estimated by subtracting each participant’s caloric intake from the estimated total energy expenditure; negative or positive values indicate a caloric deficit or surplus, respectively.p < 0.05.Given that this study was largely observational, food intake, physical activity and energy balance data were presented as mean ± standard deviation values, and no statistical testing was performed for these metrics. Dietary intake across meals was evaluated using repeated measures ANOVA. Paired samples t-tests were used to evaluate the simple main effect of time and independent samples t-tests to evaluate the simple main effect of group when significant group by time interactions were found. Normality of residuals was tested using the Shapiro-Wilks and Komolgorov-Smirnov test. Sphericity was evaluated using Maulchy’s test. During tests for normality of residuals for dietary intake one meal was determined to be a statistical outlier and was removed from the analysis because the participant’s calorie intake was approximately 3500 cal for one meal, which was 2.5 times greater than his next closest meal . Linear regression was used to determine if body weight was a significant predictor of negative energy balance. Body weight was mean centered (participants body weight – mean (body weight)) was used as the predictor for average energy balance during weeks two and three of IET. For probability testing, statistical significance was set at Physical activity data are presented in Fig. p < 0.001). There was a significant difference between all meals for calorie intake, breakfast and dinner , breakfast and lunch , dinner and lunch . Protein intake averaged 38.1 ± 11.2 g for breakfast, 33.7 ± 9.9 g for lunch, and 41.1 ± 11.0 g for dinner. There was a significant effect of meal on dietary protein intake . Post-hoc paired t-tests revealed a significant difference between breakfast and dinner , breakfast and lunch , dinner and lunch . Carbohydrate intake averaged 137.3 ± 39.7 g for breakfast, 104.5 ± 28.2 g for lunch, and 101.8 ± 30.3 g for dinner. For carbohydrate intake, Greenhouse-Geisser corrections were used because sphericity was violated. Statistical analysis revealed a significant effect of meal. Post-hoc paired t-tests revealed a significant difference between breakfast and dinner , breakfast and lunch , but no significant difference between dinner and lunch . Fat intake averaged 34.6 ± 11.2 g for breakfast, 25.9 ± 9.2 for lunch, and 27.8 ± 11.2 g for dinner. Statistical analysis revealed a significant main effect of meal . Post-hoc paired t-tests revealed a significant difference between breakfast and dinner , breakfast and lunch , but no significant difference between dinner and lunch  for fat intake.Average macronutrient intake is presented in Fig. Approximately 49/111 participants consumed less than the recommended 1.5 g/kg of body weight per day of protein intake and 78/111 consumed less than the recommended 6 g/kg of carbohydrate per day. Approximately 103/111 met the lower limit of dietary fat intake .2 = 0.54, p < 0.001). Specifically, for every 1-kg increase in body mass, energy balance became more negative by 53 cal.Energy expenditure was estimated using the most conservative MET value associated with each classification of physical activity . Figure This study evaluated the dietary intake, training load, and balance between estimated energy expenditure and energy intake in U.S. Army IET soldiers. Our primary finding was that IET soldiers expended approximately 595 more calories per day during red phase than they consumed based on our estimation of metabolic training load. It is likely that our estimation of energy expenditure is low based on three important considerations: 1) we applied conservative MET assignments to moderate, vigorous, and very vigorous intensity physical activity; 2) estimation of BMR using the Modified Harris-Benedict equation was also a conservative estimate of non-physically active energy expenditure. It assumes the participant is immobile and in a supine position, whereas over half of sedentary time for IET soldiers is standing time which has been reported to increase energy expenditure by as much as 10% ; 3) IET Quantification of training volume during IET revealed soldiers averaged approximately 3180 ± 320 min of physical activity per week with 1202 ± 291 of those minutes being moderate to vigorous physical activity across 14 weeks of IET. For comparison, research indicates only 49–53% of civilian adults 18–34 reported at least 150 min of moderate or 75 min of vigorous and only 31% reported participating in 300 min of moderate or 150 min of vigorous physical activity per week . FurtherPrevious research reported that IET soldiers spent on average 140 min per day in light, approximately 92 min in moderate, and 38 min per day in vigorous intensity exercise  . DiffereSoldiers consumed an average of 2644 cal, 114 g of protein, 352 g of carbohydrate, and 89 g of fat per day. Our results are limited as we only report week one dietary intake. A 2002 study of dietary intakes in IET soldiers found they consumed on average 3000 cal per day, whereas a 2012 study found IET soldiers consumed on average 1975 cal  , 50. DifProtein intake is crucial for provision of amino acid pools to support cellular adaptations of skeletal muscle such as hypertrophy , mitochoDiets high in carbohydrate intake are associated with increased power output , 66, proThis investigation is a first step at evaluating energy balance in the IET environment and represents only one training company at one location. One limitation to this study is our assumption that entry-level dietary intake values were reflective of eating habits throughout the 14-week duration. Additionally, we did not estimate energy expenditure during the same week in which dietary data was collected. However, training schedule and amount of time to eat are similar within red phase (weeks 1–3) and therefore dietary intake was likely similar in weeks two and three compared to what was found in week one. Additionally, energy expenditure was estimated based on the average physical activity performed by the entire company of IET soldiers. While many of the activities performed at IET are in the group setting, ideally, investigations should assign each IET soldier a monitor to wear daily throughout training to track individual energy expenditure and training load. Finally, using serial and more precise methods to track dietary intake would improve outcomes. There are known inherent errors when using food logs such as portion estimation error, therefore methodologies in which trained researchers evaluate dietary intake may improve accuracy .Our results suggest that based on the week one diet assessment, the IET soldiers in this study did not consume adequate nutrition to meet the physiological demands of training. This may impair each soldiers training response and potentially increase injury rates associated with recovery and fatigue. Additional research is needed to develop strategies to optimize soldier nutrition to improve performance and recovery.Additional file 1:Table S1. Example food log. (DOCX 20 kb)


Both berberine and metformin are well‐known antihyperglycemic agents for diabetes treatment. Adenosine monophosphate (AMP)‐activated protein kinase (AMPK) activation is often considered as the most important molecular mechanism although the mechanism has been challenged recently. Up to now, when the ambient glucose level changes dynamically, the interaction between AMPK activity and the glucose‐lowering effects of the agents remains largely unknown. To address this issue, HepG2 hepatocytes and C2C12 myotubes were preincubated at normal (5.6 mM), moderate (15 mM), or high (30 mM) glucose concentrations followed by moderate‐glucose incubation plus berberine or metformin treatment. Preincubation at high glucose concentration followed by moderate‐glucose incubation activated the AMPK pathway, but the activation was abolished with berberine or metformin treatment. In contrast, alteration from normal glucose to moderate glucose concentration in the medium suppressed AMPK activity, which was activated by berberine or metformin. Both metformin and berberine decreased the intercellular adenosine triphosphate content, enhanced glucose consumption, and lactate release under all three preincubation glucose concentrations regardless of AMPK activity. In conclusion, AMPK activated by glucose reduction is inhibited by berberine or metformin. The elevation of glucose level led to suppressed AMPK activity, which was activated with the addition of agents. The potent glucose‐lowering effects with minimal hypoglycemia of berberine and metformin may be partially due to their bidirectional regulation of the AMPK signaling pathway. Berberine and metformin promote glucose metabolism via stimulation of glycolysis, which may not be related to AMPK activity. All other reagents were, if not indicated otherwise, purchased from Sigma‐Aldrich .Dulbecco modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and other culture reagents were obtained from Gibco Life Technologies . Glucose oxidase was purchased from Shanghai Shensuo Reagents . Berberine was obtained from the National Institute for the Control of Pharmaceutical and Biological Products . Metformin was purchased from Shanghai Sangon Biotechnology Corporation . Berberine and metformin were dissolved in ddH2.22 incubator and cultured in a growth medium: DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. For differentiation of myotubes, C2C12 myoblasts were seeded into 12‐well plates in DMEM with 10% FBS for 24 hours. Then, the medium was replaced by the differentiation medium: DMEM containing 2% horse serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin for 6 days. The medium was refreshed every 48 hours. Media of different glucose concentration were prepared by adding additional glucose to low‐glucose medium (5.6 mM glucose). The osmotic pressure was adjusted with d‐mannitol . The cells were incubated with different glucose concentration for 4, 12, or 24 hours, and then treated with berberine or metformin . The cells were lysed according to the manufacturer’s instruction. After centrifugation at 12 0002.4The cells were cultured in 96‐well plates and treated with berberine or metformin in FBS‐free DMEM supplemented with 0.25% bovine serum albumin (BSA). The glucose concentration in the medium was determined by the glucose oxidase method. The amount of glucose consumption was calculated by subtracting the glucose concentration of the wells with cells from that of the blank wells.2.5The cells were cultured in 96‐well plates and treated with berberine or metformin in DMEM supplemented with 0.25% BSA. The lactate concentration in the medium was measured with a lactate reagent kit .2.6Cells were washed with ice‐cold phosphate‐buffered saline and lysed with lysis buffer , 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM EDTA, 10 μg/mL leupeptin, 1 mM phenylmethanesulfonyl fluoride (PMSF), and phosphatase inhibitor cocktail; pH 7.4). The extracted protein (35 μg) was boiled for 5 minutes, subjected to SDS‐polyacrylamide gel electrophoresis. Then, the separated proteins were transferred onto a nitrocellulose membrane. After blocking with 5% skim milk in the tris‐buffered saline with 0.1% tween‐20 (TBST) buffer for 1 hour, the membrane was incubated with primary antibody at 4°C overnight. Antibodies to AMPK, phospho‐AMPKα (Thr172), acetyl coenzyme A synthetase (ACC), phospho‐ACC (Ser79), and β‐actin were purchased from the Cell Signaling Technology . The HRP‐conjugated secondary antibodies  were used with chemiluminescence reagent  for the generation of the light signal; Gel‐Pro Analyzer 4.0 was used to quantify the Western signals.2.7t test or one‐way analysis of variance (SPSS 17.0) was used in statistical analysis of the data, with P < 0.05 considered significant.Data are presented as mean ± standard error of mean from individual experiments. Every single experiment was performed at least in triplicate. Student 33.1P < 0.05 to P < 0.001; Figures P < 0.01; Figure Effects of preincubation with different glucose concentrations on the AMPK pathway were detected in HepG2 hepatocytes and C2C12 myotubes as the processing methods shown in Figure 3.2P < 0.01 to P < 0.001; Figures The data above showed that glucose concentration increased from 5.6 to 15 mM greatly inhibited the activity of the AMPK pathway in the cells. However, under the same condition, significant phosphorylation of AMPK and ACC was observed after 10‐μM berberine or 5‐mM metformin was added Figures , 3. Berb3.3P < 0.01 to P < 0.001; Figure P < 0.01 to P < 0.001; Figure P < 0.05 to P < 0.001; Figure P < 0.01 to P < 0.001; Figure P < 0.01; Figure Although berberine and metformin caused a significant increase of AMPK phosphorylation in HepG2 cells when the glucose level increased from 5.6 to 15 mM ; when berberine and metformin were added, the depressed AMPK by glucose excess was stimulated. Conversely, while AMPK had been activated with glucose concentration descending, berberine and metformin suppressed the activated AMPK pathway significantly. For the first time, this study revealed the two‐way adjustment of the AMPK pathway by the agents. That led to precise regulation of glucose metabolism.The bidirectional regulation of AMPK activity by berberine and metformin may explain why the agents have minimal risk of hypoglycemia in clinical application. Our results suggested that berberine and metformin induced glycolysis and glucose consumption regardless of AMPK activity. When glucose concentration declined, AMPK was activated, whereas berberine and metformin inhibited the activated AMPK. That may prevent hypoglycemia by minimizing AMPK induced energy expenditure. In contrast, when glucose concentration ascended, berberine and metformin could activate the suppressed AMPK pathway and induce glycolysis simultaneously. That may maximize the antihyperglycemic effects of the agents. Thus, this study revealed that berberine and metformin exerted potent glucose‐lowering effects on hyperglycemia, and protective effects against hypoglycemia when glucose level dropped possibly via bidirectional regulation of AMPK activity.Our current study also aimed to identify whether AMPK activation was associated with glycolysis induced by berberine or metformin. Because Zhou et alIn this study, we found that both berberine and metformin significantly decreased intracellular ATP content. Our previous studies revealed that berberine and metformin promoted glycolysis, which was mainly attributed to inhibition of mitochondrial respiration.In our current study, the HepG2 and C2C12 cells were incubated at different concentration of glucose  followed by the moderate concentration of glucose to imitate different nutrient conditions including nutrient excess and nutrient deficient. AMPK has been identified as a master regulator of cellular energy status and plays a crucial role against energy‐restricted conditions.In conclusion, this study revealed a bidirectional role of berberine and metformin in the regulation of AMPK phosphorylation, depending on different nutrient conditions. AMPK, the master energy sensor, was turned on by the agents when glucose level ascended, and turned off when glucose level descended. The dramatic characteristics of berberine and metformin may lead to potent glucose‐lowering efficacy in the absence of hypoglycemia. In addition, berberine and metformin were able to induce glycolysis and glucose consumption, which was not related to the AMPK status. In the future, animal study is considered to further explore the impact of glucose alteration on AMPK activity and the efficacy of antihyperglycemic agents.The authors declare that there are no conflicts of interest.


Most people have left‐hemisphere dominance for various aspects of language processing, but only roughly 1% of the adult population has atypically reversed, rightward hemispheric language dominance (RHLD). The genetic‐developmental program that underlies leftward language laterality is unknown, as are the causes of atypical variation. We performed an exploratory whole‐genome‐sequencing study, with the hypothesis that strongly penetrant, rare genetic mutations might sometimes be involved in RHLD. This was by analogy with situs inversus of the visceral organs , which is sometimes due to monogenic mutations. The genomes of 33 subjects with RHLD were sequenced and analyzed with reference to large population‐genetic data sets, as well as 34 subjects (14 left‐handed) with typical language laterality. The sample was powered to detect rare, highly penetrant, monogenic effects if they would be present in at least 10 of the 33 RHLD cases and no controls, but no individual genes had mutations in more than five RHLD cases while being un‐mutated in controls. A hypothesis derived from invertebrate mechanisms of left‐right axis formation led to the detection of an increased mutation load, in RHLD subjects, within genes involved with the actin cytoskeleton. The latter finding offers a first, tentative insight into molecular genetic influences on hemispheric language dominance. Study design of the genome sequencing for rightward hemispheric language dominance. Images are shown from an example subject with typical left‐hemisphere language dominance, and an example subject with atypical rightward hemispheric language dominance, as assessed by functional magnetic resonance imaging. Genomic analysis was focused on rare, protein‐altering variants within genes and candidate gene‐sets. We also tested for an increased rate of rare mutations in RHLD within specific candidate gene sets, in case an increased load of mutations affecting specific biological processes might increase the chance of having RHLD. The candidate sets included genes involved in visceral laterality or the actin cytoskeleton, as well as a set of 18 genes which have been tentatively associated with human brain laterality in previous studies.22.1A total of 67 participants (33 with RHLD) were included in the present study, all of whom gave written informed consent. All RHLD subjects except one were left‐handed (Edinburgh Handedness Inventory [EHI] median = −87.50), while the controls included 14 left‐handed and 20 right‐handed participants . Summary statistics for language laterality measures and handedness are provided in Table The subjects in this study were recruited from two separate sources, that is, the BIL&GIN data set (France) and the GOAL data set (Belgium).2.2Seventeen RHLD subjects and 22 controls were drawn from a larger data set of healthy, young adults, balanced for handedness .2*‐weighted echo planar imaging acquisition  = 2 seconds; echo time (TE) = 35 ms; flip angle = 80°; 31 axial slices; 3.75mm3 isotropic voxel size).We studied hemispheric lateralization for three language tasks, namely production, reading and listening, using fMRI to calculate Global Hemispheric Functional Laterality Indexes (HFLIs), as described previously.http://www.fil.ion.ucl.ac.uk/spm/). Scans of each participant and each run were normalized to our site‐specific template, corrected for motion during the run, and then warped into the standard montreal neurological institute (MNI) space using a tri‐linear interpolation, with subsequent smoothing using a 6‐mm full width at half maximum (FWHM) Gaussian filtering. We then computed for each participant the BOLD signal difference maps and associated t‐maps corresponding to the “sentence vs word‐list” contrast for the production, reading and listening runs. For each individual and each language task, we computed a HFLI using the LI‐toolbox applied to the individual contrast t‐map of the considered language task.fMRI data analysis was performed using the SPM5 software .32*‐weighted gradient‐echo EPI sequence. Forty axial slices covering the whole brain were acquired .Sixteen RHLD participants were selected from a larger data set of healthy left‐handers N = 250)502 PartiThe 16 strongly right‐lateralized individuals all met a stringent criterion for RHLD (HFLI for language production < −50).Informed consent was obtained from all participants, and ethical approval for the study was obtained from the Ethics Committee of the Ghent University Hospital.44.1https://emea.illumina.com/systems.html). Thirteen additional subjects of European descent, who were not part of the present study, were also sequenced at the same time, and their data processed together with the 39 through preprocessing and variant calling stages  of the 39 BIL&GIN subjects was performed using Illumina's HiSeq technology by the genomics research organization and service company BGI (Hong Kong/Shenzhen)  paired‐end reads for 11 of the RHLD subjects and 14 controls, and 150 bp paired‐end reads for six RHLD subjects and eight controls. Raw reads were cleaned by excluding adapter sequences, reads with low‐quality bases for more than 50% of their lengths, and reads with unknown bases for more than 10% of their lengths. Clean reads were mapped onto the human reference genome (hg19) using the software Burrows‐Wheeler Aligner.These variants were then normalized, and variants belonging to any VQSR sensitivity tier over 99% were excluded. For the 39 BIL&GIN subjects of this study, the variant calling of SNPs and indels identified on average 4 165 806 variants per subject for the 90 bp protocol (range: 4 079 049‐4 330 101), and 4 484 638 per subject for the 150 bp protocol (range: 4 354 345‐4 657 333).5The genomics company Novogene (Hong Kong/Shenzhen) performed WGS on the 28 samples of the GOAL data set using Illumina's HiSeq Xten technology, and paired‐end sequencing with reads of 150 base pairs and 30x sequence depth. The same pipeline as that applied to the BIL&GIN data was used for alignment (build 37), variant calling, annotation and filtering . The variant calling and VQSR steps were carried out together with data from 34 European‐descent subjects who were not part of the present study, again because these steps benefit from a larger number of subjects. These variants were then normalized using the software tool vt normalize (v0.5772‐60f436c3)5.1P > .33). Inbreeding was assessed with the F coefficient estimate within each data set using PLINK (v1.9).P > .39).Within the BIL&GIN and GOAL data sets separately, population structure was assessed by calling genotypes from the sequence data for selected sets of common variants  spanning the autosomes. These were high‐confidence single‐nucleotide polymorphism (SNP) sites identified by the 1000 Genomes Project, 1000G_phase1.snps.high_confidence.hg19.vcf.gz with minor allele frequencies (MAFs) > 10% in each data set,Note that common genetic variants were only used for the purposes of assessing population stratification and inbreeding within the data sets, whereas the rest of the study was focused on rare genetic variation, which has the potential to involve highly penetrant effects.5.2http://evs.gs.washington.edu/EVS/, http://www.internationalgenome.org/home). Within the BIL&GIN and GOAL data sets separately, any variants present in at least 19 participants (case or control) were excluded as they are likely to be platform‐specific errors or else common variants not previously detected by other sequencing platforms or protocols, and would necessarily be present in at least two control subjects in BIL&GIN or three controls in GOAL (hence unlikely to be high‐penetrance mutations for RHLD).SNPs and indels were annotated using Annovar5.3Recessive: Here, we considered only homozygous or compound heterozygous mutations as potentially trait‐causal. For screening purposes, compound heterozygosity was assigned when a given gene had at least two different mutations, although allelic phase information was not usually available due to the limited sequence read lengths. Variants were excluded when they had MAF ≥ 10% on the basis of on‐line population databases (see above). At 10% MAF, assuming Hardy‐Weinberg equilibrium, the variant would be present in homozygous form at 1% in the population, that is, roughly equal to the RHLD frequency in the population. In the case that 50% penetrance might arise from L‐R randomization, as has been observed for mutations which cause situs inversus with PCD,Dominant: Here, we considered heterozygous or homozygous mutations as potentially trait‐causative. Variants were excluded as potentially causative when they had population MAF ≥ 1% in the population databases, on a similar logic as for the recessive model above, but appropriate for allelic dominance and the frequency of RHLD in the population (roughly 1%). Variants not present or with no MAF information in the population databases were retained. There were on average 196 genes per subject for the BIL&GIN‐90 bp protocol (range: 154‐215), 240 per subject for the BIL&GIN‐150 bp protocol (range: 208‐268), and 262 per subject for the GOAL data set (range: 229‐300).5.4The BIL&GIN and GOAL data sets were combined for subsequent analysis.t tests, all P > .10). Significance for single‐gene analysis was then assessed separately for individual genes and models (recessive or dominant), using the one‐tailed Fisher's exact test for a 2 × 2 contingency table, for the categories “mutated” and “not mutated” in 33 RHLD subjects and 34 controls. The minimum number of mutated RHLD subjects to achieve a nominally significant P value  was 5, that is, if a gene would be mutated in five out of the 33 RHLD subjects and none of the 34 controls, that gene would show a nominally significant P value of association with RHLD, as a putative major‐genetic effect . This approach allows for allelic heterogeneity, that is, the unit of testing is the gene, within which a variety of different mutations can be present. Note that the power and sample size considerations when modeling highly penetrant effects are different to typical genome‐wide association studies of common traits, in which large samples are screened for common variants of small effect. Here, we focus only on rare variants and interrogate the data with respect to the possibility of high penetrance. Note also that the Fisher's exact test is robust for the sample size, because the significance is assessed with respect to all of the actual possibilities that might have arisen in the contingency table in this set of subjects.We first verified that the total number of mutated genes per subject did not differ significantly between RHLD and control subjects, under either the dominant or recessive model  or 10 (recessive) of the 33 RHLD subjects, and no controls, leading to nominal P = .000186 (dominant) or P = .000373 (recessive) in the Fisher's exact test, that is, the gene would need to be a monogenic cause for roughly one third of the instances of RHLD. For these calculations, we counted how many individual genes, y, have mutations in at least x subjects, for every value of x from 1 to 67 subjects. For each value of x, we then calculated the minimum number of RHLD subjects with mutations in a given gene that would be required to produce a P value less than .05/y in the Fisher's exact test.We calculated that for an individual gene to be significant at We performed a post hoc filtering step in which we further excluded from consideration, as potentially monogenic effects, all genes which were mutated in at least one control subject, as these genes were unlikely to be causal monogenically for RHLD. Note that this filter was only applied after the statistical analysis, in order not to bias the multiple testing correction.5.5http://geneontology.org/gene-associations/goa_human.gaf.gz downloaded 16‐Nov‐2017), were “cilium” (GO:0005929), “left‐right axis specification” (GO:0070986), “actin cytoskeleton” (GO:0015629), plus two sets defined on the basis of visceral laterality phenotypes or disorders: 58 genes related to PCD and asymmetry disordersWe tested whether the RHLD cases had an increased mutational load in specific candidate gene‐sets . These candidate sets, based on the gene ontology (GO) as defined within AmiGO's direct annotationhttp://geneontology.org/gene-associations/goa_human.gaf.gz, downloaded 16‐Nov‐2017). Additional sets were investigated post hoc as child sets of the actin cytoskeleton set  per subject within the set was compared between RHLD subjects and controls by means of the one‐tailed exact binomial test, that is, considering the sum of mutated genes per subject in RHLD subjects only, the total sum across RHLD and controls combined, and the proportion of all subjects who were RHLD (33/67). Again, as an exact test, the binomial is robust for the subject sample size, and does not require assumptions on the number of mutations per individual.5.6TCTN1, rs188817098, which we initially considered a potential candidate for causing RHLD in some subjects (see P = 1), and the minor allele C had a frequency of 0.001305. Handedness (UK biobank field ID: 1707.0.0) was self‐reported and coded for the present purposes as “left‐handed” or “right‐handed”, as described elsewhere.Because the large majority of people with RHLD are left‐handed, any monogenic contributions to RHLD would likely also be strongly penetrant for left‐handedness. We checked whether a specific mutation of interest in the gene ects see , is also66.1We focused on mutations in the 33 RHLD cases which are known to be relatively rare in the general population on the basis of large‐scale genetic databases and predicted to disruptively affect protein sequence, while not being mutated in a set of 34 control subjects see . As noteP < .05), which could have arisen from being mutated in as few as five RHLD cases and no controls.Under a recessive model, no gene was even nominally significant , with five RHLD cases and no controls having heterozygous mutations . Recessive mutations in TCTN1 cause Joubert syndrome , a ciliopathy characterized by cerebellar and brainstem malformations.In the dominant model, TCTN1 missense variant , which has a maximum population frequency of 0.001199 (in ExAC non‐Finnish Europeans). This variant is present in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) as a variant of uncertain significance with potential relevance to Joubert syndrome/Meckel‐Gruber syndrome patients (SCV000634600.1). The other two TCTN1 mutations were a missense variant  and an in‐frame deletion (chr12:111070349 GATA/G), each present in one RHLD case only, and with maximum population frequencies of 0.0008 and 0.0033, respectively.Three of the five RHLD cases shared the same P = .034), with the minor allele C (frequency 0.001305) associated with left‐handedness (odd ratio = 1.24). However, this modest effect does not seem compatible with a role of this variant as a highly penetrant cause of RHLD and left‐handedness.Rs188817098 was also associated with handedness in the UK biobank data set , “left‐right axis specification” (GO:0070986), or sets defined on the basis of visceral laterality phenotypes or disorders,We investigated subsets of genes defined as belonging to specific components of the actin cytoskeleton, which included “actin filament” (GO:0005884), “myosin complex” (GO:0016459), and “cortical actin cytoskeleton” (GO:0030864), but saw no significant increase in mutation rates in RHLD in these sets Table . This maP = .04), RHLD vs left‐handed controls only (P = .004), right‐handed controls vs left‐handed controls (P = .88)  Table . This paP = .0006), while the effect was not significant in the GOAL data set (P = .4) despite having a similar trend of increased mutational load in RHLD cases  Although the TCTN1 variant showed a significant association with left‐handedness, in the expected direction , the effect size was not compatible with a highly penetrant effect. Therefore, this finding remains ambiguous.As RHLD is mostly found in left‐handed people,AR . For most of these genes, there is no clear mechanism that might link them to left‐right axis determination through chiral properties.In the present study, candidate genes that have been tentatively associated with human brain laterality in previous studies showed no evidence for an increase in mutation load in RHLD. The only gene among these that had more mutations in RHLD cases than those in controls was situs inversus of the viscera, when it occurs together with PCD, have shown normal population rates of left‐handedness and left hemisphere language dominance.We also found no evidence that candidate gene sets involved in visceral laterality or PCD have an enrichment of rare, protein‐altering mutations in RHLD. This finding is consistent with the fact that people with MAP2 have recently been associated with left‐handedness by large‐scale GWAS,Intriguingly, it may be that situs inversus of the visceral organs does associate with left‐handedness when not due to mutations affecting the nodal ciliary pathway,The possible link of RHLD to actin cytoskeleton genes will need to be replicated in larger independent data sets. Within the present study, we combined the BIL&GIN and GOAL data sets to maximize the power to detect genetic effects on RHLD, although the functional tasks used to define RHLD differed between these two data sets: hemispheric dominance was defined using a contrast at the sentence level in BIL&GIN, and a word‐level contrast in GOAL see . HoweverGiven the sample size for the present study, we focused on rare, protein‐altering mutations which had the potential to be highly penetrant effects. Whole genome sequence data, of the type produced in the present study, also contain information on noncoding variation. Rare noncoding variation has recently been implicated in neurodevelopmental disorders such as autism,Data sets based on hundreds of thousands of participants, such as the UK biobank,The authors declare no potential conflict of interest.Table S1. All putative mutations in actin cytoskeleton genes. Chr, chromosome; Ref, reference allele; Alt, alternative allele; MAF, maximum minor allele frequency across 1KG; ExAC, gnomAD populations; RS ID, refers the variant identity in dbSNP; AA, amino acid; PrD, probably damaging; PosD, possibly damaging. The RHLD and Ctrl columns show the numbers of these mutations in cases and controls . The 113 remaining genes in the actin cytoskeleton gene set, which had no mutations in this data set, were as follows: ABCB4, ABL2, ACACA, ACTA2, ACTB, ACTR2, ACTR3, ADAM17, AIF1L, ALDOA, AMPH, ANLN, ARC, ARHGAP21, ARHGAP35, ARHGAP6, ARPC1A, ARPC2, ARPC3, AUTS2, BAIAP2, BAIAP2L1, BAIAP2L2, BIN1, C10orf90, CALD1, CAPZA1, CAPZA2, CAPZB, CARMIL2, CASK, CATIP, CD2AP, CDC42EP3, CDH1, CENPQ, CFL2, CLIC4, CNN3, CNR1, CORO1C, CORO2A, CORO2B, CORO6, CRK, CTDP1, CTNNA2, DAPK1, DDX58, DHX9, DMTN, DSTN, FGR, FOXA3, FSCN1, FYB1, GABARAP, GAS2L3, H1F0, HAP1, HAX1, HNRNPC, HSPB7, IFIT5, INTS6, IPP, IQGAP2, IVNS1ABP, KANSL2, KLHL14, KLHL2, KLHL20, MARCKS, MPRIP, MSRB1, MYL2, MYLK3, MYOT, MYOZ1, MYOZ2, MYOZ3, NCOA5, ONECUT2, OPHN1, PGM1, PKNOX2, POU6F1, PPP1R12A, RAB22A, RAB5A, RARA, RND1, SEPT2, SEPT9, SORBS2, STK17B, STK38L, STOML2, TAF5, TARS, TAX1BP3, TLN2, TMEM63B, TOPBP1, TPM1, TPM2, TPM3, TRMT10A, TTC17, USH1G, WAS, WIPF1, ZNF174.Table S2. Mutation analysis of GO:0015629 “actin cytoskeleton” in relation to its child sets, and negative control gene sets of similar sizes to GO:0015629. Set size: number of genes within set. RHLD: instances of genes carrying mutations within RHLD cases; Total: instances of genes carrying mutations in RHLD cases and controls combined. The P‐value is shown from the exact binomial test, where the null probability was .4925 (33/67) participants being RHLD) and alternative hypothesis = “greater”. Negative control sets were identified which had set sizes similar to the actin cytoskeleton set, and no known links to the biology of left‐right asymmetry.Table S3. Mutation analysis of GO:0015629 “actin cytoskeleton” by different handedness groups. Test: instances of genes carrying mutations within the test group; Total: instances of genes carrying mutations in the test and contrast groups. Proportion = Test/Total. Null: null probability, given the proportion of test subjects for the contrast being tested. The P‐value is shown from the exact binomial test, with the alternative hypothesis = “greater”.Table S4. Mutation analysis of GO:0015629 “actin cytoskeleton” within each data set. Test: instances of genes carrying mutations within the test group; Total: instances of genes carrying mutations in the test and contrast groups. Proportion = Test/Total. Null: null probability, given the proportion of test subjects for the contrast being tested. The P‐value is shown from the exact binomial test, with the alternative hypothesis = “greater”.Figure S1. Multidimensional scaling (MDS) to capture overall genomic diversity among the study samples , in relation to the 1000 Genomes populations of known geographic ancestries (dots). 1KG super population codes: AFR, African; EAS, east asian; AMR, Ad Mixed American; EUR, European.Figure S2. Histogram distribution of the manual preference strength variable assessed by the Edinburgh Handedness Inventory (EHI) score across RHLD cases and controls, per data set.Figure S3. Distribution of mutations within the “actin cytoskeleton” gene set.Click here for additional data file.


L‐type voltage‐gated calcium ion channels (L‐VGCCs) have been demonstrated to be the mediator of several significant intracellular activities in excitable cells, such as neurons, chromaffin cells and myocytes. Recently, an increasing number of studies have investigated the function of L‐VGCCs in non‐excitable cells, particularly stem cells. However, there appear to be no systematic reviews of the relationship between L‐VGCCs and stem cells, and filling this gap is prescient considering the contribution of L‐VGCCs to the proliferation and differentiation of several types of stem cells. This review will discuss the possible involvement of L‐VGCCs in stem cells, mainly focusing on osteogenesis mediated by mesenchymal stem cells (MSCs) from different tissues and neurogenesis mediated by neural stem/progenitor cells (NSCs). Additionally, advanced applications that use these channels as the target for tissue engineering, which may offer the hope of tissue regeneration in the future, will also be explored. Based ov1.1 to Cav1.4, which are distributed widely among different tissues and cells, with distinct pharmacological and biophysical properties to support specific cellular tasks.v1.1, which is mainly present in skeletal muscle and expressed at low levels in other tissues, can serve as the voltage sensor mediating the process of excitation‐contraction coupling.v1.2 is widely expressed in various tissues including smooth muscle,v1.2 is indispensable for calcium ions to enter the cardiac cells and initiate cardiac excitation‐contraction coupling during the plateau phase of the action potential. Additionally, Cav1.2 is also expressed in most types of neurons, where it activates calcium‐dependent enzymes and initiates calcium‐dependent gene transcription.v1.3 is similar to that of Cav1.2.v1.3 has been shown to initiate pace‐making in dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc)v1.3 regulate the secretion of catecholamine from chromaffin cellsv1.2 and Cav1.3, Cav1.4 appears to be restricted to the retina,v1.1), Timothy syndrome (a glycine‐to‐arginine mutation at position 406 in Cav1.2), SAN dysfunction and deafness syndrome (Cav1.3), and incomplete congenital stationary night blindness (Cav1.4).According to differences of the α1 subunits, L‐VGCCs have four subtypes, ranging from CaWhile there is ample evidence supporting the vital function of L‐VGCCs in excitable cells including neurons,2Mesenchymal stem cells are multipotent cells that have a wide distribution in several tissues including bone marrow, adipose and dental tissues, and have the ability to differentiate into a large number of cell types including osteoblasts and adipocytes, as well as chondrocytes and neurons.2.1Bone marrow‐derived mesenchymal stem cells (BMSCs) are acquired mainly from bone marrow, where they account for <0.0001% of all cells. Considering the advantages of easy accessibility, multipotent differentiation and low immunogenicity, BMSCs have been widely studied and hold great promise for treating a variety of immune‐mediated diseases and/or tissue defects.2.2v1.2 can inhibit the differentiation of rDPSCs.v1.2 also plays significant roles in the odontoblastic differentiation. Using Cav1.2 shRNA to knock‐down Cav1.2 in SCAPs, they found that the formation of calcium nodule, a product of late stages of osteogenic differentiation that can reflect the ability of stem cells to differentiate into osteoblasts, was suppressed.Dental tissues are accessible resources that provide an abundant reservoir of MSCs, such as dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), stem cells from the apical papilla (SCAPs) and periodontal ligament stem cells (PDLSCs). To date, many studies have investigated the involvement of L‐VGCCs in regulating the functions of dental MSCs. DPSCs are derived from dental pulp and possess the capacity for multi‐lineage differentiation towards bone, cartilage and fat.2.32+ concentration evoked by ATP, high K+ solution, GABA and caffeine demonstrated that functional VGCCs are absent in AMSCs isolated from rats.v1.1 and defective proliferation of AMSCs. AMSCs displayed a decreased proliferation capacity and increased apoptosis after treatment with indoxyl sulphate, a digestive intermediate product that can induce chronic kidney disease, along with significantly downregulated expression of Cav1.1.In addition to the bone marrow, MSCs also exist in many other tissues, including adipose and skin tissues. The patch‐clamp analysis demonstrated that human adipose‐derived mesenchymal stem cells (AMSCs) possess functional VGCCs, although they account for <1%.32+ is needed for ESCs to differentiate towards a specific tissue fate.2+ concentration evoked by ATP, high K+ solution, GABA and caffeine.2+ entry by a high K+ solution did not change the intracellular free Ca2+ concentration.2+ currents, significantly promoted neuronal functional maturation, and this process could be inhibited by the L‐VGCCs antagonist nifedipine. Mechanistically, they discovered complete inhibition of IBMX‐induced CREB phosphorylation (cAMP‐response‐element‐binding protein) by nifedipine, which indicates that L‐VGCCs‐mediated maturation of NSCs depends on enhanced intracellular cAMP levels.2+ signalling was significantly inhibited and neurogenesis was also decreased in mESCs deficient in the Ca2+ release channel type 2 ryanodine receptors (RyR2). At the same time, the activation of L‐type Ca2+ channels or ryanodine receptors promoted neuronal differentiation in RyR2+/+ cells but not in RyR2−/− deficient mESCs. Thus, cooperation of L‐VGCCs and RyR2 is indispensable for neural differentiation of mESCs.Embryonic stem cells (ESCs) are isolated from the inner cell mass of the preimplantation embryo. During embryogenesis, elevated intracellular Ca4Neurogenesis is a process with several stages such as proliferation, fate determination, selective death/survival and maturation, in which neurons or glial cells are generated from NSCs and other neural progenitor cells. NSCs play an essential role in neural development because of their ability for pluripotent differentiation and are most active during pre‐natal neurosystem development. By contrast, adult NSCs, which are considered to be limited in their ability of differentiation,v1.2 and Cav1.3 are the predominant L‐VGCCs isoforms expressed in several regions of the central nervous system (CNS), including the cerebral cortex, amygdala, cerebellum and hippocampus. They are deeply involved in the physiological behaviours of the CNS such as cognition and memory formation.2+ influx facilitate the survival and maturation of immature neurons.v1.2 is only expressed in cells undergoing the final stages of neuronal differentiation. The neurogenesis controlled by L‐VGCCs can be enhanced by the L‐channel activator Bay K 8644, as well as by an agonist of Cav1.2/Cav1.3, FPL 64176.v1.3 knock‐down rats produced using adeno‐associated virus (AAV) or retrovirus‐based technologies showed impaired dendritic or axonal formation in vivo.Several studies suggest that the L‐type calcium current has a strong correlation with the differentiation of NSCs. Ca2+‐dependent regulatory genes in differentiation, such as BETA2/NeuroD1,B receptor can increase Cav1.3 expression in NSCs,A receptors would facilitate the Ca2+ influx through L‐type Ca2+ channels.To further study the mechanism of the regulation of neurogenic activities by L‐VGCCs, researchers investigated the involvement of Ca5v1.2 is expressed by HFSCs, where it inhibits quiescence and contributes to tissue regeneration in a calcium‐independent signal by regulating the production of the follistatin‐like 1 protein, an antagonist of BMP signalling.Cardiac progenitor cells (CPCs), which are mainly located in the myocardium, express the cardiogenic genes.6Tissue engineering is an emerging interdisciplinary field involving the use of an interactive triad of scaffolds, signalling molecules, engineering and cells, focusing on producing functional replacement tissues and creating favourable conditions for tissue regeneration.6.1v1.2 and α2D1 in undifferentiated human PDLSCs, suggesting that L‐VGCCs may be a potential target for periodontal tissue engineering.Large bone defects caused by trauma, infections and other causes6.2Heart failure, especially myocardial infarction, is one of the leading global health challenges that threaten the well‐being of more than thirty million people annually.2+ regulates excitation‐contraction coupling, as well as the localization of cardiogenic transcription factors in CPCs.v1.2 is believed to be the predominant L‐VGCCs expressed in ventricular myocytes, whereas both Cav1.2 and Cav1.3 are expressed in atrial cells as well as sinoatrial and atrioventricular node cells.2+ influx through Cav1.2 channels triggers the intercellular release of Ca2+ from the sarcoplasmic reticulum through type‐2 ryanodine receptors (RyR2), which is essential for myocardial excitation‐contraction coupling activity, and loss‐of‐function mutations of L‐VGCCs can lead to cardiac failure.Researchers have confirmed that calcium influx is an integral second messenger for the early steps of cardiomyocyte specification and commitment, leading to the differentiation of stem cells by modulating cardiac gene expression.6.3v1.2 and Cav1.3, which are widely expressed in multiple brain regions such as the cerebral cortex, amygdala, cerebellum and hippocampus,v1.3−/− mice with severe impairments in hippocampus‐associated cognitive functions,v1.3 involves in neurogenesis and cognitive function formation, giving promising future for neurological recovery enhancement through L‐VGCCs.Since adult NSCs are limited in differentiation and distribution, neurons are refractory to replication. The reprogramming or regeneration of human neural tissue2+ channels, especially Cav1.2 and Cav1.3.2+ influx and a higher percentage of responsive neurons results in increased proliferation and neural differentiation. The exposure can also stimulate CREB phosphorylation, which mediates the effects of intracellular Ca2+ signals on gene expression and is thus involved in neuronal differentiation and neurogenesis.2+ channels, which in turn led to neural differentiation, as well morphological and functional maturation of neural stem cells.Considering the indispensable role of L‐VGCCs in neurogenesis, researchers are striving to find ways to modulate neurogenic activities by targeting L‐VGCCs, with some success. The exposure to extremely low‐frequency (50‐Hz) electromagnetic fields (ELFEFs) was found to increase the expression and modulate the function of voltage‐gated Ca7In this review, we discussed not only the important roles of L‐VGCCs in the functional regulation of several stem cell types, but also the potential application of drugs targeting L‐VGCCs in tissue engineering. As discussed above, L‐VGCCs partake in the regulation of osteogenic, myogenic and neural differentiation, which makes their agonists or antagonists highly promising candidates for the repair and regeneration of bone, heart and neural tissue.2+ influx by the overexpression of L‐VGCCs modulated the processing of the beta amyloid (Aβ) precursor protein, leading to a positive feedback loop and ultimately the development of Alzheimer's disease.Notwithstanding, there are still considerable challenges within this field. First, functional L‐VGCCs are the precondition for the efficacy of the drugs targeting them, but they are present only in a portion of stem cells. Secondly, the role of L‐VGCCs in osteogenic differentiation still remains controversial, partly due to the varying characteristics of different stem cell types. Thus, more basic research is needed before attempting the therapy targeting L‐VGCCs for bone regeneration. Thirdly, it has been reported that L‐VGCCs can be detrimental to possibly cause severe neurodegenerative diseases. For example, researchers found that increasing CaThe authors have declared that no competing interest exists.Yi‐zhou Tan, Dong‐dong Fei and Xiao‐ning He: Conception and design, manuscript writing; Ji‐min Dai, Rong‐chen Xu and Xin‐yue Xu: Manuscript writing; Jun‐jie Wu and Bei Li: Conception and design, financial support, final approval of manuscript.


Sensitization is often work‐related. We report a case of an anaphylactic reaction to chloramine‐T.Chloramine‐T . She was diagnosed with anaphylaxis, and observed and treated with 0.5 mg of intramuscular adrenaline and 2 mg of intravenous clemastine at the hospital. Some hours later, she was discharged, with only diffuse mild erythema remaining. She had performed cleaning activities at a butchery for 17 years without using gloves. She had regularly developed localized wheals after skin contact with chloramine‐T. We performed prick tests with an in‐house preparation of 10 mg/mL of the patient's product. Readings were performed after 15 minutes. Physiological salt as a negative control caused no wheal or flare. Histamine, as a positive control, and chloramine‐T caused erythematous wheals and flares with mean diameters of 6 and 12.5 mm, respectively (Figure This is the first report of an anaphylactic reaction caused by immediate‐type hypersensitivity to chloramine‐T. On review of the literature, we found several case reports describing urticaria, rhinitis and asthmatic bronchial obstruction caused by chloramine‐T after skin contact or airborne exposure.The authors have no conflicts of interest to report.


This study aimed to compare intubation performances among i-gel blind intubation (IGI), i-gel bronchoscopic intubation (IBRI), and intubation using Macintosh laryngoscope (MCL) applying two kinds of endotracheal tube during chest compressions. We hypothesized that IGI using wire-reinforced silicone (WRS) tube could achieve endotracheal intubation most rapidly and successfully. p < 0.05 between two devices or p < 0.017 in post hoc analysis of the comparison among three devices.  In 23 emergency physicians, a prospective randomized crossover manikin study was conducted to examine the three intubation techniques using two kinds of endotracheal tubes. The primary outcome was the intubation time. The secondary outcome was the cumulative success rate for each intubation technique. A significant difference was considered when identifying p < 0.017) than that of using IBRI and MCL in both endotracheal tubes  tube; 14.6 vs. 27.4 vs. 19.9 in WRS tube; sec). There were no significant (p < 0.05) differences between PVC and WRS tubes for each intubation technique. The intubation time to reach 100% cumulative success rate was also shorter in IGI (p < 0.017) than that in IBRI and MCL in both PVC and WRS tubes.  The mean intubation time using IGI was shorter ( IGI was an equally successful and faster technique compared with IBRI or MCL regardless of the use of PVC or WRS tube. IGI might be an appropriate technique for emergent intubation by experienced intubators during chest compressions. Airway management in cardiac arrest is essential for successful resuscitation . GloballIn CPR for OHCA patients, advanced airway device insertions such as SAD or endotracheal intubation (ETI) are not prior to chest compressions (CCs). When CCs are performed properly, advanced airway device insertion should be considered for optimal oxygenation. Especially in OHCA caused by serious respiratory causes such as acute respiratory distress syndrome or airway obstruction, ETI can be more appropriate than SAD insertion to correct hypoxia and to improve survival ultimately .Nevertheless, ETI using direct laryngoscope during CPR could not be easily achieved even if it is performed by experienced emergency physicians . The verETI through inserted i-gel has already been attempted by anesthesiologists in the form of fiberoptic bronchoscope-guided ETI –10. HoweWe assumed that IGI could minimize interruptions of CCs and achieve successful ETI during CPR. In addition, when using conventional polyvinyl chloride (PVC) endotracheal tube (ETT) in IGI, the PVC tube can be folded and compressed when passing through i-gel. We thought it might result in an increase of hands-off time and decrease of ETI accuracy. So, we assume that the use of wire-reinforced silicone (WRS) tube instead of PVC tube could overcome this problem .This study aimed to compare intubation performances among IGI, i-gel bronchoscopic intubation (IBRI), and intubation using Macintosh laryngoscope (MCL) applying two kinds of ETT during CCs. We hypothesized that IGI using WRS tube could achieve ETI most rapidly and successfully.NCT02411422).We conducted a randomized crossover manikin study to examine intubation performance using two intubation techniques through i-gel (IGI and IBRI), direct laryngoscopy (MCL), and two ETTs during CCs. This study was performed at Hallym University's simulation center in February 2015. The local ethics committee approved this study in February 2015  of Hallym University Kangnam Sacred Heart Hospital). We registered the study protocol in Clinical Trials before study initiation  and i-gel™ , flexible intubation scope  for ETI with fiberoptic guidance. We use two types of ETT, e.g., PVC tube  and WRS tube .Participants performed IGI and IBRI using size-4 i-gel  for medium adult (50–90 kg). A flexible intubation scope was used to guide ETT in IBRI. For direct laryngoscope, Macintosh blade (MCL) was used; this device has a size-4 curved blade with a Satin Slip Stylet . Two types of ETTs with an internal diameter of 7.0 mm were used in this study.We used a high-fidelity manikin  to perform CCs and ETI. The normal (nondifficult) airway setting was maintained in the manikin during the study.The bed-height setting of this study was simulated using a bed  with a foam mattress . A backboard  was placed on the bed. The height of the stretcher bed was adjusted to 88.6 cm (bed height: 80 cm + foam mattress: 7.6 cm + backboard: 1 cm) for both ETI and CCs.Instructors gave 1-hour lecture and 2-hour practice for high-quality CPR, ETI using i-gel as blind conduit (IGI), and ETI using i-gel as conduit with fiberoptic guidance (IBRI) for 23 subjects and 4 chest compressors. All instructors were advanced cardiovascular life support (ACLS) instructors certificated by AHA (American Heart Association). They also had more than 500 times experience for ETI and more than 50 times experience for IGI and IBRI. The CPR lecture was constituted with the appropriate chest compression (CC) rate (100–120 bpm), CC depth (5–6 cm), and complete chest recoil and avoiding hyperventilation. The lecture for IGI and IBRI was constituted with the method of i-gel insertion, how to use flexible intubation scope, and the confirmation method of successful intubation. In the IGI and IBRI practice, the subjects were required for more than 10 times drill for IGI and IBRI, respectively. The successful intubation was confirmed by chest rise during bag mask ventilation via ETT. All chest compressors during ETI were AHA-BLS providers. They performed 2 min high-quality CC during ETI on an ALS simulator to prevent fatigue. In the CPR practice, they were requested more than 5 times drill for 2 min hands-only CPR under guidance by ACLS instructors. The CPR quality was monitored by the feedback system of high-fidelity manikin.http://www.random.org) was used for random order generation in order to minimize learning effect.After lecture and practice session, subjects were randomly divided into 2 groups by kinds of ETT firstly. For dividing participants to 2 groups, we used drawing lots. The sequence generator  provider performed chest compression to the ALS simulator at a rate of 100 to 120 per minute and 5 to 6 cm depth with complete chest recoil. We use airway lubricant  when i-gel was used as a conduit for smooth insertion.st ventilation and chest rising were achieved through ETT by bagging bag-valve mask . , when ETT passed the vocal cord  and when 1e, FVT). .In the procedure of IGI, subjects inserted i-gel to the ALS simulator, and then ETI was performed using i-gel as a blind conduit. In that order, the subject gave a ventilation through inserted ETT by bagging of bag-valve mask and verified chest rising. Before the subject performed IGI, instructors gave information for successful IGI such as sniffing position and counterclockwise rotation of ETT.In the procedure of IBRI, we mounted an ETT #7.0 on the bronchoscope before the procedure began. Firstly, subjects inserted i-gel to the ALS simulator, and then subject inserted the bronchoscope to 15 mm connector of i-gel and checked the vocal cord while watching the screen monitor. Then, subjects passed the bronchoscope through vocal cord and verified carina. We pushed the mounted ETT through the bronchoscope and removed it and gave a ventilation through ETT with a bag-valve mask and verified chest rising.When randomization of three kinds of ETI methods was finished, the subject performed ETI with another type of tube. The sequence of ETI methods was rerandomized.All recordings were fulfilled by one instructor. We verified successful ETI by inserting the bronchoscope to ETT for visual, confirming carina.We established intubation time as a primary outcome and cumulative intubation success rate as a secondary outcome.We regarded esophageal intubation and exceeding 2 minutes from starting intubation to first ventilation as failed intubation. In all ETI attempts, verification of successful ETI using the bronchoscope was done.p value was less than 0.05 between two devices and less than 0.017 was considered as significant difference in post hoc analysis of the comparison among three devices.The data were collected and arranged using a standard spreadsheet application . Statistical analysis was carried out with the 22.0 version of the Statistical Package for the Social Sciences (SPSS) program for windows . We described statistics as frequencies and percentages for demographic data and mean ± standard deviation (SD) for continuous data. We used the Shapiro–Wilk test for verifying normal distribution, and we used the Wilcoxon signed-rank test because the result was not according to normal distribution. In addition, we used the Friedman test for comparing three intubation methods and applied Bonferroni's method for post hoc analysis. Kaplan–Meier analysis was performed to analyze the cumulative success rate for intubation time. A significant difference was considered when Twenty-three subjects participated in this study. The baseline characteristics of the participants are shown in p=0.277; IGI vs. MCL, p=0.001). (IIT in IGI (4.1 ± 1.4 sec) was equal to that in IBRI (4.3 ± 1.2 sec) and significantly shorter than VET in MCL (5.8 ± 2.3 sec) regardless of kinds of ETT . .p=0.001; IGI vs. MCL, p=0.007). In WRS tube, IIT-TPT in IGI (7.3 ± 2.3 sec) was equal to that in MCL (7.1 ± 4.0 sec) and shorter than that in IBRI (18.8 ± 17.5 sec) . There were no differences between PVC and WRS tubes in the comparison of each of the three intubation techniques .  was significantly shorter than that of IBRI (20.0 ± 12.2 sec) and longer than that of MCL (7.0 ± 8.9 sec) . .p < 0.001; IGI vs. IBRI, p < 0.001). Similarly, in WRS tube, TPT-FVT in IGI (3.3 ± 0.9 sec) was also shorter than that of IBRI (4.6 ± 2.0 sec) and MCL (7.0 ± 2.0 sec) . There were no significant differences between PVC and WRS tubes in the comparison of each of the three intubation techniques .  was significantly shorter than that of IBRI (4.6 ± 0.9 sec) and MCL (7.0 ± 2.0 sec) . .p=0.016; IGI vs. IBRI, p < 0.001). In the WRS tube, FVT in IGI (14.6 ± 3.4 sec) was also shorter than that of IBRI (27.4 ± 19.1 sec) and MCL (19.9 ± 6.7 sec) . There were no significant differences between PVC and WRS tube in the comparison of each of the three intubation techniques .  was shorter than that of IRBI (29.3 ± 12.5 sec) and MCL (20.2 ± 9.7 sec) . .p=0.002; IGI vs. IBRI, p < 0.001).  to reach 100% cumulative success rate . .This study demonstrated that the IGI was equally successful and faster technique compared with IBRI or MCL regardless of the use of PVC or WRS tube. To our knowledge, this is the first study which compares the intubation performance of IGI with that of IBRI during chest compressions.According to the instruction manual for i-gel, IBRI is recommended for i-gel-guided intubation , 15. IBRThe blind intubation through SAD such as IGI is not a usual method for emergent intubation in arrest patients . ClinicaRegarding the nature of blind technique of IGI, the success rate for IGI has been reported from 75% to 100% despite the high speed of IGI , 18. To In this study, we compared the efficacy of the PVC tube compared with that of the WRS tube in IGI and IBRI. The WRS tube can be more advantageous than the PVC tube, because the WRS tube is more flexible and noncompressible during the passage through i-gel , 20. NevThis study has some limitations. First, the results of this study were based on manikin simulation. In a clinical setting, there will be several significant factors affecting the intubation performance. These factors include the dynamic hindrance for glottic view during chest compressions, the anatomical variation of airway, and the hindrance by blood or vomitus of arrest patients . Thus, tSecond, this study is a small sample pilot study for IGI and IBRI. Recently published regarding studies showed similar study design with this study –24. FurtThird, although all participants had more than 50 times intubation experiences, the possibility of the experience difference still exists among junior and senior residents and EPs. These unequal experiences for intubation could affect the intubation performance such as intubation time or success rate.IGI was an equally successful and faster technique comparing with IBRI or MCL regardless of the use of PVC or WRS tube. Therefore, IGI might be an appropriate technique for emergent intubation by experienced intubators during CCs.


Pre‐mRNA splicing is a complex process requiring the identification of donor site, acceptor site, and branch point site with an adjacent polypyrimidine tract sequence. Splicing is regulated by splicing regulatory elements (SREs) with both enhancer and suppressor functions. Variants located in exonic regions can impact splicing through dysregulation of native splice sites, SREs, and cryptic splice site activation. While splicing dysregulation is considered primary disease‐inducing mechanism of synonymous variants, its contribution toward disease phenotype of non‐synonymous variants is underappreciated.F9 exonic point variants including both synonymous and non‐synonymous for their effect on splicing using a series of in silico splice site prediction tools, SRE prediction tools, and in vitro minigene assays.In this study, we analyzed 415 disease‐causing and 120 neutral The use of splice site and SRE prediction tools in tandem provided better prediction but were not always in agreement with the minigene assays. The net effect of splicing dysregulation caused by variants was context dependent. Minigene assays revealed that perturbed splicing can be found.Synonymous variants primarily cause disease phenotype via splicing dysregulation while additional mechanisms such as translation rate also play an important role. Splicing dysregulation is likely to contribute to the disease phenotype of several non‐synonymous variants. The current version of Center for Disease Control and Prevention's (CDC) Hemophilia B Mutation Project (CHBMP) database (f9‐CHBMP‐v5‐5‐5‐15) contains 1,131 F9 variants , nonsense variants , intronic variants causing splice site changes , frameshift variants , large structural changes of >50 base pairs , small structural changes , promoter variants , 3′Untranslated region variants , and synonymous variants . Of these, missense variants and synonymous variants in exonic regions are point variants that involve single‐nucleotide substitution and make up majority (59%) of F9 variants.The partial or complete absence of FIX leads to the blood clotting disorder, hemophilia B Bowen, . Based oSynonymous variants do not alter the underlying aa sequence and are considered to primarily cause disease by altering splicing of pre‐mRNA transcript , SpliceSiteFinder‐like (SSF‐like), and Human Splicing Finder (HSF) splice site prediction tools that are included in the Alamut visual software . These tools employ different methods to predict splice sites: NNSPLICE employs two “neural networks” that were trained on consensus splice sites, while also considering dinucleotide frequencies due to the strong correlation between neighboring nucleotides in splice site consensus sequences  combines the predictions of several individual SRE prediction tools  for the presence of ESEs and ESSs and assigns scores based on their relative density  plasmid . Total RNA was isolated 24 hr posttransfection using RNeasy Plus Mini Kit  and reverse transcription was performed using High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). PCR amplification was performed with Platinum Hot Start PCR Master Mix (Thermo Fisher Scientific) using Alfa2‐3 forward primer; 5′‐CAACTTCAAGCTCCTAAGCCACTGC‐3′ and Bra2 reverse primer; 5′‐GTCACCAGGAAGTTGGTTAAATCA‐3′  on FIX global translation rates was assessed by performing in vitro translation assay. Assay was performed with Rabbit Reticulocyte Lysate (RRL) cell‐free system (Promega) supplemented with calf liver tRNAs as described earlier  also reported relatively lower scores for acceptor site of exon 4 (2.56 vs. 9.9 ± 1.50 for other corresponding sites) and donor site of exon 5 (6.49 vs. 11.84 ± 1.17 for other corresponding sites), suggesting that these sites are possibly weak and less conserved.We employed NNSplice, MES, SSF‐like, and HSF tools to assess the effect of exonic disease‐causing and neutral variants on disruption of native splice sites and/or activation/creation of cryptic splice sites. A total of 535 variants from our curated database Table  that incls Table . Our anals Table . An addiF9 exonic disease‐causing and neutral variants analyzed in this study are provided in Table In our study, as previously described in the Materials and Methods section, variants were considered to affect splicing upon weakening of native site (≥10% score decrease) or activation/creation of a comparably strong cryptic site (≥90% of native score). Complete details of splice site score changes reported by in silico splice site prediction tools for 3.2To validate the predictions of the in silico splice site tools, in vitro minigene assays were performed for selected variants. Details of variants selected for this assay, in silico predictions by splice site tools, and results from minigene assays are shown in Table In exon 4, we evaluated four disease‐causing non‐synonymous variants: c.278A>G, c.316G>A, c.365G>T, and c.373G>A in comparison with wild‐type exon 4 sequence. In the minigene assays, exon 4 reporter constructs should yield a 360 bp PCR product upon normal splicing (exon inclusion) and 246 bp PCR product in the event of exon skipping. In our study, exon 4 wild‐type sequence and c.278A>G variant, that was predicted to increase native acceptor site strength and positively influence splicing, showed normal splicing with 100% exon inclusion. C.316G>A and c.373G>A variants, that were predicted to create a new cryptic acceptor site of significant strength by SSF‐like and HSF tools, showed 0 and 1.74% of exon skipping, respectively. C.365G>T variant, that was predicted to activate a cryptic acceptor site by all four tools, showed 5.59% exon skipping.In exon 5, we evaluated the following variants: c.423C>T, c.459G>A, c.465C>T, c.479G>T, c.479G>C, c.484C>A, and c.513A>G in comparison with wild‐type exon 5 sequence. In the minigene assays, exon 5 reporter constructs should yield a 375 bp PCR product upon normal splicing and 246 bp PCR product in the event of exon skipping. In our study, exon 5 wild‐type construct showed 34.31 exon skipping Figure . The exoIn exon 6, we evaluated the following variants: c.535G>A, c.677G>A, c.711A>G, c.715C>T, c.719G>T, and c.723G>A in comparison with wild‐type exon 6 sequence. In the minigene assays, exon 6 reporter constructs should yield a 449 bp PCR product upon normal splicing and 246 bp PCR product in the event of exon skipping. Of the variants analyzed, c.535G>A, c.677G>A, c.715C>T, and c.719G>T are non‐synonymous disease‐causing variants that were predicted to create/activate cryptic acceptor sites predominantly by SSF‐like and HSF tools. In minigene assay, these variants showed normal splicing with 100% exon inclusion similar to wild‐type sequence. In exon 6, c.711A>G and c.723G>A are synonymous disease‐causing variants. Of these, c.711A>G was predicted to activate a new cryptic donor site by all four tools. In our study, this variant induced mis‐splicing with the loss of 17 nucleotides in the exon , the predominant effect on splicing was exon skipping rather than mis‐splicing from the use of predicted cryptic splice sites. Also, c.484C>A, a disease‐causing synonymous variant that was not predicted to effect splicing by all splice site prediction tools showed significant exon skipping. These results suggested a possible role of SREs in the dysregulation of splicing by these variants. To assess the dysregulation of SREs by the exonic variants under study, we calculated the score changes for variant sites in comparison with native sequence using ESRseq, HEXplorer methods, and the EX‐SKIP tool  and 120 neutral variants (64 non‐synonymous and 56 synonymous) for their effect on splicing using in silico splice site prediction tools and further evaluated selected variants using in vitro minigene assays and in silico SRE prediction tools.In this study, we analyzed a curated dataset of 535 F9 gene indicated that exon 4 and 5 carry weak acceptor and donor site, respectively  showed increased exon skipping relative to respective wild‐type sequences. Interestingly, in addition to exon skipping, c.479G>T also induced mis‐splicing through activation of cryptic donor site. These results underscore how splicing dysregulation can contribute partially or wholly to disease phenotype induced by non‐synonymous variants. All the neutral variants tested by minigene assays were synonymous and located in exon 5. Of these, c.423C>T and c.513A>G variants showed increased exon skipping relative to wild‐type sequence. The partial dysregulation of splicing induced by these neutral variants may not be sufficient to lower the serum FIX levels (to <40%) to induce hemophilia B. Overall, results from our study show that An important observation from the minigene assay data was that some of the variants that were predicted to activate a cryptic donor site showed mis‐splicing , whereas none of the variants that were predicted to activate a cryptic acceptor site showed mis‐splicing . Branch point recognition plays an important role in acceptor site selection during pre‐mRNA splicing . For some variants, the predicted cryptic splice sites did not translate into mis‐splicing and a subsection of these showed variable levels of exon skipping . For the variants that showed relatively higher levels of exon skipping , in silico SRE prediction tools predicted exon skipping due to disruption of ESEs and/or creation of ESSs. The exon skipping observed for c.484C>A seems to be clearly due to disruption of SREs as no cryptic splice site activation was predicted by splice site tools. However, for remaining variants, whether the observed exon skipping was primarily due to disruption of SREs or a combination of creation/activation of a competitive cryptic splice sites and disruption of SREs were not clear. Overall, our results indicate that splice site and SRE prediction tools used in tandem provide a better prediction of splicing dysregulation induced by exonic variants. However, as one would expect these tools were not always correctly predictive of the splicing dysregulation as we observed for several of the exon 6 variants. Also, the net outcome of splicing dysregulation differed for variants across exons and not always predictable by in silico tools as it will depend on several additional factors including location and strength of cryptic splice sites, strength of corresponding native splice sites, and density of SREs (Caminsky et al., F9. The in silico splice site and SRE prediction tools when used in tandem more accurately predicted spicing dysregulation induced by such variants. The net effect of point variants on splicing dysregulation is context dependent and can be difficult to predict by in silico prediction tools. When primary tissue samples are not available, as is often the case, minigene assays remain a straightforward approach to assess novel variants for splicing effects. While the findings in this work were drawn from studying variants in F9, we anticipate that these findings may be recapitulated upon careful analysis of variants in other genes as well.In conclusion, our study suggests that splicing dysregulation may contribute to the pathogenicity of majority of synonymous variants and several non‐synonymous variants in None declared.Our comments/our contributions are an informal communication and represent our best judgment. These comments do not bind or obligate FDA. Click here for additional data file. Click here for additional data file. Click here for additional data file.