The woman’s gut microbiota during pregnancy may support nutrient acquisition, is associated with diseases, and has been linked to infant health. However, there is limited information on gut microbial characteristics and dependence in pregnant women. In this study, we provide a comprehensive overview of the gut microbial characteristics of 1479 pregnant women using 16S rRNA gene sequencing of fecal samples. We identify a core microbiota of pregnant women, which displays a similar overall structure to that of age-matched nonpregnant women. Our data show that the gestational age-associated variation in the gut microbiota, from the ninth week of gestation to antepartum, is relatively limited. Building upon rich metadata, we reveal a set of exogenous and intrinsic host factors that are highly correlated with the variation in gut microbial community composition and function. These microbiota covariates are concentrated in basic host properties  and blood clinical parameters, suggesting that individual heterogeneity is the major force shaping the gut microbiome during pregnancy. Moreover, we identify microbial and functional markers that are associated with age, pre-pregnancy body mass index, residency status, and pre-pregnancy and gestational diseases. The gut microbiota during pregnancy is also different between women with high or low gestational weight gain. Our study demonstrates the structure, gestational age-associated variation, and associations with host factors of the gut microbiota during pregnancy and strengthens the understanding of microbe–host interactions. The results from this study offer new materials and prospects for gut microbiome research in clinical and diagnostic fields. The woman’s gut microbiota is essential during pregnancy3 due to its crucial roles in nutrient acquisition4, immune remodeling5, and protection against infection6. In recent years, many studies have investigated the temporal variation in the gut microbiota in pregnant women and revealed a dramatically altered structure of the gut microbiota at different stages of pregnancy8 or, conversely, a relatively stable gut microbial profile during pregnancy9. The inconsistent observations of these studies could be due to the complexity and mutability of the gut microbiota and the limited sample sizes. In addition, a range of parameters in women, including their physical condition before and during pregnancy 11 and psychological 12 and environmental factors 13, affect their gut microbiota over the course of pregnancy. In turn, alterations in the gut microbiota may have subsequent impacts on the occurrence of gestational diseases15, fetal status, pregnancy outcomes16, and even the immune development of the offspring18. A series of gut microbes have been identified as participators in these processes. For example, an anti-inflammatory commensal Faecalibacterium is negatively correlated with gut permeability during pregnancy19, decreased in women with gestational diabetes mellitus (GDM)20, and involved in infant gut microbial maturation21. Based on the current status of research, however, these findings have not always been validated by related studies18. Therefore, it is still necessary to extensively investigate the dynamics of the gut microbiota during normal pregnancies and to quantitatively assess bacterial-level variations resulting in host heterogeneity, especially in a large cohort-based study.The gut microbiota has been associated with wellness and diseaseIn the current study, we performed a population-level investigation of the gut microbiotas of 1479 pregnant women of Chinese origin, with a broad range of gestational ages, from the 9th week of gestation to antepartum (>36th week). We used 16S rRNA gene sequencing technology and bioinformatic analyses to identify microbial diversity and compositional and functional characteristics across pregnancy. Integrative analyses of 132 host parameters were also performed to investigate microbe–host associations.n = 204 ± 95) but few participants in the first trimester  and at the antenatal period .The study cohort consisted of 1479 pregnant women with a mean age of 30.6 ± 4.3 years . All participants were recruited from the Guangzhou Women and Children’s Medical Center in Guangzhou, a large modern city located in Guangdong Province in South China. The exclusion criteria included participants who had taken antibiotic treatment or probiotic supplements in the 4 weeks prior to sample collection, strict vegetarians, and individuals with alcoholism or with other unusual dietary habits. The basic characteristics of all participants are summarized in Table 2) and 7% of the women were overweight/obese (BMI ≥ 25 kg/m2). A total of 36.8% (934/1479) of the women had at least one disease before or during pregnancy; in particular, 18% of the women had GDM.To investigate the host variables that are associated with the microbiota during pregnancy, we analyzed 132 exogenous and intrinsic host factors of the participants, including 22 intrinsic host properties, 63 biomedical indices (52 blood parameters and 11 urine parameters), 20 self-reported or clinically diagnosed diseases, 7 hepatitis virus infection statuses, and 20 parameters of pregnancy outcome and neonatal information , and 86% could be annotated into a genus or a family .The gut microbiotas of 1479 fecal samples of pregnant women were characterized by sequencing the V4 variable region of the bacterial 16S rRNA gene, generating a total of 75.8 million high-quality sequences . A total of 7701 operational taxonomic units (OTUs) were identified and taxonomically annotated based on an open source, universal microbiome bioinformatics platform (QIIME2)24, despite the occasional perturbance of the low-abundance phyla25. To test whether the compositional pattern of pregnant women is stable at lower taxonomic levels, we then compared our cohort to the Guangdong Gut Microbiome Project (GGMP) cohort26  at the bacterial genus level. In the GGMP cohort, there were 1048 nonpregnant women (age 18–44 years) included in the current comparison. The pregnant women yielded 29 dominant genera with a mean relative abundance >0.5%, and 25 (86.2%) of these genera were also presented among the 32 dominant genera in the GGMP nonpregnant women  and Bacteroidetes 11.9%) , with a slight reduction in Ruminococcus-type in the last stage of pregnancy.The primary structure of the human gut microbiome is described by enterotypecus Fig. . This en23. These 489 OTUs comprised 95.2% of the total abundance  during different gestational stages, representing a relatively stable “core microbiota” across pregnancy. Notably, only 1.2% (6/489) of these OTUs were significantly different  when comparing different gestational stages  and was also markedly separated among individuals with three enterotypes  orthologs (KOs), which were further integrated into 525 modules . Of these, 1517 KOs and 272 modules were core functions with a mean relative abundance cutoff of 0.01%, representing 94.3% and 66.9% of the total abundance, respectively (Supplementary Data α (within-sample) and β (between-sample) diversity in terms of the OTU profiles. No significant differences in α diversity were detected during the pregnancy period .To investigate differences in the gut microbiota at different stages of pregnancy across their subjects, we first assessed the microbial adonisq = 0.026) and 0.14% (adonisq = 0.049) of the gut microbiota variance at the OTU and genus levels, respectively. This effect size was significant but quite smaller than that of the enterotype stratification and was relatively smaller than those of other host parameters (see sections below). Unconstrained redundancy analysis based on Bray–Curtis distance between microbial genera did not show any trends across gestational age , represented the major contributors to the gestational age-constrained axes , suggesting that these genera may be potential microbial markers.We produced the correlation network of 29 dominant genera based on the relative abundance in subjects at different stages of pregnancy Fig. , indicatR2 = 0.11%, q ≤ 0.05; Fig. We identified 25 host parameters  that significantly correlated with overall microbial community variation at the OTU level  and residency status  were most prominent in the functional profiles, confirming that the effect of host heterogeneity on gut microbial functions is notable. Likewise, the combination of 6 function-correlated parameters explained 1.46% of the gut functional variation, similar to the scale of the variation at the OTU and genus levels.The half (52%) of these parameters were also validated at the genus level of microbial composition at 28, which allowed us to capture the correlations between each parameter and microbial genus by deconfounding the effects of other unrelated parameters. We identified 137 significant inter-associations  between the host parameters and microbial genera  and blood parameters (n = 38) composed the major percentage of these associations, followed by the gestational age parameters (n = 19). One parameter, the pregnant women’s residency status, correlated with the largest number of genera including several important members of Lachnospiraceae . Our population-level data validated the previous observation of a potential correlation between host parameters and gestational gut microbial composition in small datasets (see “Discussion”) and found novel associations such as the negative correlation between PBMI and Bifidobacterium ers n = 1. One parn = 369; younger group: ≤27 years, n = 359) and PBMI , as well as compared their types of residency . Among these groups, the older and obese women, as well as the younger and lean women, were statistically overlapped , in agreement with the previous population study showing that the BMI of reproductive age women is continuing to increase with age29.To identify the host property-associated OTUs that could play an exact role in shaping the gestational gut microbiome, we compared the gut microbial community compositions and functions of pregnant women between the top 75% and bottom 25% of individuals in terms of age , Dialister (F0194 and F0285), Mogibacteriaceae (F0308), Oscillospira (F0147), Clostridiales (F0323), Faecalibacterium prausnitzii (F0008), and Prevotella copri (F0293), while the younger-enriched OTUs included Veillonella , P. copri (F0011 and F0329), Enterobacteriaceae (F0048), Haemophilus parainfluenzae (F0082), Bacteroidales_S24-7 (F0349), Clostridium perfringens (F0216), Dialister (F0369), Lactobacillus (F0426), Rothia mucilaginosa (F0230), and Turicibacter (F0179). The negative correlation of Enterobacteriaceae with women’s age was in agreement with the observation at the genus level  and F0021/F0022 (Blautia obeum) and one younger-enriched OTU F0011 (P. copri), were dominated in their abundance (>1%) in the women’s gut microbiota  were associated with age, of which 11 were enriched in older women and 13 were enriched in younger women , Roseburia faecis (F0017), Adlercreutzia (F0103), and Anaerostipes (F0189), while the lean-enriched OTUs included Oscillospira (F0199 and F0263), Ruminococcaceae (F0111 and F0334), [Ruminococcus] gnavus (F0020), Clostridium ramosum (F0185), Bifidobacterium (F0033), and Streptococcus (F0051). The negative correlation between Bifidobacterium and PBMI was consistent with the observation at the genus level. Three dominant OTUs with a minimum mean abundance >1% in all individuals were F0008 (F. prausnitzii), F0017 (R. faeces), and F0020 ([Ruminococcus] gnavus)  were associated with PBMI, including 12 that were enriched in obese women and 8 that were enriched in lean women , Megamonas (F0001), Blautia (F0012), Lachnospiraceae (F0007), and Clostridium (F0023), as well as several immigrant-enriched OTUs, including F. prausnitzii (F0004), Gemmiger formicilis (F0006), P. copri (F0011 and F0031) and Ruminococcus bromii (F0025), were of the dominant taxa (>1%) in the women’s gut microbiota. Functionally, 81 core KEGG modules were associated with residency status  were associated with women’s type of residency, including 43 that were enriched in native women and 60 that were enriched in immigrants  of 0.67 for the classification of older and younger women in our cohort Fig. , and the30. GWG was positively associated with two important short chain fatty acid (SCFA) producers32, Faecalibacterium and Roseburia  and an AUC of 0.75 for women with the highest 10% GWG  and 29–32 weeks . These results suggested that the gut microbial composition may be a good indicator for the weight status of pregnant women. Noticeably, adding the PBMI to the regression model increased its predictive power  and the bottom 25%  of GWG. Only 8 OTUs were associated with GWG at P ≤ 0.01 , Dialister (F0204), and Clostridium (F0165), while the highest-GWG-individual-enriched OTUs were Clostridiales (F0160 and F0176), R. faecis (F0003), Paraprevotella (F0432), and Lachnospiraceae (F0406). Noticeably, lowest-GWG-individual-enriched OTU F0049 and the highest-GWG-individual-enriched OTU F0003 were also determined as important features in the random forest regression model based on continuous GWG mentioned above , Methanobrevibacter (F0286), and Blautia (F0333), while the GDM-depleted OTUs included Streptococcus (F0010 and F0051), Lactobacillus (F0426), Turicibacter (F0179), Peptostreptococcaceae (F0043), Lactococcus (F0282), Ruminococcaceae (F0101), and Lachnospiraceae (F0261). The Streptococcus genus was the signature taxa that was lower in GDM patients  were associated with GDM, including 3 that were enriched in GDM women and 8 that were enriched in non-GDM women , F. prausnitzii (F0321), Roseburia (F0272), Ruminococcaceae (F0080), Dialister (F0285), and Clostridiales (F0259), were associated with hepatopathy, all of which were enriched in women with hepatopathy , including P ≤ 0.01; however, none of these OTUs passed the multiple test correction  and Anaerostipes (F0102), were disease enriched, while 3 control-enriched OTUs were Bilophila (F0122), Streptophyta (F0374), and Clostridiales (F0176).Five core OTUs were identified at ion Fig. . Two of P ≤ 0.01, corresponding to q < 0.336; Fig. Coprococcus (F0059 and F0195), Oscillospira (F0314), Ruminococcaceae (F0326 and F0334), Lachnospiraceae (F0370), and Clostridiales (F0156), while the control-enriched OTUs were two Bacteroides (F0053 and F0089) members.Nine core OTUs (1.8%) were associated with hypothyroidism, including 7 that were enriched in women with the disease and 2 that were enriched in non-hypothyroidism women  the negative association between blood thrombin time (a coagulation indicator) and Bacteroides, which can be explained by Bacteroides  being the main producer of an important blood-clotting factor, vitamin K2, in the human body42; and (2) the frequent associations between blood total bile acid or uric acid (UA) and gut microbial members , facilitated by the fact that many gut microbes are important participates in host bile acid and UA metabolism43. Intriguingly, some associations such as the negative correlation between PBMI and Bifidobacterium have not been previously reported. In addition to these examples, there were many correlations among the considerable unexplained variance between host parameters and the gut microbiome, requiring further study to understand their significance.Previous population-based reports had estimated the effect size of host factors on healthy gut microbiota and explained a considerable proportion of microbial variation based on their collected parametersF. prausnitzii and B. obeum were enriched and those of P. copri were diminished. An increase in F. prausnitzii is beneficial for gut health, mostly due to its functions in butyrate production and anti-inflammatory effects45, while gut P. copri can induce insulin resistance by producing branched-chain amino acids46. Faecalibacterium was also positively correlated with GWG in our cohort. In addition, in this cohort, only 11 OTUs and no functional modules were altered between pregnant women with GDM and normoglycemic pregnant women. Rare gut taxonomic and functional variations in GDM have also been reported in women in the third trimester of pregnancy20. Consistently, the enrichment of Blautia OTUs was identified in both the previous report and our cohort, suggesting their potential role in GDM development.Our comprehensive data on the gut microbiome of pregnant women enabled us to identify the taxonomic and functional signatures of age, PBMI, and residency status, as well as pre-pregnant and gestational diseases in a sufficient number of samples. For example, in older pregnant women, the OTUs of 26, in south Chinese population, the host location was the strongest associations with gut microbiota variations comparing with other phenotypes. Our result thus suggested that the effect of host location on gut microbiota can partly be retained in immigrants. This strong “ethnic origin” effect on gut microbiota was also widely observed in European35 and first-generation American immigrants47.In our cohort, the large difference of pregnant women’s gut microbiota between native residents and immigrants may be contributed by numerous factors, such as lifestyle, economic level, education, and disease epidemiology. As reported in a recent studyParaprevotella/Lachnospiraceae and GWG, partly overlapped with the observations in previous studies10. Moreover, there was significant correlation between the observed and the predicted values for GWG regression models based on samples at two gestational stages (13–16 and 29–32 weeks), but not the others. This suggested a good potential of the gut microbiota for the prediction of the energy and nutrient metabolism of women at particular gestational age. Noticeably, PBMI may be necessary for building more accurate GWG prediction models based on microbiome composition.The association between weight-associated parameters and the gut microbiota is of particular interest. In our cohort, several parameters, including women’s weight before pregnancy and at delivery, GWG, and infant weight parameters, were associated with the overall gut microbiota as well as some gut microbes. The correlation of some bacteria with GWG, e.g., a negative correlation between Because our study design was limited in that each participant provided one fecal sample for analysis, we lacked longitudinal data to fully determine the dynamic variation in each pregnant woman throughout gestation. In addition, the current study does not provide any mechanistic explanation for the variation in host heterogeneity and gut microbiota.Clostridium difficile or an unknown agent; (6) chronic constipation; (7) unusual dietary habits such as individuals with alcoholism and strict vegetarians; and (8) conventional antibiotic treatment or probiotic supplement in the preceding 4 weeks. Fecal samples were obtained from participants by using a sterile toilet and a fecal collection bag  during the hospital stay. For each participant, 3 parallel samples were subpackaged and frozen in −80 °C freezers within 30 min of collection.This study received approval from the Ethics Committee of Guangzhou Women and Children’s Medical Center (No. 2018030306). Written informed consent was obtained from all participants, and no financial compensation was provided. The methods were carried out in accordance with the approved guidelines. All pregnant women who visited the prenatal clinics at Guangzhou Women and Children’s Medical Center from January 2017 to September 2017 were asked to participate in our study. The recruited pregnant women were invited to fill in a questionnaire, including their basic information , lifestyle data, medical history, and a self-administered food frequency questionnaire. Our exclusion criteria included (1) usage of the following drugs in the previous 6 months: systemic antibiotics, corticosterone, cytokines, methotrexate or other immunotoxic drugs, hormonal contraceptives, and high dose of commercial probiotics; (2) presence of risky diseases: serious cardiovascular disease, inflammatory bowel disease, irritable bowel syndrome, and celiac disease; (3) human immunodeficiency virus infection; (4) intestinal surgery within 5 years; (5) chronic diarrhea caused by 48, pregnant women were diagnosed with GDM if ≥1 of the following criteria were met: BS ≥ 5.1 mmol/L, BS60 ≥ 10.0 mmol/L, or BS120 ≥ 8.5 mmol/L. Pre-pregnancy weight was based on pregnant women’s self-report at the first clinic visit . Pregnant women’s body measurement index , gestational disease status, hepatitis virus infection, pregnancy outcomes, and neonatal information were documented from hospital records.Blood and urine samples were obtained from the pregnant women on the same day as the fecal sample collection, and the blood and urine parameters were measured by standard procedures and obtained from hospital records. Except that the blood glucose parameters BS (fasting blood glucose), BS60 (1 h blood glucose), and BS120 (2 h blood glucose) were obtained by a standard 2-h 75 g oral glucose tolerance test between 23 and 29 weeks of gestation for GDM diagnosis. Based on the criteriaTotal bacterial DNA was extracted from the fecal samples using the MOBIO PowerSoil® DNA Isolation Kit 12888-100 protocol and stored at −80 °C in Tris-EDTA buffer solution before use. We amplified the V4 region of the 16S rRNA gene using the universal 515F (5’-GTGYCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACNVGGGTWTCTAAT-3’) primers, along with barcode sequences for each sample. Each reaction mixture contained 10 μM each of forward and reverse primers, 1000 ng of template DNA, 200 μM of dNTPs, 5 μL of 10× EasyPfu Buffer, and 2.5 units Easy Pfu DNA Polymerase in 50 μL reaction. The PCR conditions were as follows: 95 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 40 s, and a final extension step at 72 °C for 4 min. PCR products were separated by agarose gel electrophoresis, and the expected band size for 515F–806F was approximately 300–350 bp. DNA quantification was conducted using a Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher/Invitrogen cat. no. P11496) following the manufacturer’s instructions. The amplicon library was combined in equimolar amounts and subsequently quantified (KAPA Library Quantification Kit KK4824) according to the manufacturer’s instructions. In addition, 150 bp paired-end sequencing was performed on the Illumina MiniSeq platform at Promegene Co. Ltd. .49. All reads were truncated at the 150th base with a median Q score >20 to avoid sequencing errors at the end of the reads. Noisy sequences, chimeric sequences, and singletons in the sequence data were removed by DATA250. Denoised paired-end reads were joined, setting a maximum mismatch parameter of two bases. The representative sequences  were defined at 100% similar merged sequences. We used the term “operational taxonomic unit (OTU)” instead of “feature” in the whole article for convenience. Then the taxonomy of the features was identified using the classify-sklearn classification methods based on the Greengenes 13.8 database (https://data.qiime2.org/2018.11/common/gg-13-8-99-515-806-nb-classifier.qza) via the q2-feature-classifier plugin. The phylogenetic analysis was performed in QIIME2 with “qiime alignment mafft,” “qiime alignment mask,” and “qiime phylogeny fasttree” commands, based on the tutorials at https://docs.qiime2.org/2019.1/tutorials/moving-pictures/. The phylogenetic tree of the core OTUs was visualized using iTOL v451. To measure the gut microbiota diversity  and quantify the taxonomic composition of the samples, all samples were rarefied to an even sampling depth of 20,000 sequences.16S rRNA amplicon sequences were processed using QIIME226 from the European Nucleotide Archive database (accession no. PRJEB18535). Only data from nonpregnant women aged 18–44 years were included. Data were processed using the same method as mentioned above.To compare the gut microbiota of pregnant women with that of nonpregnant women, we downloaded the raw 16S rRNA gene sequencing data of the GGMP projectAlpha and beta diversities were calculated in the QIIME2 platform with the “qiime diversity core-metrics-phylogenetic” command. The Shannon’s diversity index, observed OTUs, Faith’s phylogenetic diversity , and Pielou’s evenness were used to reflect community richness and evenness. The Jaccard distance, Bray–Curtis distance, and unweighted and weighted UniFrac distances were implemented to assess the similarity or dissimilarity between individuals.http://enterotype.embl.de/enterotypes.html)27. Fecal samples were delineated into three enterotypes based on their genus-level relative abundance profiles using the Jensen–Shannon divergence and reference-based alignment algorithm.Enterotype assignment was performed based on the published tutorial .Statistical analyses were implemented at the R v3.4.2 platform  and was calculated using the vegdist function in the vegan package. Distance-based redundancy analysis (dbRDA) was performed on OTUs and taxonomic composition profiles with the vegan package, based on the Bray–Curtis distance, and visualized using the R ade4 package. Procrustes analysis was used to determine similarity between two multivariate axes  and was performed with the R vegan package. The Procrustes P value was generated based on 1000 permutations. MaAsLin28 analysis was performed using default parameters (http://huttenhower.sph.harvard.edu/maaslin).Principal coordinate analysis (PCoA) was carried out using the adonis function within the R vegan package. The combined effect size of multiple host parameters was also calculated using the adonis function.We estimated the effect size of each variable (host parameter) on microbiome variation using the randomForest and pROC packages in R. OTUs (or KOs) with significantly different abundances between groups were selected to build the random forest model , and the performance of the model was assessed with leave-one-out cross-validation. The AUC was calculated using the roc function.This analysis was carried out using the t test, Wilcoxon rank-sum test, and one-way ANOVA test, were performed on the R platform. Spearman correlation test was assessed using the cor.test function with “method=spearman” parameter. The q value was used to evaluate the false discovery rate for correction of multiple comparisons and was calculated based on the R fdrtool package. For a single test, P ≤ 0.05 was considered statistically significant. For multiple test, unless otherwise mentioned, q ≤ 0.05 was considered significant. For MaAsLin analysis, the threshold q ≤ 0.2 was considered significant54. For the association analyses between gut microbial/functional compositions and host parameters , the threshold P ≤ 0.01 was used for presenting the results, and simultaneously the corresponding q-values was shown for estimating the significant level.All statistical tests, including Student’s Further information on experimental design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Supplementary Data 14Supplementary Data 15Supplementary Data 16Supplementary Data 17Reporting Summary


Sub-Saharan women use smokeless tobacco (SLT) more than smoked tobacco. Among Western African countries, the estimated weighted prevalence of SLT use in rural women was found to be the highest in Burkina Faso (after Sierra Leone). This study aimed to assess the prevalence of SLT use and its associated factors among rural women in Burkina Faso by using nationally representative data.We used data from the 2013 STEPwise approach to Surveillance (STEPS) study, which provided sociodemographic, clinical , biological , and tobacco and alcohol consumption data. Data for 1730 rural women were used, and we performed Student’s chi-squared and logistic regression analyses.p < 0.001), undernourishment , decreased waist circumference , decreased DBP , increased SBP , and increased differential blood pressure . The co-use of alcohol was also a significant risk factor .The prevalence of current SLT use was 13.8% (95% CI: 12.2–15.5). Significant risks for SLT use were the presence of dental symptoms (adjusted odds ratio [aOR] = 2.59; The prevalence of current SLT use was high among rural women in Burkina Faso, and significant concerns for users included alcohol co-use, the occurrence of dental symptoms, undernourishment, and an increase in differential blood pressure. National Public Health interventions are needed to reduce SLT use and its health-related concerns. Smokeless tobacco (SLT) and smoked tobacco (ST) are both used in sub-Saharan African (SSA) countries, and SLT is used more than twice as often in rural areas than in urban areas . The preWe used data from the first Burkina Faso national survey conducted in 2013, which was based on the WHO STEPS methodology . The pro2, in kg/m2; a BMI < 18.5 kg/m2 was considered underweight, a BMI of 18.5–24.9 kg/m2 was considered normal weight, and a BMI ≥ 25 kg/m2 was considered overweight/obese) and waist circumference (cm). The biological characteristics tested in blood were total cholesterol (mmol/l), high-density lipoprotein cholesterol , and fasting sugar (mmol/l). Blood pressure  was measured three times, and we kept only the mean value for each indicator; levels ≥140/90 mmHg defined high blood pressure (HBP), and the difference between the mean values of SBP and DBP (SBP - DBP) was used to determine the differential blood pressure. Lifestyle factors were determined by using self-reporting techniques and included current (past month) alcohol consumption and current (past year) smokeless  or smoked tobacco consumption. Dental problems were also recorded by using a self-reported method to report having the following during the past 12 months: i) difficulties chewing food, ii) difficulties with speech/trouble pronouncing word or iii) pain or discomfort in the teeth/mouth.Participants’ demographic variables included age, marital status, education level, and occupation. We also reported whether the women were living in households with at least one member aged ≥18 years. The anthropometric characteristics were weight (kg), height (m), body mass index  to analyse the data. The quantitative variables were expressed as the means ± standard deviations, and the categorical variables were expressed as percentages (%), with a confidence interval of 95% . Student’s t-test was used to compare quantitative variables, and the chi-squared test was used for categorical variables. Logistic regression analysis was performed to identify the clinical, biological, and lifestyle factors associated with SLT after adjusting for sociodemographic features. The model was determined by backward elimination . For all analyses, a 2 vs 21.7 ± 3.6 for BMI and 74.6 ± 10.7 cm vs 76.4 ± 11.7 for waist circumference , respectively. The mean SBP was significantly higher in SLT users than in nonusers , as was the differential blood pressure . However, there was no significant difference in the mean DBP between SLT users and nonusers . The mean glycaemia was also higher for SLT users , decreased DBP  and increased SBP . When replacing both the SBP and DBP variables with the single differential blood pressure (SBP-DBP in mmHg) in the multivariable model, the differential unit increase was also significant . Concerning sociodemographic variables, significant risk factors were old age  and lack of education .The results of the logistic regression are reported in Table The prevalence of current SLT use was high among rural women in Burkina Faso, and given the current state of epidemiologic transition, the relationship of SLT use with noncommunicable diseases should make SLT use a public health concern.Sarcocephalus latifolius (a plant that provides synthetic tramadol), or non-medical tramadol, in Cameroon [The prevalence of current SLT use among rural women in Burkina Faso was 13.8% (95% CI: 12.2–15.5), while only one woman used ST. Such statistics suggest a socially permissive attitude towards SLT use by females. A lower standardized prevalence estimate of 3.86% (95% CI: 3.22–4.48) was noted 2 years earlier (in 2011 and only in the sample of women aged 15–49 years) , as wereCameroon  and khatCameroon , 25.p = 0.001 for those with alcohol experience; aOR = 4.84; p = 0.007 for episodic binge drinkers) [Current SLT users were also frequently current alcohol users , as has been reported for women in Cambodia   and in Krinkers) . SLT andrinkers) , and therinkers) . Thereforinkers) . Unforturinkers) , and therinkers) . Althougp < 0.05) and differential blood pressure  (Table p < 0.05) was consistent with this effect and was consistent with the findings of Onwuchekwa in rural Niger  [p < 0.01)  Table . Substansidents) . Furthersidents) , 35; thuSLT use was associated with the presence of some dental symptoms . Cheema et al. reported an association with poor oral status  in Qatar . Users op < 0.01), as it was in rural Ghanaian women  [The prevalence we found (16.0%) was similar to the results reported in previous studies in Burkina Faso  . Tobacco= 0.002) . There i= 0.002) , and hun= 0.002) .p < 0.001) and not by overweight/obesity , which potentiates the psychological and physical effects and, unfortunately, includes adverse effects. Addictive habits may also include the expectation of immediate hunger suppression in an environment with low food availability. Thus, worsening undernourishment in SLT users might be connected to insufficient food intake resulting from the appetite-suppressing effects of the nicotine delivered by SLT used over the long term. The increases in SBP and differential blood pressure suggest serious cardiovascular concerns associated with SLT use. Furthermore, an overview of the chemical compounds and their levels in locally produced tobacco leaves should be given attention. The lack of increases in waist circumference and total cholesterol may be due to the severity of the undernourishment status of SLT users. Regarding ST use, National Public Health interventions are needed to reduce SLT use and its health-related concerns.The efficacy of a brief dental office intervention for the general population of smokeless tobacco users was previously described . Neverth


Paramesotriton chinensis was sequenced. The complete mitogenome of P. chinensis is a circular double-stranded DNA sequence that is 16,361 bp long and was biased toward A + T content at 61.3% . The complete mitogenome of P. chinensis consists of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 1 ribosomal RNAs (16S rRNA), and 1 putative control region. This study presented the complete mitogenome of P. chinensis and provided essential and important DNA molecular data for further phylogenetic and evolutionary analysis for genus Paramesotriton.The complete mitochondrial genome of the Asian warty newt  Paramesotriton, currently consists of 14 nominal species endemic to southern China, except for P. deloustali which is mainly distributed in northern Vietnam. This genus commonly known as the Asian warty newts, consists of two species groups, which have been recovered as reciprocally monophyletic sister groups in molecular phylogenetic analyses , which may verify the phylogenetic position of this species.The salamandrid genus P. chinensis was obtained from Zhejiang and deposited at the Zhejiang Museum of Natural History, Hangzhou, Zhejiang, China. Genomic DNA extraction and next-generation sequencing were described in previous publication  to produce a single, circular form of complete mitogenome with about an average of 122 × coverage. The mitogenomic sequence has been deposited into GenBank under the accession number KY609177.Specimen of P. chinensis is a circular double-stranded DNA sequence that is 16,361 bp long and was biased toward A + T content at 61.3% . DOGMA  and clade B   . Our mol


Histoplasma capsulatum is the most common endemic mycosis in the United States and frequently presents as an opportunistic infection in immunocompromised hosts. Though liver involvement is common in disseminated histoplasmosis, primary gastrointestinal histoplasmosis of the liver in absence of lung involvement is rare. Similarly, cholestatic granulomatous hepatitis in liver histoplasmosis is rarely seen.We present a rare case of primary gastrointestinal histoplasmosis manifesting with acute granulomatous hepatitis and cholestasis in a 48-year-old female with psoriatic arthritis, receiving methotrexate and infliximab. The epidemiology, risk factors, clinical presentation, diagnosis, and treatment of histoplasmosis is discussed. Furthermore, we review the published cases of biopsy-proven disseminated histoplasmosis with cholestatic jaundice to highlight histoplasmosis involvement in the liver.Histoplasmosis should be considered in immunosuppressed patients with fever, chills, abdominal pain and cholestasis with progressive jaundice, particularly in subjects without evidence of biliary obstruction. Future studies are needed to accurately assess the risk of this fungal infection, specifically in patients on immunomodulatory therapy for autoimmune disease. Histoplasma capsulatum, a dimorphic fungus found in barns, old houses, and soil rich in bird and bat droppings. Direct inhalation of spores is the primary mode of infection. Symptomatic infection with H. Capsulatum is most likely in patients who are immunosuppressed and unable to develop an effective cell-mediated immunity against the organism , AST 181 U/L [ref. range 0–35 U/L], ALP 1138 U/L [ref. range 35–105 U/L], and LDH 406 U/L [ref. range 118–225 U/L]. On admission to our hospital, additional laboratory investigation was notable for elevated white blood cell count of 13.5 K/μL (ref. range 4–10 K/μL), lymphocyte count 8.5 K/μL (ref. range 1.2–3.7 K/μL), GGT 885 U/L (ref. range 5–36), total bilirubin 2.5 mg/dL (ref. range 0–1.2), and ferritin 1229 ng/ml (ref. range 10–120 ng/ml). An abdominal ultrasound showed non-specific post-cholecystectomy changes; a hepatobiliary iminodiacetic acid (HIDA) scan was negative for biliary leak or obstruction; computed tomography (CT) of abdomen and pelvis with contrast was without focal liver lesions or fluid collections; and an magnetic resonance cholangiopancreatography (MRCP) was without intrahepatic biliary ductal dilatation. The patient was then transferred to our institution for further evaluation.Serologic testing for hepatitis viruses A, B and C, Epstein-Barr virus, cytomegalovirus, herpes simplex virus and human immunodeficiency virus were negative. A high titer of anti-nuclear antibodies (ANA) 1:640 was detected while Ig immunoglobulins and rheumatoid factor were within normal range. F-actin IgG and anti-Histone antibodies were weakly positive at 32 [ref. range 0–19 units] and 2.6 [ref. range 0–0.9], respectively. Anti-neutrophil cytoplasmic, anti-RNP, anti-Smith, anti-SSA/SSB and anti-dsDNA autoantibodies were negative.The direct bilirubin reached 6.0 mg/dL (ref. range 0–0.3 mg/dL) on day 6 of admission despite normal indirect bilirubin of 0.5 mg/dL (ref. range 0.1–1.0 mg/dL). A liver biopsy was obtained on Day 7. This found numerous non-necrotizing granulomas with sinusoidal congestion, mild predominantly microvesicular steatosis (~ 20%) without significant ductitis or ductular reaction, no fibrosis on H&E stain, rare small budding yeast on GMS stain, negative acid-fast stain, negative PAS-D stain, negative iron stain, and minimal pericellular and periportal fibrosis on trichrome stain . The incidence of histoplasmosis in adults aged 65 years and older in the U.S. is 3.4 cases per 100,000 population and up to 6.1 cases per 100,000 population [Histoplasmosis is the most common endemic mycosis in the United States, in the environment in areas surrounding the Ohio and Mississippi River valleys. Between 60 and 90% of people who live in this area have been exposed to the fungus at some point during their lifetime , anorectal diseases, and GI bleeding associated with superficial or deep ulcers with and without perforation are more common compared to immunocompetent individuals [Gastrointestinal (GI) tract involvement has been reported in histoplasmosis but is usually asymptomatic or presents with vague abdominal pain in the majority of patients . GI maniividuals . Immunosividuals .H.capsulatum var. capsulatum appears as ovoid yeast cells measuring 2 to 4 μm with narrow based budding. Hematoxylin and eosin (H&E) stain may reveal portal tract infiltration with lymphocytes or histiocytes but is often too insensitive to detect the presence of fugal organisms. Additional staining with Gomori methenamine silver (GMS) and periodic acid-Schiff (PAS) best detects H.capsulatum by highlighting the yeast cell wall. As the presence of yeast cells consistent with H.capsulatum may not necessarily indicate active infection, culture data should be interpreted in the appropriate clinical context. Fungal culture can take up to 8 weeks for colony identification and its sensitivity for progressive disseminated histoplasmosis and chronic pulmonary disease is 74.2 and 66.7%, respectively [The diagnosis of histoplasmosis involves a multifaceted approach involving clinical and laboratory evidence of disease . The golectively . FurtherH.capsulatum, voriconazole and posaconazole. As mentioned previously, serum and urine antigen levels are useful for monitoring treatment with most decline appreciated in the first 2 weeks of successful therapy and continued for 12 weeks.Since disseminated histoplasmosis is fatal without treatment, prompt initiation of antifungal therapy is essential. Amphotericin B and itraconazole are the two agents most commonly used to treat for disseminated histoplasmosis . HoweverOur patient case illustrates an uncommon presentation of histoplasmosis that began as acute granulomatous hepatitis associated with cholestatic jaundice and progressed to disseminated histoplasmosis. Despite starting the appropriate treatment with IV Amphotericin and subsequently switched to oral Itraconazole, our patient developed worsening cholestasis as well as systemic symptoms such as pleural effusions and cardiac tamponade. After multiple thoracenteses and an urgent pericardiocentesis, patient recovered with full normalization of liver function tests by the 68th day of treatment.With an extensive laboratory and imaging work-up, making the diagnosis of histoplasmosis is often challenging. In our case, the liver biopsy showed the pathognomonic findings of histoplasmosis including granulomatous hepatitis and ovoid yeast elements.H.capsulatum by highlighting the yeast cell wall. Furthermore, Kothadia and colleagues described a case of granulomatous hepatitis in an HIV patient who initially presented with a total bilirubin of 10.2 mg/dL and succumbed to severe infection with multiorgan system failure within 3 weeks of admission [Cholestasis has received little attention in case reports of disseminated histoplasmosis. This is likely because not all cases of hepatic histoplasmosis manifest with significant cholestasis . Thus fadmission . The sevTwo days prior to presentation, our patient underwent an elective laparoscopic cholecystectomy for right upper quadrant pain thought to be biliary colic. In retrospect, her presentation may have been secondary to disseminated histoplasmosis. An important learning objective from our case is to have high index of clinical suspicion for histoplasmosis among immunocompromised patients who present with cholestasis in the absence of other causes by standard laboratory and imaging evaluation. The case presented by Kothadia et al. also describes a patient who underwent laparoscopic cholecystectomy for unclear etiology shortly before being diagnosed with disseminated histoplasmosis . AlthougHistoplasmosis should be considered in immunosuppressed patients presenting with fever, chills, abdominal pain and cholestasis with progressive jaundice, particularly in subjects without evidence of biliary obstruction. The full spectrum of hepatic manifestations of this disease is unknown but runs the gamut in the literature from mildly abnormal liver enzymes to severe icteric cholestasis with fever and pain. Future studies are needed to accurately assess the risk of this fungal infection specifically in patients on immunomodulatory therapy for autoimmune disease.


To investigate the hyperreflective foci (HRF) as an inflammatory biomarker using optical coherence tomography angiography (OCTA) in neovascular age-related macular degeneration (AMD) patients after antivascular endothelial growth factor (anti-VEGF) treatment and its association with the retinal microcapillary density.  Twenty-five eyes from 25 patients with neovascular AMD were included in the study. All eyes were imaged with OCTA at baseline (M0) and after 3 consecutive injections  of anti-VEGF. The number of HRF in the superficial capillary plexus (SCP), deep capillary plexus (DCP), and outer retina was counted. The vascular density of the fovea, parafovea, and the whole macula, as well as the area of the foveal avascular zone (FAZ), was measured. P < 0.01) and outer retina  after treatment. There was no significant difference for HRF number in the SCP, the vascular density , and FAZ area before and after treatments.  The mean interval between baseline and follow-up with OCTA was 93.08 ± 5.00  days. Compared with the baseline, the number of HRF significantly decreased in DCP (7.52 ± 3.06 vs. 3.76 ± 1.48,  The number of HRF in DCP and outer retina might serve as an inflammatory biomarker in patients with neovascular AMD. The reduced HRF possibly represents the alleviation of inflammation after anti-VEGF treatment in patients with AMD. Age-related macular degeneration (AMD) is the main cause of irreversible severe vision loss in elderly individuals, with a prevalence that exponentially increases with aging. It has been estimated that about 288 million people worldwide will suffer from AMD by 2040 . AMD is μm [With the advancement of noninvasive retinal imaging technologies, such as optical coherence tomography (OCT), the hyperreflective foci (HRF) can be imaged, which attracted the researchers' attention , 5. HRF μm , 7. HRF μm . Subsequμm –11. AlthNowadays, many studies that support the hypothesis that activated microglial cells induced by an inflammatory response in the retina are a pivotal origination of HRF , 13. MicIn this study, we investigated whether HRF could serve as a possible biomarker of retinal inflammation in patients with neovascular AMD and its possible association with retinal microcapillary density. The number of HRF, vascular density in different retinal layers, and the foveal avascular zone (FAZ) area was measured and quantified with OCT angiography (OCTA) in neovascular AMD patients before and after 3 consecutive anti-VEGF injections.In this retrospective study, we enrolled 25 consecutive treatment-naïve eyes from 25 neovascular AMD patients who were diagnosed with comprehensive ophthalmologic examinations in the Department of Ophthalmology, Shanghai General Hospital affiliated to Shanghai Jiao Tong University, Shanghai, China, between January 1, 2020, and July 30, 2020. The participants with three consecutive monthly intravitreal injections of anti-VEGF agents were enrolled. This study was approved by the Clinical Research Ethical Committee of Shanghai General Hospital affiliated to Shanghai Jiao Tong University (Permits No. 2020KY205) and adhered to the principles of the Declaration of Helsinki. All individual participants provided written informed consent.The exclusion criteria were as follows: (a) previous treatments for neovascular AMD ; (b) other vitreoretinal diseases, including dry AMD, vitreous hemorrhage, subretinal fibrosis, and retinal vascular disease; (c) high myopia ; and (d) history of ocular surgery, uveitis, and apparent media opacification.All participants underwent full ophthalmologic examinations, including best-corrected visual acuity (BCVA), intraocular pressure (IOP) measurement, and anterior segment evaluation using slit-lamp biomicroscopy, fundus photography, and OCTA imaging at the baseline (M0) and 1 week after treatment (M3).One experienced surgeon performed all intravitreal injections. The intravitreal injection was performed at the temporal limbus through the eyeball's pars plana under aseptic conditions. All patients received three once-monthly intravitreal injections of aflibercept (2 mg/0.05 mL) using a 30-gauge needle. A 1-week variation was allowed for every injection interval.2 sections centered on the fovea. Enface images of the superficial capillary plexus (SCP), deep capillary plexus (DCP), and FAZ area were automatically recorded and analyzed by the OCTA autosegmentation software. The SCP was segmented as 3 μm below the internal limiting membrane and 15 μm below the inner plexiform layer (IPL); the DCP was set between 15 μm and 70 μm beneath IPL, while the outer retina was located beneath the DCP. In the SCP and DCP, two central circles divided capillary plexus into the “fovea area” (a disc with a 0.6 mm diameter) and “parafoveal area”  as shown in Retinal microvasculature was imaged using the RTVue XR Avanti OCT system , and measurements were acquired using the manufacturer's AngioVue software. The scan covered an area of 3 × 3 mmμm to 50 μm range in order to exclude small counting noise signals as HRF noise signals and prevent large hyperreflective clumps, which are presented as typical hard exudates in fundus photography. Poor quality images with a signal strength index < 4/10 were excluded from further analysis. The quantification of HRF was carried out by two experienced physicians independently.The number of HRF was manually counted within a 3 mm diameter centered on the foveal area using a fovea-spanning horizontal B-scan. HRF were subdivided into 3 categories according to different retinal layers: the SCP, the DCP, and the outer retina, as described above. The maximal diameter of HRF was limited within the 20 t-test was used to compare the number of HRF, the vascular density of SCP and DCP, and the FAZ area between the baseline (M0) and after 3 consecutive monthly anti-VEGF injections (M3). P value <0.05 was considered as statistical significance.The data were analyzed by using SPSS 22.0 software. All values are presented as a number or mean ± standard deviation. The visual acuity was presented as the logarithm of the minimum angle of resolution (logMAR). A paired Twenty-five eyes from 25 patients  with neovascular AMD were consecutively enrolled in the study, as shown in P < 0.01) and outer retina , compared to baseline; the representative images of HRF were shown in P = 0.12).Analysis of the OCTA parameters revealed that the HRF was distributed across all retinal layers, including SCP, DCP, and outer retina, in neovascular AMD patients. As shown in Regarding retinal microvascular density in the SCP and DCP analyzed with OCTA, no significant difference was found for the microvascular density in the foveal, parafoveal, and whole macular sectors before and after treatment as shown in 2  and 0.31 ± 0.08 mm2 , respectively, with no statistically significant differences (P = 0.11).The distribution of FAZ areas in the retina is depicted in Microglial cells are considered to have a vital role in the inflammatory response in the pathogenesis of AMD , 17, 18.The introduction of high-resolution OCTA has facilitated the study of different capillary plexus in vivo. Previous studies have suggested that HRF may be used as a marker of inflammation in AMD patients , 20. It In our study, the HRF was localized in almost every retinal layer, especially the outer retina of patients with neovascular AMD, indicating the migration and aggregation of microglia cells in the outer retina. Our results were consistent with the previous observations , 22, whiPrevious experimental studies have demonstrated that microglia-induced retinal inflammation is a relatively early process during AMD development, which precedes neovascularization, microvascular dysfunction, and retinal neurodegeneration . In thisPrevious studies have also indicated that HRF could be an inflammatory hallmark, contributing to retinal dysfunction in many retinal diseases. For example, in neovascular diabetic retinopathy, HRF was significantly higher in diabetic patients compared with healthy controls . FurtherHsia et al.  comparedThe present study suggested that HRF could be used as an indicator or sensitive biomarker for retinal inflammation in neovascular AMD. After anti-VEGF treatment, the reduced HRF number might indicate the alleviation of inflammation in the retina and, possibly, improvement in the visual function in patients with neovascular AMD. Yet, long-term observation and a study with a larger sample size should be performed to compare the relationship among HRF, visual outcome, intraretinal cysts, and similar.


Staphylococcus aureus infective endocarditis (IE), especially in rodents, are commonly used to investigate the underlying pathogenesis, disease progression, potential diagnostic approaches, and therapeutic treatment. All these models are based on surgical interventions, and imply valve trauma by placing a polyurethane catheter at the aortic root. While the influence of endothelial damage and inflammation on the induction of IE has been studied intensively, the role of the catheter, as permanent source of bacteremia, and the interplay with bacterial virulence factors during the formation of IE is poorly understood. In our study, we aimed at identifying which set of preconditions is required for induction and formation of IE: (1) tissue injury, (2) permanent presence of bacteria, and (3) presence of the full bacterial repertoire of adhesion proteins. We investigated the manifestation of the disease in different modifications of the animal model, considering different degrees of endothelial damage and the presence or absence of the catheter. In four infection models the induction of IE was assessed by using two bacterial strains with different expression patterns of virulence factors – S. aureus 6850 and Newman. In vivo magnetic resonance imaging showed conspicuous morphological structures on the aortic valves, when an endothelial damage and a continuous bacterial source were present simultaneously. Cellular and inflammatory pathophysiology were characterized additionally by histology, real-time quantitative polymerase chain reaction analysis, and bacterial counts, revealing strain-specific pathogenesis and manifestation of IE, crucially influenced by bacterial adherence and toxicity. The severity of IE was dependent on the degree of endothelial irritation. However, even severe endothelial damage in the absence of a permanent bacterial source resulted in reduced valve infection. The spread of bacteria to other organs was also dependent on the pathogenic profile of the infectious agent.Animal models of  Staphylococcus aureus (S. aureus) infective endocarditis (IE) has continuously increased  was used to depict the manifestations of bacterial vegetation on the valves, histology to confirm tissue damage and bacterial presence, colony forming units (CFU) counts to quantify bacterial distribution and burden, and clinical scores to represent the overall disease severity.Thus, within the frame of our study we systematically investigated the influence of the degree of valvular tissue damage, the presence of foreign material, as well as the different virulence profiles on the induction of n = 144) with a body weight of 16–25 g (average weight: 21.5 ± 1.7 g) and an age of 8–12 weeks were used in this study.Female C57BL/6 mice  injection through the tail vein  24 h after surgical intervention. The four interventions differed in site and duration of catheter placement , assuming that no sustained damages  occurred at the valves. This model was used to assess the burden of surgery and systemic infection without IE formation.5 CFU was used, as it was previously shown to reliably induce IE . The infectious dose of 10nduce IE . On the nduce IE . Organs All animals were scored and examined daily for health status  defined by the approved animal use protocol. Each day, the corresponding score points for each health feature were added up and summarized as clinical score, representing a quantitative measure of the severity of the disease. Design of the clinical score was based on previous studies  and typiS. aureus strain 6850 (S. aureus 6850 strong expression (p < 0.05) of cytotoxic and hemolytic toxins such as α-toxin (hla) could be demonstrated  and stored at −80°C until use. Colony forming units were enumerated after serial dilution and overnight growth at 37°C on agar plates. Antibiotic-sensitivity testing was performed on fresh S. aureus isolates (Automated VITEK® 2 System Version 9.02 (BioMérieux Inc.)  .hla), staphylococcal protein A (spA), extracellular adherence protein (eap), Clumping factor A (clfA) and Fibronectin binding protein A (fnbA), the gene for gyrase B (gyrB) was taken as the housekeeping gene. All data were normalized to this gene. Primers used in this study are listed in Extraction of bacterial RNA and real-time quantitative polymerase chain reaction (RT-qPCR), including complementary DNA synthesis and real-time amplification was performed as described previously . In orde2 mixture), and core body temperature and respiration rates were monitored using a MRI compatible monitoring system . A self-gated CINE-UTE sequence was used to visualize the infected aortic valves over the cardiac cycle , MTX: 256 × 256, section: 1 mm, scan duration: 12:08 min). 20 images per cardiac cycle were reconstructed retrospectively.Measurements were performed at 9.4T on a BioSpec 94/20 small animal MRI system  equipped with a 1 T/m gradient insert  and ParaVision software version 5.1 (Bruker). A mouse body quadrature volume coil with an inner diameter of 35 mm  was used for image acquisition. During MRI, the mice were anesthetized with isoflurane  according to standard protocols. Hematoxylin and eosin staining (HE) was used to detect basophilic components (DNA/RNA) and eosinophilic structures (collagen/muscle), and Weigert’s Elastica-van-Gieson (EvG) staining was used to label collagen to indicate changes in the blood vessel . In brief, heart valves or catheters were fixed in 2% (v/v) formaldehyde and 2.5% (v/v) glutaraldehyde in 100 mM sodium cacodylate buffer, pH 7.4, at 4°C overnight. For heart valves: After washing in PBS, samples were postfixed in 0.5% (v/v) osmium tetroxide and 1% (w/v) potassium hexacyanoferrate III in 100 mM sodium cacodylate buffer for 2 h at 4°C followed by washing with distilled water. After dehydration in an ascending ethanol series from 30 to 100% ethanol, samples were two times incubated in propylenoxide each for 15 min and embedded in Epon  using a flat embedding mold. For catheters: After dehydration in ethanol up to 70%, samples were embedded in LR White medium , and polymerized using UV light according to the manufactures instructions. Ultrathin sections were cut with an ultramicrotome (Leica Ultracut UCT), collected on copper grids and negatively stained with 2% uranyl acetate for 20 min. Electron micrographs were taken at 60 kV with a Phillips EM-410 electron microscope using imaging plates .The 2 μm thick sections of the heart valves were dewaxed and boiled in citrate buffer (pH 6) (2 × 2 min). Subsequently, the primary antibody von-Willebrand-factor (vWF)  was added and incubated overnight at 4°C. Next, unbound primary antibody vWF was removed from the slice and secondary antibody goat-anti-rabbit (Biotin) (B2770 life technologies dilution 1:600) was added to the sections and incubated at room temperature for 1 h. Subsequently the label Alexa Fluor 546 with Streptavidin-Tag (dilution 1:800) was incubated for 45 min at room temperature to bind to the biotin. Finally, DAPI staining, incubating slices for 6 min at room temperature, was used to display the cell nuclei.p < 0.05 was considered as significant.Statistical analyzes were performed with the software GraphPad software version 5 . For each IE model (I–IV) bacterial titers were analyzed statistically using a one-way ANOVA calculation. Bacterial load on the valves was compared to that in all other organs after Bonferroni correction. A value of All animal experiments were approved by the North Rhine-Westphalia Agency for Nature, Environment, and Consumer Protection .S. aureus strain 6850 or Newman and were examined with respect to abnormal valve formations by in vivo MRI  were investigated in all four models of IE (I–IV) 24 h post infection by counting CFUs  was counted in the aortic arch. Local infection was higher than on the aortic valves , representing a significant difference in bacteria counts in Newman infections . Lung and spleen showed the strongest infection  that did not receive any pathogen infection showed mild to moderate clinical scores in the range of 4–12 . In NewmS. aureus strains. Several animal models have been established over the last years , CS , HV (microbiology), SN (PCR), UH (TEM), VHös , VHoe , and YT  performed experiments. AJ (histology), CS , HV (microbiology), MK (histology), SN , UH (TEM), VHös , and VHoe  analyzed data. CF, CS, MW, and VHoe wrote the manuscript. All authors interpreted data, and revised, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Atrial fibrillation (AF) and other arrhythmias are prevalent and often encountered by general practitioners (GPs). In response to the growing prevalence and to assist practitioners in the diagnosis and management of AF, the Cardiac Society of Australia & New Zealand and Heart Foundation of Australia published the first Australian AF Guidelines in 2018. We aimed to examine (a) the proportion of GPs who performed any form of AF screening and identify the methods they applied, (b) GPs’ awareness of the AF Guidelines and approaches to arrhythmia screening, (c) the roles of conventional 12-lead ECG and mobile health devices, and (d) GPs’ confidence in ECG interpretation and need for training.GPs Screen their patients for Atrial Fibrillation and othEr aRrhythmia (GPSAFER)” was conducted from October 2018 to March 2019. The participants were recruited via various GP networks across Australia. Ethics approval was granted by The University of Sydney.A cross-sectional online survey titled “p <  0.001). Factors significantly and positively associated with AF screening were “awareness of the AF Guidelines” (p <  0.001), “number of years working in general practice” (p <  0.001), and “confidence in ECG interpretation of AF” (p = 0.003). Most GPs reported that they were very or extremely confident in interpreting AF  and complete heart block . Many GPs  would like to receive online ECG interpretation training.A total of 463 surveys were completed. Many GPs  performed some forms of AF screening and applied at least one AF screening method, most frequently pulse palpation . Some  GPs considered assessing their patients for other arrhythmias . Most GPs  were not using mobile ECG devices in their practice but some  were contemplating it. One third  of GPs were aware of the Australian AF Guidelines; those aware were more likely to perform AF screening (98.9% vs 76.7%, Assessment of arrhythmias is common in general practice and GPs are open to further training in ECG interpretation and using mobile ECG devices to aid their clinical practice. Increasing awareness of AF Guidelines and improving confidence in ECG interpretation may increase AF screening. Atrial fibrillation (AF) is a common arrhythmia, with its prevalence estimated at 33.3 million people globally . It is oEarly diagnosis of AF and treatment with anticoagulant medication can reduce the risk of stroke by over 60% –9, and 4These guidelines recommend GPs obtain a medical-quality electrocardiogram (ECG) trace and hence highlight the importance of GPs being able to interpret ECGs for AF and other common arrhythmias. Both conventional 12-lead ECG and single-lead handheld ECG are appropriate in this setting for screening AF .We aimed to examine (a) the proportion of GPs who performed any form of AF screening and identify the methods they applied, (b) GPs’ awareness of the AF Guidelines and approaches to arrhythmia screening, (c) the roles of conventional 12-lead ECG and mobile health devices, and (d) GPs’ confidence in ECG interpretation and need for training.This was a cross-sectional online survey aiming to recruit a diverse sample of GPs and GP registrars practising in urban and rural settings across Australia. The survey was distributed from 19th October 2018 to 31st March 2019 via GP networks and organisations. Participation was voluntary and required informed consent. We identified organisations with GP networks across Australia by searching the Australian Department of Health website  which prGeneral Practitioners’ Screen their patients for Atrial Fibrillation and othEr aRrhythmias (GPSAFER)” (see supplementary file). The following topics were investigated in the survey: arrhythmia screening, awareness of the Australian AF Guidelines, use of conventional 12-lead ECG and handheld single-lead ECG devices, and practitioners’ confidence in ECG interpretation and need for training. We asked how they usually screened asymptomatic patients for AF ; how frequently they screened AF in asymptomatic patients with hypertension, diabetes, valvular heart disease, heart failure, stroke, and those prescribed antidepressants ; were they aware of the Australian AF Guidelines and had they incorporated the guidelines into their clinical practice for patients aged 65 years or older ; how they usually obtained an ECG ; how time-consuming was obtaining a conventional 12-lead ECG (extremely/ very/ moderately/ slightly/ not time-consuming); did they use a mobile device to obtain a single-lead ECG on patients (regularly/ sometimes (such as six-monthly)/ occasionally/ never but contemplating using one/ never but not contemplating using one) and how confident were they (extremely/ very/ moderately/ mildly/ not confident) that a mobile handheld ECG device could provide adequate quality information to detect AF, complete heart block and prolonged QT; did they consider assessing their patients for arrhythmias other than AF and specified the type of arrhythmia/ conduction disorder ; how confident were they in diagnosing ECG abnormalities (extremely/ very/ moderately/ mildly/ not confident): AF, complete heart block, distinguished between Mobitz I and II second degree heart block, left bundle branch block, trifascicular block and prolonged QT-interval; were they aware of the automatic ECG interpretation provided by conventional 12-lead ECG machine and how helpful was it (extremely/ very/ moderately/ slightly/ not helpful); had they attended ECG interpretation training in the last 3 years, did they want to attend one and their preferred mode of training delivery (hardcopy such as RACGP Check magazine/ webinar (seminar via the internet)/ online education module/ face to face workshop/ other, specify). The initial survey questions were developed by the study team and piloted with six practitioners to assess suitability and clarity. Through consultations with GP peers the survey was substantially shortened to enable completion in 5 mins. and thereby minimise the GP's workload and maximise participation. Data collection also included participant's demographic information, the number of years working in general practice (less than one year/ 1–4 years/ 5–9 years/ 10–19 years/ 20 years and more), the number of hours per week working in general practice  and their practice location postcode. We converted postcodes to state/territory using JavaScript codes [We designed a survey titled “pt codes  and catept codes . The surpt codes . Ethics P-value < 0.05. Analysis was performed using SPSS version 25 [Assuming a conservative estimation that 50% of GPs performed any form of AF screening, the study would require a sample size of 385 for estimating the expected proportion with 5% absolute precision and 95% confidence. That is, we would be 95% confident that between 45% and 55% of the GP population did some form of AF screening . Categorrsion 25 . With rersion 25 .A total of 83 organisations were approached via emails. The response rate was 51.8% i.e. 43 of the organisations agreed to disseminate the survey via their electronic newsletters and websites; 32 (38.6%) didn’t reply to the first and second email requests, and 8 (9.6%) declined the request  didn’t complete the consent question and 87/218 (39.9%) consented but didn’t complete the survey of whom 79 didn’t answer any questions at all and eight answered some questions but terminated before the demographic questions. These incomplete responses were excluded from analysis.Among the 463 participants, 328/463 (70.8%) were female, 378/463 (81.6%) were fellows of RACGP or ACRRM, 265/463 (57.2%) had less than ten years work experience in general practice and 325/463 (70.2%) usually worked 17–40 h per week, and 209/463 (45.1%) worked in regional rural areas  reported that they performed some forms of AF screening and applied at least one AF screening method  and 69  reported that they would not screen asymptomatic patients for AF. Among the 394 respondents, 151  applied two or more AF screening methods. The AF screening methods reported were as follows: pulse-palpation , 12-lead ECG , Holter monitor , mobile screening device , and use of blood pressure measuring device to detect AF .GPs reported high rates of screening for AF for patients with comorbidities: patients with heart failure (95.9% screened), stroke (94.4% screened), valvular heart disease (93.7% screened), diabetes (90.1% screened), hypertension (89.8% screened) and patients prescribed antidepressant (49.5% screened)  reported they considered assessing their patients for arrhythmias other than AF. Among these, 237/299 (79.3%) assessed for complete heart block, 236/299 (78.9%) long-QT, 221/299 (73.9%) second degree heart block, 208/299 (69.6%) left bundle branch block, 99/299 (33.1%) trifascicular block, and 33/299 (11.0%) for other arrhythmias .Respondents were asked to select all methods that they used to acquire an ECG: 416/463 (89.8%) obtained a 12-lead ECG in their practice, 110/463 (23.8%) referred to a pathology service, 14/463 (3.0%) referred to a cardiologist, 11/463 (2.4%) used a mobile handheld ECG device, 7/463 (1.5%) referred to hospital, and 2/463 (0.4%) used another method (e.g. ECG done by emergency department nurse). Many practitioners  found it was moderately to extremely time consuming to obtain a conventional 12-lead ECG. Most of them  were aware of the automatic ECG interpretation provided by a conventional 12-lead ECG machine but many of them  found the auto-interpretation slightly helpful or not helpful at all, while some of them  found it very or extremely helpful and remainder of them  found it moderately helpful.When asked whether the practitioners used a mobile health device to obtain a single-lead ECG on patients, 424/463 (91.6%) never used one: 277/463 (59.8%) replied “never and not contemplating using one”, while 147/463 (31.7%) replied “never but contemplating using one”. However, some respondents reported that they were very or extremely confident that a mobile handheld ECG device could provide adequate quality of information to detect the following conditions: AF 104/463 (22.5%), complete heart block 52/463 (11.2%), and long-QT 30/463 (6.5%).Only 110/463 practitioners 23.8%) reported that they had attended ECG interpretation training in the last 3 years. GPs reported that they were very or extremely confident in interpreting the following ECG abnormalities: AF , complete heart block , left bundle branch block , distinguishing between Mobitz I and II heart block , prolonged QT-interval  and trifascicular block   reported that they performed some forms of AF screening and applied at least one AF screening method and 46.9% versus 24.0% (p <  0.001) applied two or more AF screening methods . Use of mobile screening devices was limited in both groups i.e. 12/175 (6.9%) and 4/288 (1.4%) (Table p <  0.001), “number of years working in general practice” (p <  0.001), and “confidence in ECG interpretation of AF” (p = 0.003). Location of practice i.e. urban and rural (p = 0.360) and gender of GP (p = 0.510) were not significant predictors.The survey was launched approximately 2 months after the release of the Australian AF Guidelines and opened for participation for about 6 months. Approximately one third 175/463, 37.8%) of respondents were aware of the Australian AF Guidelines and among them 143/175 (81.7%) had applied the AF Guidelines with most or all their patients aged 65 years and older. Comparing those who were aware and unaware of the AF Guidelines, we found 98.9% versus 76.7%  in patients with heart failure, stroke and valvular disease. Screening for AF was significantly and positively influenced by awareness of the AF Guidelines, the number of years working in general practice and their confidence in ECG interpretation of AF. Taggar and colleagues reported that improving practitioners’ skill in diagnosing AF had a role in AF screening . Our finIn 2017, the Economist Intelligent Unit (EIU) [Despite mobile devices being referred to in the AF Guidelines, only 3.5% used a mobile device in practice, though about a third were contemplating using one, and about 23% were confident that a mobile device could provide adequate quality of information to detect AF. Obtaining an ECG trace is important in confirming the diagnosis of AF and about 90% of GPs reported being able to obtain a 12-lead ECG in their practice. However, GPs reported that they infrequently used 12-lead ECG for screening. Nearly half of GPs reported that 12-lead ECGs were time consuming, only about 80% reported they were confident in interpreting AF on ECGs and about half reported that ECG auto-interpretation was unhelpful or minimally helpful. These observations reflect some barriers to GPs using ECG to aid their AF screening.Mobile health devices are emerging technologies. Yet, the rising availability and interest from consumers in mobile ECG devices , 21 coupWith the increasing pressure on general practice in Australia, and indeed on primary care internationally, it is not surprising that time is a barrier to obtaining an ECG trace. In a study conducted by Somerville and colleagues, the average time taken to obtain a conventional 12-lead ECG in general practice was 10.6 min (including the time taken for preparing the patient and placing electrodes correctly) . While tPatients with other arrhythmias could present with similar symptoms to AF e.g. palpitations, syncope and dizziness. Hence, it is important that GPs are capable of diagnosing AF and other arrhythmias. Our findings show that most GP respondents were confident in diagnosing AF using ECGs. While several GPs considered assessing their patients for other arrhythmias, there was lower confidence in interpreting these less common but critical ECG abnormalities such as heart blocks and prolonged QT. Some guidelines do recommend ECG screening for prolonged QT in patients prescribed tricyclic antidepressants . At presRecruiting GP participants in research has been reported by several studies as challenging , 31. OurWe limited the number of questions and cut questions during our pilot as we wanted the survey to take less than five minutes to complete. Therefore our study did not obtain information on the accuracy of ECG interpretation, the correlation between confidence in and correctness of ECG interpretation and the further details about the types of patients they actually saw in practice, and the actual frequency of AF screening among patients seen in their practice.Many GPs performed some forms of AF screening and applied at least one AF screening method, but there were barriers to the timely and efficient acquisition of medical-quality ECG traces to aid their diagnoses and a lack of uptake of newer technologies that could help to address these time barriers. Our findings suggest that raising awareness of AF Guidelines and improving confidence in ECG interpretation may increase uptake of AF screening. Also identifying and researching ways to enable efficient AF screening in primary care may assist. GPs were interested in ECG interpretation and keen for further knowledge, especially in the interpretation of other ECG abnormalities, and hence supporting this with accessible continuing education programs should be prioritised.Additional file 1. General Practitioners Screen their patients for Atrial Fibrillation and othEr aRrhythmias (GPSAFER) – Survey questionnaire.


Isometric and isokinetic torque, muscle soreness, Z-band streaming, muscle heat shock protein (HSP) 25 and 72, plasma creatine kinase (CK), and plasma interleukin-6 (IL-6) were measured over 96 h post-RE to assess various direct and indirect markers of muscle damage. Results: Integrated MyoPS increased ~72% over 96 h after RE (p < 0.05), with no differences between groups (p = 0.98). Isometric, isokinetic, and total peak torque decreased ~21% by 48 h after RE (p < 0.05), whereas total peak torque was ~10% greater overall during recovery in LEAAs compared to placebo (p < 0.05). There were moderate to large effects for peak torque in favour of LEAAs. Muscle soreness increased during recovery with no statistical differences between groups but small to moderate effects in favour of LEAAs that correlated with changes in peak torque. Plasma CK, plasma IL-6, and muscle HSP25 increased after RE (p < 0.05) but were not significantly different between groups (p ≥ 0.13). Consistent with a trend toward attenuated Z-band streaming in LEAAs (p = 0.07), muscle HSP72 expression was lower (p < 0.05) during recovery in LEAAs compared with placebo. There were no correlations between MyoPS and any measures of muscle damage (p ≥ 0.37). Conclusion: Collectively, our data suggest that LEAAs moderately attenuated muscle damage without concomitant increases in integrated MyoPS in the days following an acute bout of resistance exercise in free-living recreationally active men.Background: Leucine-enriched essential amino acids (LEAAs) acutely enhance post-exercise myofibrillar protein synthesis (MyoPS), which has been suggested to be important for muscle repair and recovery. However, the ability of LEAAs to concurrently enhance MyoPS and muscle damage recovery in free-living humans has not been studied. Methods: In a randomized, double-blind, placebo-controlled, parallel-group design, twenty recreationally active males consuming a controlled diet (1.2 g/kg/d of protein) were supplemented thrice daily with 4 g of LEAAs (containing 1.6 g leucine) or isocaloric placebo for four days following an acute bout of lower-body resistance exercise (RE). MyoPS at rest and integrated over 96 h of recovery was measured by D Muscle protein synthesis (MPS) is elevated for up to 48 h after resistance exercise (RE) as a means to remodel and repair skeletal muscle ,2,3. It Unaccustomed RE results in structural perturbations of the muscle cytoskeletal architecture  that is commonly characterized as muscle damage ,21, althMuscle damage induced by unaccustomed RE is also commonly assessed through surrogate markers including loss of force , increasAccordingly, the purpose of this study was to determine the impact of LEAA supplementation on muscle recovery over a 96 h post-RE period in free-living conditions through assessments of integrated MyoPS and various markers of muscle damage and performance. This allowed us to investigate the relationship between integrated MyoPS and muscle damage recovery following RE. We hypothesized that the provision of LEAAs would increase post-RE integrated 96 h MyoPS compared to an isocaloric placebo, and that this would be associated with reductions in muscle damage outcomes.https://www.clinicaltrials.gov as NCT03319147. Participants completed a Physical Activity Readiness Questionnaire [Twenty recreationally active and healthy young men  providedionnaire  to verif2O). A second saliva sample was obtained two hours following the ingestion of D2O. A fasted saliva sample was obtained on Day 2, and both fasted saliva and blood samples were obtained on Day 3.In a randomized, double-blind, placebo-controlled, parallel-group design, participants underwent an 8 day exercise trial after being assigned to the LEAA or placebo group. Participants were instructed not to perform any structured physical activity during the 8 day trial. Daily physical activity was tracked via accelerometer , which allowed us to confirm that accelerometer−based measures of total energy expenditure, moderate to vigorous physical activity, and total step count were not different between groups. An overview of the 8 day exercise trial is outlined in On Day 4, while fasted, a baseline muscle biopsy was obtained from the non-dominant leg along with blood and saliva samples. A standardized breakfast  was consumed immediately post-biopsy, and a minimum of one hour elapsed post-breakfast before participants underwent the RE protocol. Saliva and blood samples were obtained directly post-RE, and then participants immediately ingested either a LEAA or placebo drink (time 0 h). A second muscle biopsy from the same leg was obtained at 4 h post-RE, along with saliva and blood samples. On Days 5–8, fasted saliva and blood samples were obtained at 24, 48, 72, and 96 h post-RE, whereas third and fourth muscle biopsies from the same leg as the previous biopsies were obtained at 48 and 96 h post-RE, respectively. This unilateral biopsy approach was designed to minimize any biopsy induced discomfort from impacting our secondary outcomes of muscle strength and soreness (see below). In addition, as acute, low-load exercise can stimulate muscle protein synthesis , the comMaximal muscle strength and muscle soreness were assessed pre-RE and 4, 24, 48, 72, and 96 h post-RE on the contralateral leg from which biopsies were obtained. Maximal muscle strength was assessed by maximal voluntary isometric contraction and isokinetic shortening contractions (MVC) at slow (60°/s) and fast (270°/s) angular velocities using an isokinetic dynamometer  . Each coSubjective ratings of muscle soreness  were collected from participants using a 100 mm visual analogue scale. Extremes of 0 and 100 mm were anchored as “no pain” and “worst possible pain”, respectively, and participants drew a line on the scale indicating their perceived soreness. Total perceived muscle soreness was calculated as the sum of thigh, hamstring, and calf muscle soreness.Participants were on a controlled diet throughout the duration of the exercise trial in the form of commercially available, pre-packaged foods that provided energy to cover 1.6*REE. The macronutrient distribution was 1.2 g/kg/d of protein, 4 g/kg/d of carbohydrate, and the remaining calories from fat. Although this protein intake is slightly lower than the habitual dietary intake of our participants  and the Test drinks were consumed three times per day on Days 4−7 and subjects were informed to separate meals and test drinks by at least 1.5 h. LEAA drinks were prepared individually using crystalline amino acids  and the placebo drink was energy matched with carbohydrates from Polycal . The LEAA mixture consisted of 4 g of essential amino acids . In total, 20 g of Tang , 5 g of Splenda , and 2 g of PFD-1  were added to all drinks to mask the taste and ensure blinding.The RE protocol was composed of 45° inclined bi-lateral leg press and leg extension. Each exercise consisted of five sets of 9–12 maximum bilateral repetitions at 75% 1RM with 90 s of rest between sets [Muscle samples were obtained from the vastus lateralis of the non-dominant leg only though percutaneous muscle biopsy with manual suction . The pro2O ingestion to assess changes in myofibrillar fractional synthesis rates (FSR) integrated over 96 h post-RE [2O ingestion was determined through isotope exchange to acetone using GC−MS as previously described [Ala is deuterium enrichment of protein-bound alanine, EBW is mean deuterium precursor enrichment of body water between time points, and t is the time (h) between biopsies. The single biopsy approach was used to estimate resting (baseline) MyoPS [MyoPS was determined by oral D post-RE . Muscle  post-RE ,41. Muscescribed . The fraescribed ,43. In be) MyoPS .Muscle HSP was assessed by Western blotting for muscle HSP25 and HSP72 content using primary polyclonal antibodies specific to muscle HSP25  and muscle HSP72 , diluted 1:1000 in TBST and 2% milk, with secondary antibody diluted 1:20,000 in 3% milk. The overall Western blotting protocol has been previously described .After being frozen, muscle tissue samples were fixed in osmium tetroxide, dehydrated in graded ethanol baths and embedded in epoxy resin (Spurr’s) with fibers oriented longitudinally. Each block was sectioned (0.5 mm) and marked with toluidine blue. Individual fibers were mainly examined at ×1000 magnification  by electron microscopy for areas of Z-band streaming and images were obtained. Z-band streaming was scored by analyzing fibers for the percentage area occupied by Z-band streaming per total fiber area in the image, as well as the percentage of fibers containing any Z-band streaming . A seconBlood samples were withdrawn into tubes containing ethylene diamine tetraacetic acid (EDTA) disodium salt and centrifuged for 10 min at 2500 rpm. Plasma was subsequently aliquoted and stored at −80 °C.Quantification of plasma CK activity was performed with a commercial kit  and quantification of plasma IL-6 concentration was performed with a commercial ELISA kit  in accordance with the manufacturer’s instructions.Witard et al.  reportedt-tests were used to compare participant characteristics between groups. Due to approximately half (45%) of participants not displaying any Z-band streaming post-RE, the Z-band streaming data violated assumptions of normality and were thus analyzed by linear mixed models analysis with group and measurement time as fixed factors and subjects as random factors. Values with two standard deviations from the mean were classified as outliers, and sensitivity analyses were performed with the outliers winsorized [p < 0.05.All variables were examined by two-way ANOVA with treatment and time as factors. When a significant main effect or interaction was observed, differences were examined by Sidak’s multiple comparisons test. Independent samples nsorized  to the nnsorized  but withnsorized . Therefonsorized  was calcnsorized , outliernsorized  to the nDue to potential placebo effects on performance and perceptual outcomes, it was important to blind participants to their supplement group. When asked to guess the test drink they received, participants had the option to choose from, “Protein”, “Carbohydrate”, or “Don’t know”, and then rated their confidence in their choice on a scale from 1 (not confident) to 5 (very confident). Out of the 20 participants, 8 correctly guessed the test drink they received, and 6 of these correct guesses were confident (3 or higher). We therefore believe that participants were well blinded, and that potential knowledge of their treatment condition was not a factor in study outcomes.p < 0.001) and was not different between groups  with no differences between groups .Body water enrichment decayed over the 8 day trial (p > 0.05), slow isokinetic , fast isokinetic , and total  peak torque (expressed in N∙m). Expressed as a percent of baseline, all MVC measures significantly changed throughout the 8 day trial in both groups , slow isokinetic (p = 0.095) and fast isokinetic (p = 0.138) peak torque, and total peak torque significantly differed between groups , with that of the LEAA group being greater than that of the placebo group. There were no interaction effects for all MVC measures (p > 0.05). Notable results from sensitivity analyses of outlier correction include a larger effect of group on isometric peak torque (p = 0.078) and total peak torque (p = 0.036) with that of the LEAA group being greater than that of the placebo group. There were overall moderate to large effects of LEAAs compared to placebo on post-RE MVC measures (There were no baseline group differences for mean (±SD) isometric , whereas calf soreness remained unchanged , but there were small to moderate effect sizes favouring LEAAs over placebo , there was a trend for placebo to be greater than LEAAs for both measures .The scores of percentage of muscle fiber area with Z-band streaming and percentage of muscle fibers with any Z-band streaming are presented in p = 0.437), significantly increased over the 8 day trial (p < 0.001). Although muscle HSP25 was not significantly different between groups (p = 0.668), muscle HSP72 was significantly lower with LEAAs relative to placebo . Notable results from sensitivity analyses of outlier correction include a larger effect of time (p = 0.008), group (p = 0.020), and interaction (p = 0.097) on muscle HSP72 for LEAAs to be lower than placebo.Expressed as a percent of baseline, muscle HSP25, but not muscle HSP72 (p = 0.029), but there were no effects of group or interaction . Notable results from sensitivity analyses of outlier correction include a larger effect of time (p = 0.004), group (p = 0.206), and interaction (p = 0.261) on plasma CK for LEAAs to be lower than placebo, and a larger effect of group (p = 0.053) on plasma IL-6 for LEAAs to be higher than placebo.Expressed as a percent of baseline, plasma CK activity significantly increased over the 8 day trial (p > 0.05). Total muscle soreness correlated with isometric , fast isokinetic , and total  peak torque. Muscle HSP25 and HSP72 only correlated with each other . Plasma IL-6 only correlated with fast isokinetic peak torque . Aside from correlations within MVC measures, no other correlations were apparent for all variables. Figures for the correlative data presented here can be found in The main finding of this study was that, in contrast to our hypothesis, LEAA supplementation three times per day was not more effective than placebo at enhancing integrated MyoPS over 96 h following an unaccustomed bout of RE in recreationally active men consuming a controlled diet with 1.2 g/kg/d of protein. However, consistent with our hypothesis, LEAAs moderately improved recovery from post-RE muscle damage, which interestingly did not correlate with changes in MyoPS. 2O. We speculate that this may be related to our lower D2O dosing protocol, which was based on previous seminal work [2O) for measurements over shorter incorporation times [Resistance exercise increased integrated rates of MyoPS over the 96 h post-RE recovery period in our recreationally active young men. This is consistent with the ability of RE to stimulate MyoPS for at least 48 h ,52 and pnal work  but has on times ,55. NeveTo increase the ecological validity of the study, participants in the present study consumed a controlled diet of 1.2 g/kg/d, which is slightly lower than the North American average  but is iStudies investigating protein supplementation and muscle damage recovery following lower-body RE in untrained individuals have yielded inconsistent findings. For example, studies have reported positive effects of protein supplementation on post-RE muscle soreness but not muscle strength , muscle The underlying mechanism for the attenuation of muscle force losses with LEAA supplementation is unknown. Previous authors have suggested that the greater bioavailability of amino acids, particularly the BCAAs, may reduce the extent of myofibrillar breakdown following eccentric exercise and thus preserve muscle function ,67. HoweDamaging eccentric exercise elevates the content of HSP25  and HSP72 in skeletal muscle ,73,74. MThe present study employed a parallel-group design to assess the impact of LEAAs on post-RE muscle damage in order to avoid the prohibitively long washout that would otherwise be required to allow for an attenuation of the repeated bout effect, which might persist for up to 12 weeks . In addiIt has been suggested that increased MyoPS following unaccustomed RE and protein ingestion facilitates enhanced skeletal muscle remodeling and therefore may improve the recovery from exercise-induced muscle damage , but thiIn summary, the increase in integrated MyoPS over 96 h post-RE was not enhanced by thrice daily LEAA supplementation (containing 1.6 g leucine each) compared to placebo. However, LEAAs preserved muscle force production and moderately attenuated muscle soreness, which was accompanied by a reduced muscle HSP72 response and a trend toward decreased Z-band streaming. Although muscle soreness and strength were correlated, no relationship was observed between changes in MyoPS and any markers of muscle damage. Collectively, our data suggest that LEAAs have the ability to mitigate muscle damage in the days following an acute bout of resistance exercise independent of augmenting free-living MyoPS. Future research examining the mechanisms of inflammation and muscle damage recovery in response to amino acid supplementation and unaccustomed exercise is warranted.