Hepatitis C virus (HCV) infection is a widespread problem in prisons. The present study aimed to assess the prevalence of HCV seropositivity, HCV genotypes, factors associated with HCV seropositivity in newly incarcerated prisoners and to report experiences of treatment with pegylated interferon/ribavirin for HCV-positive inmates.Vanglate meditsiiniline infosüsteem) databases.Patient data were extracted from the Estonian prison medical information system  and 1b . Twenty-five prisoners received HCV treatment: 60% (n = 15) were cured, 16% (n = 4) relapsed , and 12% (n = 3) were unresponsive .Among 1845 prisoners newly incarcerated from January 2014 to January 2015, the overall prevalence of HCV was 56.3% (95% CI: 54 to 59), and 25.5% (95% CI: 23.5 to 27.6%) had HIV (39.0% had neither). The all-inclusive HCV testing strategy identified 37.7% more HCV infected prisoners than the risk-based  case finding. Factors associated with HCV seropositivity included history of drug use (aOR 6.51 95%CI 5.12–8.28), HIV co-infection (aOR 2.56 95%CI 1.92–3.43), previous incarceration (aOR 3.61 95%CI 2.48–4.04), and increasing age. The main HCV genotypes were 3a (HCV seropositivity rate is high and HCV tretment rate is very low in Estonian prisons. Optimizing case finding and scaling up treatment is critical to addressing the health needs of prisoners and meeting public health goals. Hepatitis C virus (HCV) infection is an escalating global health issue and prisoners are emerging as an important high-risk group. HCV prevalence in this subpopulation ranges from 3 to 38% , 2. AlthHCV prevalence is higher among prisoners than in the general population . IntravePrisoners with human immunodeficiency virus (HIV) are more likely to be infected with HCV probably because of the similarity in the routes of transmission of these blood-borne infections , 3. AccoAnother risk factor related to HCV infection is repeated incarceration , 13, an In some studies HCV infection was observed more frequently in female inmates than in males. . In partDirect-acting antivirals (DAAs) are relatively new and expensive, but cost-effective when well utilised as interventions for chronic hepatitis C, and preliminary results suggest that DAAs may eradicate HCV from the blood . The effect of long-term treatment with DAA  is yet to be convincingly demonstrated . Still, Most HCV infected people are unaware of their condition . PrisonsThe prison population in Estonia has stabilised at nearly 3000 individuals in recent years but the incarceration rate, with 222 prisoners per 100,000 population, still remains very high. According to the Council of Europe’s annual penal statistics, the highest incarceration rates in Europe are recorded in the former Soviet Union countries  .Needle and syringe exchange programmes (NSP) in Estonia were launched in 1997. In addition to NSP, condom distribution, different counselling services incl. social, health, psychological, legal, experience counselling), basic humanitarian aid, primary hygiene possibilities , naloxonAccording to the Estonian Ministry of Justice, there were 3580 prisoners in Estonia’s four prisons during the period between 1 January 2014 and 1 January 2015 . A study conducted in Estonia in 2012–2013 found that, of all prisoners, one-third had a history of drug use, 15.6% were infected with HIV and, of those that were HIV-positive, 54.1% were receiving antiretroviral therapy (ART) .The implementation of the project “Improvement of Prevention and Treatment of Infectious Diseases in Estonian prisons”  targetinThe aim of this study was to assess the prevalence of HCV seropositivity and its related factors among newly incarcerated prisoners and to describe the effects of treatment of HCV infection in Estonian prisons.This study was carried out from 1 January 2014 to 1 January 2015. The term ‘newly incarcerated prisoner’ is used for all people, including adults and juveniles, detained during the investigation of a crime, while awaiting trial, after conviction, before sentencing and after sentencing, and incarcerated in one of the four Estonian prisons.For this study we relied on the secondary data available in the administrative and health databases of the Estonian prison medical information system.The prison electronic patient database is designed for prison healthcare and contains electronic health records. For those tested for HCV, the following data were extracted from prisoners’ health records: (i) HCV test result, (ii) HCV genotype, (iii) age, (iv) gender, (v) HIV serostatus, (vi) history of drug use , and (vii) HCV treatment results. HCV in prisons is treated by prison infectious diseases physicians who also make HCV-genotype testing decisions.For all samples collected in the prison medical information system over the study period, HIV testing , anti-HCV antibodies testing , and HCV genotyping (The VERSANT® HCV Genotype 2.0 Assay) were performed at Synlab Estonia .prisoners’ register). The prisoners’ register is an electronic database containing selected information on the details of prison sentence as well as personal data. Information on tattoos included in the register is based on inspection of prisoners’ skin at prison entry.Data on tattoos and previous incarceration were extracted from the databases of prisoners, detained persons, persons in custody and probationers  and relative frequencies (%)) and continuous variables . A chi-squared test was used to explore differences in categorical variables and the Wilcoxon rank sum test for age comparisons between the HCV infected and non-infected groups. Associations between antibodies to HCV (the outcome of interest) and covariables were explored bivariably and multivariably  by logistic regression analyses. Odds ratios (OR) and adjusted OR (AOR) together with 95% confidence intervals (CI) are presented. Associations between covariables were also tested to assess the reliability of the multivariate models (according to the possibility of multicollinearity). The statistical software program SPSS version 16.0  was used.n = 1242) were tested for HCV with the exception of 112 prisoners (26 prisoners declined testing and the reason for not testing was unknown for the other 86).Between 1 January 2014 and 1 January 2015, 1957 new prisoners were admitted to the Estonian prison system. Of those, 603 were already infected with HCV (based on data from previous testing in prison). As per the new testing strategy, the rest of the newly imprisoned individuals (n = 1742), 58.1% had previously used illicit drugs (n = 1034), 28.1% were tattooed (n = 519), and 68.9% had been previously incarcerated (n = 1272). The prevalence of HCV seropositivity was 56.3%  and the prevalence of HIV 25.5% (95% CI 23.5–27.6%) (n = 469). One fifth  of the newly incarcerated prisoners had HCV-HIV co-infections. The majority  of the HIV-positive prisoners were also infected with HCV . Of these prisoners, 94.4% were male (p < 0.001), to have a history of drug use , to be HIV-infected , and to have previously been incarcerated . There were no gender differences in HCV seropositivity (Table The old testing strategy – high-risk based case finding - would have identified only 37.7% (164/435) of the HCV infected newly incarcerated prisoners.While comparing HCV infected and non-infected newly incarcerated prisoners, we found that HCV-infected prisoners tended to be slightly older, and were more likely to have a tattoo . The risk of HCV seropositivity was also significantly higher among those who were HIV-positive (AOR 2.56 95% CI 1.92–3.43) and those previously incarcerated (AOR 3.16 95% CI 2.48–4.04). The risk of HCV positivity increased with age. While tattooing was associated with the HCV rates in the bivariable analysis, this association was not found in the multivariable analysis.n = 172) and 1b . A further 17% (n = 65) had genotype 1a, and 2.6% prisoners (n = 10) had genotype 2. Only one prisoner had HCV genotype 4.Of the 1038 prisoners who tested positive for HCV, 383 (36.9%) underwent HCV genotype testing. The main genotypes identified were 3a , received HCV treatment (pegylated interferon (PEG-INF) and ribavirin). The decision to treat was reached after an in-depth, individualized process including informed consent and considering other factors such as co-morbid addiction and mental illnesses. For patients with a history of drug use, a program-based addiction treatment  was requNone of the patients had previous experience with interferon and ribavirin. The average age of treated patients was 35.5 years (range 29–45). Fifteen of 25 (60%) treated patients underwent ultrasound-guided liver biopsies. The Metavir scoring system was used to classify fibrosis levels . 11  among newly imprisoned individuals in Estonia. HCV seropositivity among newly incarcerated and tested prisoners (with either unknown HCV status or previous negative HCV test) was 35%. These prevalence figures are consistent with those from Italy (38%)  and ScotThe risk factors for HCV seropositivity we observed  are in accordance with previous research , 14, 36.Previous incarceration has been significantly associated with HCV infection in studies from Australia, England, and Wales –38. MultIn our study, the seropositivity of HCV was higher among those with tattoos. However, this association was lost while adjusting for potential confounders. It is known that tattooing is common in prisons and jails  and prisWe found the presence of hepatitis C antibodies in 12 individuals (0.6%) who were HIV negative and had no records indicative of drug use. While we do not have data on other HCV risk factors , all of the 12 prisoners had tattoos. Eight of them had acquired their tattoos while in prison during the Soviet era . One might speculate that the traditions of tattooing in prisons have changed .Data on the relationship between HCV and gender in prison populations is also conflicting. In some studies, HCV infection was observed more frequently in incarcerated women than in incarcerated men. , 3. OtheScreening strategies based on risk factors have traditionally failed to identify all patients at risk of infection, leaving a sizable proportion of patients unaware of their infection. We observed that a screening system for HCV case finding relying only on known risk factors misses a sizeable proportion of newly incarcerated individuals with HCV infection. The all-inclusive HCV testing strategy identified 37.7% more HCV infection cases among the newly incarcerated than would have been detected on the basis of known risk factors  screening.n = 172, 44%) and 1b . HCV genotypes have geographically distinct distributions. HCV genotype 1 and 3 infections are globally more prevalent than all other genotypes. Those genotypes, more commonly found in lower-income countries, account for a significant proportion of HCV cases worldwide [In our study, the main HCV genotypes were 3a  do not differ from that in the community.Reducing HCV prevalence among prisoners has the potential to reduce the burden of disease in the general public in the community. When developing HCV screening strategies in prisons, policy-makers should consider the risk factors that we have highlighted in our study.Our findings have several implications: they show that (i) universal HCV testing at prisonentry has the potential to increase the number of people who are aware of their infection, (ii) there is a need to scale up comprehensive HCV care to those infected. We believe that the results of this study might be applicable to other countries in the region that have witnessed injection drug use and related HIV epidemics in the late 1990s and early 2000s.


While hyperthermic intraperitoneal chemotherapy (HIPEC) after cytoreduction surgery (CRS) has been shown to improve patient survival and disease-free progression in peritoneal carcinoma (PC) patients, the procedure relates to a high postoperative infection rate. Herein, we report the bacterial and fungal infections after CRS and HIPEC from a single institution in Saudi Arabia.  A prospective observational study was conducted on 38 patients with PC selected for CRS/HIPEC procedure between 2012 and 2015 in our centre. Escherichia coli was the most frequent isolate, followed by multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa. Lower preoperative albumin and a prolonged preoperative activated partial thromboplastin time (APTT) are associated with postoperative infections, while a prolonged preoperative hospital stay (hazard ratio (HR) = 1.064; confidence interval (CI) = 1.002–1.112; P=0.042) and more intraoperative blood loss (>10%)  were independent risk factors for postoperative infections. Three cases died during the follow-up period; all were due to infection.  Postoperative bacterial and fungal infection within 100 days was 42.2%, bacterial infection was reported always, and fungal infection was reported in 5 (13.2%) cases. Infections from the surgical site were considered the most common infection site. Multidrug-resistant extended-spectrum beta-lactamase (ESBL)  The infection rate in our centre compared to previous studies of comparable patients was matching. Effective management of postoperative infections should be considered, and identified risk factors in this study can help to focus on effective prevention and treatment strategies. In the past, peritoneal carcinomas (PCs) were considered an untreatable situation, with poor prognosis and short life expectancy following diagnosis. Recently, cytoreduction surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) is being used more and more in selected PC patients and offers a promising prolonged survival or disease-free progression to PC patients , 2.CRS combined with HIPEC was developed first by Spratt in 1980 as a locoregional treatment for primary or secondary peritoneal tumours. The surgical procedure for CRS/HIPEC is described elsewhere . In summCRS/HIPEC has been widely existing for more than 20 years. However, this treatment has received a heavy criticism for its cost , high raAll patients with PC who were eligible for CRS/HIPEC at King Abdullah Medical City, Mecca, Saudi Arabia, from March 2012 to May 2015, were included in our study, and the study was approved by the institutional review board .2 dissolved in 1 litre normal saline) inside the peritoneal cavity. Heat is applied after an administration of the drug from 41 to 42°C for 90 min. Drains of a circuit pump placed inside the peritoneal cavity were used to extend and circulate the heated chemotherapy. HIPEC provides a high locoregional concentration of the chemotherapeutic agents with low systemic effect as the high temperature increases the infiltration ability of the drug without affecting the cell viability, and it also reduces side effects on the cardiovascular system, oxygen consumption, and coagulation. Herein, CRS/HIPEC surgeries were done by the same surgical team and using the same procedure. The hemodynamic status was controlled by using cooled infusions and replacement of fluid loss. To protect against postoperative infection, 2 gm cefazolin and 500 mg IV metronidazole were infused to patients, half an hour before surgery, and continued for 72 hours thereafter. Thoracic epidural analgesia was done to reduce the postoperative pain and the duration of postoperative ventilation. Immediate postoperative intensive care unit (ICU) admission for at least 48 hours was scheduled for all patients to provide postoperative organ or vasopressor support. Follow-up of patients was done monthly for the first 100 days and then every six months until the last visit or death. Our data included the first 100 days only. Clinical assessment and pathological investigation of tumour markers and CT scan's assessment were done to diagnose recurrence.The surgical procedure for HIPEC is described elsewhere . In summDetection of pathogens was done as described before . SamplesEmpiric antimicrobial treatment in the form of cefazolin, cefuroxime, cefepime, imipenem, ciprofloxacin, erythromycin, tazocin, amikacin, and metronidazole was initiated until culture results were released. Antibiotics were later adjusted or changed by the culture results. In case of fungal infection, fluconazole was given at 5–10 mg/kg/day IV over 1 hour; if there was no improvement, amphotericin B was administered as a continuous drip at a rate of 0.25–1 mg/kg/day IV. Miconazole oral gel was used daily, and the wide-spectrum antibiotic was stopped.t-test, and qualitative data were analyzed by the chi-square test to differentiate between the two studied groups: group 1 (complicated with infections) and group 2 (not complicated with infections). Data are presented as mean ± standard deviation or percentage where appropriate. The potential of several preoperative, operative, and postoperative variables for postoperative infection was measured by Cox's proportional hazard regression models with a 95% confidence interval, and the variables that gave P value of less than 0.05 in univariate analyses were used in a logistic regression model to determine the independent risk. A statistically significant relationship was indicated by P value of less than 0.05 by using Package for the Social Sciences for Windows statistical software (version 21).Quantitative data were analyzed by the Student's 2. The number of female patients was more than that of male patients: 23 and 15, respectively. Mucinous adenocarcinoma (15 (39.5%)) was the most commonly encountered histopathology; the colorectal tumour was the primary site of tumour in 20 (52.6%) patients. Tumour grading, organ metastasis, and KRAS mutation in the primary tumour were detected and compared between the studied groups. The total hospital stay follow-up period was (37.6 ± 38.6) (10–100) days, and the ICU stay was . Only new infections that occurred following the procedure were included; preoperative infections were excluded. There were only 3 mortalities, and all had an infection. Herein, a total of 38 patients who underwent CRS/HIPEC procedures from March 2012 to May 2015 met the inclusion criteria and were included in our cohort prospective study. The mean age of patients was 52 ± 14 (24.5–74.9) years, and the median body mass index was 26.63 (16.41–40.42) kg/mEscherichia coli were the most frequent bacterial pathogens, followed by multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecium isolates. Vancomycin-resistant Enterococci were isolated from 2 samples. In total, 9 fungal infections were caused by 7 yeasts other than Candida albicans (YOTCA) species and 2 by Candida albicans isolates. The exact details of the pathogens, site, and time occurrence are in Tables During the postoperative follow-up period, bacterial and/or fungal infection was reported in 16 42.1%) cases, bacterial infection was reported always, and fungal infection was reported in 5 (13.2%) cases. Patients with infection showed 43 episodes of infection, and infection rate was 2.8 per patient. In total, 45 bacterial and fungal pathogens were isolated from 2 polymicrobial and 41 monomicrobial episodes. The bacterial isolates were 36 pathogens. Gram-negative isolates were higher than Gram-positive organisms (Gram-positive/Gram-negative ratio is 7/29). Extended-spectrum beta-lactamase (ESBL) .1% casesn = 23, 53.5%), followed by respiratory tract , urinary tract , and bloodstream .Patients' investigations revealed forty-three episodes of bacterial and fungal infections. Surgical site represented the major focus  were further tested by multivariate analysis; a prolonged preoperative hospital stay (hazard ratio (HR) = 1.064; confidence interval (CI) = 1.002–1.112; P=0.042) and more intraoperative blood loss  independently predicted occurrence of postoperative infection , a prolonged preoperative hospital stay, and more intraoperative blood losses were potential risk factors for the occurrences of postoperative infections. Those potential predictors that showed significance (nfection .CRS and HIPEC are a promising therapeutic modality for PC patients; however, postoperative infections still represent the main cause for mortality, prolonged hospital stay, and healthcare costs in those patients , 9, 24. Owing to the obtained results, we suggest the use of a standardized protocol for the infection prevention, monitoring, and treatment in all patients enrolled for cytoreductive surgery and HIPEC based on the use of antibiogram presented in Tables


Patients with a Tac C/D ratio <1.05 ng/mL × 1/mg at three months after RTx were characterized as fast metabolizers and those with a C/D ratio ≥1.05 ng/mL × 1/mg as slow metabolizers. Five-year patient and overall graft survival were noticeably reduced in fast metabolizers. Further, fast metabolizers showed a faster decline of eGFR (estimated glomerular filtration rate) within five years after RTx and a higher rejection rate compared to slow metabolizers. Calculation of the Tac C/D ratio three months after RTx may assist physicians in their daily clinical routine to identify Tac-treated patients at risk for the development of inferior graft function, acute rejections, or even higher mortality. Tacrolimus (Tac) is a part of the standard immunosuppressive regimen after renal transplantation (RTx). However, its metabolism rate is highly variable. A fast Tac metabolism rate, defined by the Tac blood trough concentration (C) divided by the daily dose (D), is associated with inferior renal function after RTx. Therefore, we hypothesize that the Tac metabolism rate impacts patient and graft survival after RTx. We analyzed all patients who received a RTx between January 2007 and December 2012 and were initially treated with an immunosuppressive regimen containing Tac (Prograf Tacrolimus (Tac) is recommended by The Kidney Disease: Improving Global Outcomes (KDIGO) guideline as the immunosuppressant of choice after renal transplantation (RTx) . AlthougWe recently proposed a classification of patients receiving Tac into two major metabolism groups. Our stratification is based on the calculation of the C/D ratio . A C/D ratio <1.05 ng/mL × 1/mg identifies fast metabolizers, whereas a C/D ratio ≥1.05 ng/mL × 1/mg indicates a slow metabolism . AlternaPrior to analysis, all patient data was anonymized and de-identified. The local ethics committee  approved the study. The methods used in this study were carried out in accordance with the current transplantation guidelines and the Declarations of Istanbul and Helsinki. Written informed consent with regard to recording their clinical data was given by all participants at the time of transplantation.®), mycophenolate mofetil, prednisolone, and induction therapy. Oral CMV-prophylaxis with valganciclovir was administered for 100 days for D+/R+, D−/R+ and D+/R− recipients, and none if both the donor and the recipient were negative for CMV. Recipients aged < 18 years, with combined transplants, and for whom the three month C/D ratio could not be adequately calculated ) were excluded. The Tac target trough level was 6–10 ng/mL. Recipient and donor data was collected from the patient files. The following parameters were examined: Patient and donor demographics, recipient body mass index (BMI), recipient history of hypertension or diabetes mellitus, cause of end-stage renal disease (ESRD), number of prior kidney transplants, time on dialysis, donor type of transplantation, degree of human leukocyte antigen (HLA)-mismatching, current panel-reactive antibodies (PRA), cold and warm ischemia time and incidence of new-onset diabetes after transplantation (NODAT) and cytomegalovirus (CMV) DNAaemia ). CMV screening was performed monthly during the first six months after RTx, every second month during months 6–12, and on indication.We retrospectively analyzed all patients who underwent RTx between January 2007 and December 2012 at the University Hospital Münster and were initially treated with an immunosuppressive regimen containing Tac  by the corresponding daily Tac dose (D), as published before ,16.C/D As inpatient values are more prone to errors due to coexisting factors like diarrhea, anaemia and CYP3A4 interfering drugs as azoles, e.g., only outpatient tacrolimus concentrations were considered. Measurements with exceptional high Tac trough concentrations (>15 ng/mL) were not considered to exclude false-high values due to Tac ingestion prior blood sampling. For 50 randomly selected patients we additionally calculated the Tac C/D ratio at one and six months as an average C/D ratio and compared it to the three-month C/D ratio to account for further potential factors that can influence the C/D ratio and might affect single-time point measurements. As the 3-month C/D ratio strongly correlated with the average C/D ratio at month one and six, we applied the 3-month C/D ratio for the following patient categorization: As defined previously, patients with a Tac C/D ratio <1.05 ng/mL × 1/mg were categorized as fast metabolizers. Patients with a C/D ratio of 1.05–1.54 ng/mL × 1/mg or a C/D ratio ≥1.55 ng/mL × 1/mg were defined as intermediate metabolizers and slow metabolizers, respectively . For simThe main outcome measures were patient and overall graft survival. Patient survival was defined as time from RTx to death (from any cause) or last contact for alive patients. Overall graft survival was defined as the time from RTx to death (from any cause), graft failure, or last contact, whichever occurred first. Graft failure was defined as the reinitiation of dialysis treatment. Further outcome parameters were serum creatinine and estimated glomerular filtration rate (eGFR) at years one to five after transplantation as well as the frequency of biopsy-proven acute rejection episodes (defined by Banff classification) and the rejection-free survival. Patients were subjected to kidney biopsy in case of a relevant rise in creatinine (≥0.3 mg/dL). Biopsies were evaluated by two pathologists. Whole blood was analyzed for creatinine  and Tac (automated tacrolimus (TACR) assay; Dimension Clinical Chemistry System; Siemens Healthcare Diagnostic GmbH; Eschborn; Germany). Only 12 h Tac trough levels were used for analysis. Renal function was determined by calculating the eGFR using the CKD-EPI equation.® Statistics 25 for Windows . Normally distributed continuous variables are shown as mean ± standard deviation (SD) and non-normally distributed continuous variables as median and 1st and 3rd quartiles . Absolute and relative frequencies have been given for categorical variables. Pairs of independent groups were compared using the Student’s t-test for normally distributed data, Mann–Whitney U test for non-normal data, and Fisher’s exact test for categorical variables. To compare paired data, we used the Wilcoxon test for continuous variables and the McNemar test for categorical variables. Statistical analysis was performed using IBM SPSSp-value of the score test ≤ 0.05, exclusion: p-value of the likelihood ratio test > 0.1). Results have been presented as hazard ratios (HR) with 95% confidence interval (95% CI) and p-value of likelihood ratio test. The p-value of score test is given for non-selected variables in multivariable analyses.Survival analyses were based on a maximum follow-up of five years after RTx. Patient survival, overall allograft survival as well as rejection-free survival were analyzed using the Kaplan-Meier method , and thep-value of Wald test ≤0.05/>0.1).Mixed models with AR (1) covariance structure were fitted to analyze the impact of biological and clinical markers on the time course of eGFR between year one and five after the transplantation based on the eGFR values observed at annual intervals during this period. Univariable analyses included each marker separately along with its interaction with time since baseline measurement (at year one after transplantation) in order to assess (i) the baseline eGFR and/or (ii) whether potential time trends of eGFR differ between the subgroups defined by the marker. Multivariable models were built using a stepwise variable selection procedure in order to assess the impact of C/D ratio metabolism status on baseline eGFR and time trends of eGFR while adjusting for potential confounding factors. Models included (i) C/D ratio metabolism status and its interaction with time since baseline measurement in a first block and (ii) potential confounding factors along with their interactions with time since baseline measurement in a second block with forwards variable selection  as well as multivariable analysis (HR 5.749 (95% CI 1.556–21.242), p = 0.004) ) for fast metabolizers in univariable Cox regression and 2.715  after adjustment for potential confounders  . Overallfounders . Causes of death are given in p = 0.040) and multivariable analysis (p = 0.032)  a,b.p = 0.032, p = 0.035) as well as multivariable analysis (HR 1.622 (95% CI 1.085–2.424), p = 0.020)   . Table 7ctively) B, whereaHerein, we first described a significant influence of the Tac metabolism type on mortality after renal transplantation in a study population with a long-term observation period. A higher five-year mortality in fast metabolizers was accompanied by a higher rejection rate and inferior kidney function. Our study highlights the importance of a risk stratification strategy of RTx patients including information on individuals’ Tac metabolism rate which turned out to be an independent risk factor for a lower patient survival after renal transplantation. The C/D ratio is a simple tool that can be easily applied for this purpose. Based on our previous findings revealing an impact of fast Tac metabolism (C/D ratio <1.05 ng/mL×1/mg) on inferior renal function in a two-and three-year follow-up after RTx or LTx ,16 we heIn congruence, Kuypers et al. observed that patients with high early Tac dose requirements  had a significantly reduced kidney function at three-months post-RTx . This waIn both aforementioned studies, rejection rates were calculated but not related to the C/D ratio or the dose requirements. However, as Kuypers et al. observed significantly higher rates of graft failure (32.3% vs. 13.7%) and lower rates of patients discontinuing steroids (5.8% vs. 23.7%) in patients requiring higher Tac doses, one can assume a higher rejection rate in these patients. We herein firstly describe a significant effect of the C/D ratio on acute rejections in a long-term follow up. In our study, rejection-free survival was increased in slow metabolizers, with higher frequencies of humoral and mixed rejections in fast than in slow metabolizers. In multivariable regression analysis, the BMI and the number of prior transplantations were associated with rejection as well. Recently, Barraclough et al. stated that the outcome of RTx patients depends on the immunosuppression within the first week after transplantation, although a relation between the AUC or Tac trough level and rejection was not detected . Of noteMortality in fast metabolizers over the five-year observation period was consistently higher than in slow metabolizers, despite a tendency towards an older age in slow metabolizers. Overall, graft failure was low in both groups but aligned with the data from the literature ,22. In aWe recognize that a study of this nature has limitations because of its retrospective design and potential errors inherent to maintaining a single-center database. Moreover, due to the relatively small patient size, inaccuracies in the data collection might affect the results; though data acquisition was performed thoroughly to avoid inconsistency or entry errors. The analyses are based on the assumption that coding errors and missing data are stochastic. Although we attempted to include as many relevant confounding parameters as possible there might still be residual factors that were not accounted for like the non-adherence of patients for example, which is difficult to measure. Prospective studies are needed to confirm our findings. We conclude from our data that the calculation of the C/D ratio, as a simple, cost-effective tool, can assist physicians in their daily clinical routine to identify Tac-treated patients at risk of developing an inferior graft function, acute rejections, or even higher mortality. This information should be used to individualize and optimize immunosuppressive therapy.


The aim of this study was to evaluate the correlation between endothelial nitric oxide synthase (eNOS) polymorphism (−786T>C) and migraine susceptibility in a meta-analysis.I2 index and quality assessment was performed using the Newcastle–Ottawa scale.A literature search was performed for case–control studies from inception to July 30, 2018 focusing on eNOS polymorphism (−786T>C) and risk of migraine. From 454 full-text articles, 6 were included in this study. Heterogeneity was assessed with the P = .17; I2 = 18%). However, subgroup analysis showed CC variant increase the risk for migraine compared with TT+ TC genotypes in Caucasian populations , which could not be observed in non-Caucasian populations . There was no significant difference for other genotypes and alleles between patients with migraine and healthy controls .CC genotype was not related to higher susceptibility of migraine compared with TT+ TC genotypes with significant difference (fixed effects model; OR = 1.27; 95% CI = 0.90–1.80; This meta-analysis indicated that CC variant increases the risk for migraine compared with TT + TC genotypes in Caucasian populations. Migraine is clinically diagnosed according to the criteria set out by the International Headache Society (IHS). Migraine often begins with premonitory symptoms hours or days before the onset of pain. The most common premonitory symptoms include fatigue, impaired concentration, and neck stiffness. Furthermore, other psychological , arousal (drowsiness), neurological (photophobia), and cranial parasympathetic symptoms (lacrimation), and general symptoms  can occur before the onset of pain. The IHS classifies migraine as migraine with aura (MA), representing about one-third of all migraine, or migraine without aura (MO) that accounts for approximately two-thirds of all migraine in the population.,4,5 Although researches have been performed for many years, the complex pathogenetic mechanisms of migraine still remain unclear. Migraine is considered to be as a result of combination of environmental and genetic aspects.Migraine is a chronic paroxysmal neurological disorder characterized by attacks of moderate or severe headache and reversible neurological and systemic symptoms that affects approximately 12% to 16% of the population. It exerts a variety of physiological effects such as regulating blood pressure via smooth muscle relaxation, and functioning as a neurotransmitter. NO in the migraine pathogenesis has been reported in several studies.,9 Polymorphism of NOS could affect production of NO, which may be connected with migraine. The associations between a single nucleotide polymorphism (SNP) of NOS and migraine were widely studied.–20 Although nNOS is predominately expressed in neurons of the central and peripheral nervous systems, studies evaluating the association between nNOS polymorphism and migraine were limited. There was no marked association between nNOS polymorphisms  and migraine susceptibility.,11 Conversely, a series of studies reporting the relationships between SNPs of iNOS, eNOS genes, and risk of migraine.–20 There was no association between bi-allelic tetranucleotide polymorphism of iNOS and migraine. The 2087G>A (rs2297518) and the −1026C>A (rs2779249) polymorphisms in the iNOS gene affect the susceptibility to MA when their effects are combined within haplotypes, whereas the 2087G>A affects the susceptibility to aura in migraine patients. A significant interaction between iNOS 2087G>A and eNOS 2512+242G>A/C (rs743506) polymorphisms in migraine patients compared to control group, suggesting that this combination affect migraine susceptibility. Three clinically relevant polymorphisms of eNOS have been studied: SNP in the promoter region , an SNP in exon 7 , and the variable number of tandem repeats (VNTR) in intron 4.,22 Borroni et al reported that eNOS (+894G>T) polymorphism was an independent risk for MA. However, a later study could not verify the conclusion. A recent meta-analysis indicated that “T” allele of the eNOS +894G > T variant increased the risk of migraine among non-Caucasians. The substitution of thymine by cytosine in the 5′ promoter region at nucleotide—786 (−786T>C) results in a significant reduction in eNOS gene promoter activity and in decreased basal NO production. Similarly, there are some controversies for the connection between eNOS polymorphism (−786T>C) and migraine risk.,18–20 This meta-analysis was carried out to evaluate the relationship between eNOS (−786T>C) polymorphism and migraine susceptibility.Nitric oxide (NO) is a signaling molecule with a short half-life, which is synthesized from L-arginine by 3 isozymes of nitric oxide synthase (NOS), including neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS).22.1Two authors independently performed literature search for published studies from inception to July 30, 2018 using PubMed, Cochrane Database, EMBASE, Web of science, Google scholar, and China National Knowledge Infrastructure (CNKI). The keywords used were “nitric oxide synthase,” or “NOS,” or “endothelial nitric oxide synthase,” or “NOS3,” or “eNOS” and “migraine,” or “migraine disorder.” The titles and abstracts of the resulting articles were examined and unrelated articles were excluded. If an article was selected for inclusion, the references were reviewed for additional studies. This study was approved by the ethics committee of the First Hospital of Jilin University.2.2,5,14,19 or whose health was established by medical diagnosis.,20 Studies were excluded if one of the following existed: studies with insufficient genotyping data of patients or control group; case reports; review articles; experiment researches; case-only studies. Disagreements between the 2 reviewers regarding inclusion of a study were resolved through consensus.An article was relevant if it reported original data from case–control study, regardless of language, investigating the correlations between eNOS polymorphism (−786T>C) and migraine susceptibility. Controls were healthy individuals without migraine randomly selected from general populations after excluding those with underlying diseases ,2.3Two independent reviewers extracted the data and assessed the quality of each study using the Newcastle–Ottawa scale (NOS). In this scale 9 points represent the highest quality. In case of disagreements, the original article was evaluated by a third reviewer.2.4I2 statistic: values of 25%, 50%, and 75% represent mild, moderate, and severe heterogeneity, respectively. Based on results of the heterogeneity test, a fixed effect model was used if P > .10, while a random effects model was performed if P ≤ 0.10. Begg's funnel test and Egger's test were applied to evaluate publication bias across studies with Stata (version 12.0). A P < .05 was considered statistically significant.RevMan 5.2 (Cochrane database) was used to analyze the data. The results were reported as odds ratios (OR) with 95% confidence intervals (CI). Heterogeneity between studies was assessed by using the 33.1We followed the PRISMA  statement for reporting our results. Six studies of full-text articles were selected for inclusion in this meta-analysis. Our selection process for studies included in the analysis was shown in Figure 3.2,19 2 from Turkey,,5 1 from the United Kingdom, and the last one was from Iran. A total of 763 patients with migraine and 560 healthy controls were included in this meta-analysis. The frequencies of genotypes and alleles were summarized in Table The characteristics of the included studies, which were published between 2005 and 2017, were presented in Table 3.3P = .12; I2 = 86%). CC genotype was not associated with higher susceptibility of migraine compared with TT+ TC genotypes with significant difference  . However, subgroup analysis showed CC variant increase the risk for migraine compared with TT+ TC genotypes in Caucasian populations , which could not be observed in non-Caucasian patients   and C allele  did not increase the risk of migraine compared with TT genotype and T allele, respectively.Considering heterogeneity existed in this study, we used Galbraith plots to explore the heterogeneity sources Fig. . Low New3.5P = .452 and 0.317, respectively)  Fig. .4 NO activates soluble guanylate cyclase and subsequently increases cGMP. cGMP can induce gene expression via the induction of transcription factors such as mitogen activated protein (MAP) kinases or NF-κB. Although the aetiology and pathogenesis of migraine remain unclear, NO is believed to play a key role in migraine pathogenesis. Migraine was initiated by NO donors, glyceryl trinitrate (GTN), or isosorbide dinitrate, which indicated that NO may participate in the pathological process of migraine.,25 GTN did initiate migraine headaches but not the aura in migraineurs with aura in most studies. This indicated that NO is involved in the pain mechanism in both types of migraines but not in the initiation of the aura. Further study demonstrated that not NO itself but the elevation of cGMP by NO or other mediators is necessary to induce migraine. Inhibition of NOS in spontaneous migraine attacks leads to the attenuation of symptoms in two-thirds of the patients. Thus, NO seems not only be involved in the initiation of migraine attacks but also in the maintenance of these attacks. Calcitonin gene related peptide (CGRP) is a key player in migraine pathophysiology. CGRP levels were elevated following GTN-induced migraine. Pretreatment of trigeminal ganglia cultures with NO donors was also found to increase CGRP release in response to both depolarization and inflammatory mediators, which may have occurred due to NO donors increasing CGRP promoter activity.,31 While NO donors have some functions independent of CGRP, their function in migraine may strongly relate to promoting CGRP production and release, adding to the CGRP-influenced pathways in migraine.NO regulates a host of physiological functions, including vascular tone, pain sensation, neurotransmission, and as an immune defence mechanism.NO was synthesized by NOS from L-arginine. The activity of NOS has an important impact on the production of NO, and polymorphism of NOS gene leads to variant activity of NOS. In this study, we carried out a meta-analysis to evaluate the correlation of eNOS polymorphism (−786T>C) and migraine risk. The overall results did not indicate associations between eNOS polymorphism and migraine. However, subgroup analysis showed CC variant increase the risk for migraine compared with TT + TC genotypes in Caucasian populations.P = .58; I2 = 0%). Second, using Galbraith plots, source of heterogeneity was identified. After removal of studies with significant heterogeneity, fixed model showed that both CC + CT genotypes and C allele did not increase migraine susceptibility compared with TT genotype and T allele, respectively.When compared between CC + TC genotypes and TT genotype, between T allele and C allele, significant heterogeneity existed. Random effects model indicated that there was no significant difference between patients and healthy controls. First, we carried out subgroup analysis by populations. Lack of association was observed between eNOS polymorphism and migraine susceptibility in non-Caucasians . Although statistical significance could not be acquired with current studies, a larger sample may reach a positive result. Hence, further study is still needed.Our study has several strengths. First, this is the largest study to date evaluating the correlation between eNOS −786T>C polymorphism and risk of migraine. Second, patients and controls of different genetic backgrounds made it possible to analyze the associations in different populations. However, our study also has several limitations. First, due to apparent heterogeneity across studies in some cases, the findings from our study should be dealt with some caution. Second, we did not include academic dissertations and conference papers, so there may have been bias in provision of data. Third, there are other factors that affect the incidence of migraine, for example, gender,Although the association of eNOS −786T>C polymorphism and migraine susceptibility were reported in previous studies. This meta-analysis indicated that CC variant increase the risk for migraine compared with TT+ TC genotypes in Caucasian populations. More studies are still needed to verify such conclusion.We thank the Department of Geriatric Medicine, the First Hospital of Jilin University, for their assistance in this work. This work was supported by the Program from Health commission of Jilin Province (2014Q030).DH, WZH, DB, HYN, and ZHY conceived the study. DH, WZH, DB, HYN, and ZHY designed the study and analyzed the data. DH, DB, and ZHY wrote this manuscript. All authors discussed and revised the manuscript before submission.Conceptualization: Han Dong.Data curation: Han Dong, Bin Dong, Ya Nan Hu, Hui Ying Zhao.Formal analysis: Han Dong, Zhi Hao Wang, Bin Dong.Funding acquisition: Han Dong.Investigation: Han Dong, Bin Dong, Ya Nan Hu, Hui Ying Zhao.Methodology: Han Dong, Zhi Hao Wang, Bin Dong, Ya Nan Hu, Hui Ying Zhao.Resources: Zhi Hao Wang.Software: Han Dong, Zhi Hao Wang, Bin Dong.Supervision: Hui Ying Zhao.Validation: Ya Nan Hu.Writing – original draft: Han Dong.Writing – review & editing: Han Dong.


Data are lacking regarding the prevalence of benzodiazepine long-term use in the general population. Our aim was to examine the prevalence of prescribed benzodiazepine long-term use (BLTU) according to sociodemographic and clinical factors in the French general population.Data came from 4686 men and 4849 women included in 2015 in the French population-based CONSTANCES cohort. BLTU was examined using drug reimbursement administrative registries from 2009 to 2015. Analyses were weighted to provide results representative of the French general population covered by the general health insurance scheme. Weighted prevalence of BTLU and weighted Odds Ratios (OR) of having BTLU were computed with their 95% Confidence Interval (95% CI) according to age, education level, occupational status, occupational grade, household income, marital status, alcohol use disorder risk and depressive symptoms. All the analyses were stratified for gender.Weighted prevalence of BLTU were 2.8%(95% CI:2.3–3.4) and 3.8%(95% CI: 3.3–4.5) in men and women, respectively. Compared to men, women had an increased risk of having benzodiazepine long-term use with OR = 1.34(95% CI = 1.02–1.76). Aging, low education, not being at-work, low occupational grade, low income, being alone and depressive state were associated with increased risks of having BTLU.BLTU is widespread in the French general population, however this issue may particularly concern vulnerable subgroups. These findings may help in raising attention on this public health burden as well as targeting specific at-risk subgroups in preventive intervention.The online version of this article (10.1186/s12889-019-6933-8) contains supplementary material, which is available to authorized users. Benzodiazepines are the most prescribed drugs worldwide and they are primarily used for their anxiolytic properties . For insHowever, data are lacking in the literature regarding the prevalence of benzodiazepine long-term use in the general population . When avThe CONSTANCES cohort includes a large randomized sample of the French population, from various sociodemographic status . Its linThe CONSTANCES cohort is a national population-based cohort of randomly recruited participants, including volunteers aged 18–69 years at baseline in 22 selected health screening centers from the principal regions of France . To be rThe CONSTANCES cohort benefits from its systematic linkage to the SNIIRAM  database . This nan = 81), without professional activity (n = 18), other situations (n = 16)); occupational grade /intermediate worker /executive ); household income in euros per month (< 2100/from 2100 to 2800/from 2800 to 4200/> 4200); marital status ; education level based on the 2011 International Standard Classification of Education (ISCED) with a categorical variable corresponding to the highest obtained degree [n = 83, n = 18 and n = 21, respectively), thus providing a binary variable regarding at-risk alcohol use.From the baseline questionnaires, we used the following sociodemographic variables: age ; gender; occupational status   and 3.8%(95% CI = 3.3–4.5) in men and women, respectively. The distributions of the prevalence of benzodiazepine long-term use according to sociodemographic and clinical variables are displayed in Table p < 0.05; Table Compared to men, women had an increased risk of benzodiazepine long term use with OR = 1.34(95% CI = 1.02–1.76). In both men and women, the following conditions were associated with increased risks of benzodiazepine long-term use: age over 35 compared to less; not being in couple; low education; low income; not being in employment; depressive state , that are not well adapted to epidemiology given the very high cost of monitoring thousands of subjects in this way . Second,Our findings regarding differences of prevalence according to sociodemographic and clinical factors are in line with results from other cohort studies that found associations between benzodiazepine inappropriate use and gender, age, education, occupational status, occupational grade, income, marital status and depressive symptoms , 25, 38.The present study shows that benzodiazepine long-term use concerns a huge number of subjects in the French general population. This issue does not seem to be specific to France, with warnings in several other countries such as UK, Norway, Sweden, Germany, Netherlands, Denmark, USA and Australia , 14, 25.To prevent from detrimental consequences of chronic use, several guidelines have been published to help practitioners, including how to perform screening and managing withdrawal –45. HowePrevalence of prescribed benzodiazepine long-term use in the French general population were 2.8%(95% CI = 2.3–3.4) and 3.8%(95% CI = 3.3–4.5) in men and women, respectively. Aging, poor sociodemographic conditions and depressive state were significantly associated with increased risks of benzodiazepine long-term use. These findings, based on weighted analyses from participants included in 2015 in the national population-based CONSTANCES cohort, informed not only on significant associations but also on the number of concerned subjects. Thus, benzodiazepine long-term use is widespread in the French general population, however this issue may particularly concerns vulnerable subgroups. These findings may help in raising attention on this public health burden as well as targeting specific at-risk subgroups in preventive intervention.Additional file 1:Table S1. Prevalence of benzodiazepine long-term use in men of the French general population in 2015. (DOCX 26 kb)Additional file 2:Table S2. Prevalence of benzodiazepine long-term use in women of the French general population in 2015. (DOCX 26 kb)


Outcomes of interest included milk yield (kg/d) and milk components, serum concentration of glucose, and non-esterified fatty acids (NEFA), resumption of cyclicity, time-to-first artificial insemination (AI), conception at first AI, and conception within 150 DIM, disease occurrence, culling, mortality.Negative energy balance during the transition period is a concern for both conventional and certified organic dairy systems. During early lactation, supplemental strategies are used to cope with nutrient deficiencies that are associated with impaired health, production, and reproduction. As organic certified dairies in the United States face restricted access to dietary supplements, the evaluation of products especially formulated for organic production is needed. Our objective was to assess the effect of supplementing 0.45 kg/d of an organic rumen-protected fat (RPF) on health, metabolic status, and productive and reproductive performance of organic certified Holstein cows from 1 to 150 days in milk (DIM). Two-hundred and two cows were enrolled in a randomized blocked controlled trial conducted from January to July 2017 in a certified organic dairy located in Northern Colorado (USA). Two groups were randomly assigned to be individually supplemented with organic RPF . During grazing, multiparous (MP) ORG cows had greater milk yield compared to MP CON cows, whereas no effect was found in primiparous (PP) cows. Health outcomes, serum metabolite concentrations, and reproductive performance were not affected by the inclusion of RPF. Body condition loss was smaller in the ORG group up to 80 DIM; however, there was no effect on body condition during the grazing season and in the overall study period.A significant effect for the inclusion of RPF was found in daily milk yield; RPF supplemented cows had greater milk yield (1.6 kg/d) compared to CON cows up to 150 DIM (These results indicate that supplementation of RPF increased daily milk yield and prevented body condition loss during at 90 DIM. However, RPF supplementation did not affect health, serum metabolite concentration, milk components, and reproductive outcomes. Transition cows are challenged by nutrient deficits to support milk production, which triggers mobilization of fat, labile protein, and calcium . These mNutritional management of transition cows is commonly reported as a preventative strategy , 12, 13 Fats are energetically denser than carbohydrates . NonetheMost commercial RPF contain calcium soaps of palmitic and oleic FA , with suIn this study, we hypothesized that the supplementation of an organic RPF, in a form of treatment pellets (ORG group), will improve productive performance, metabolic status, reproductive performance, and health when compared to control cows (CON group) fed with a control pellet elaborated to match all ingredients except the RPF. Thus, the general aim of this study was to assess the effect of supplementing 0.45 Kg/d of an organic RPF from 1 to 150 DIM on health and metabolic, productive and reproductive status of lactating organic Holstein cows.P = 0.03) up to 150 DIM. Additionally, parity and DIM (P < 0.0001) and the interaction term between treatment group and DIM had significant effects (P = 0.015). Milk yield by study group across time can be observed in Fig. Milk yield (kg/d) was evaluated until 150 DIM, as well as during grazing season and by 305 DIM. Cows in the ORG group produced 1.6 kg/d more milk compared to CON cows . Average daily milk yield for the 7-d before grazing was considered as baseline. Overall, MP ORG cows receiving the rumen-protected fat produced more milk the week before the start of grazing, compared to MP CON cows . During the first week of grazing no differences were observed between ORG and CON among MP cows . In contrast, in the following weeks MP ORG cows had greater milk yield compared to MP CON cows. However, these differences were not observed among PP cows . As depicted in Table Fat, protein, and 3.5% fat corrected milk (FCM) were compared from the farm test day performed every 15 days during the whole study period. Milk components were tested for a total of seven times. However, depending on the calving dates, there was some variation in DIM among the study cows at the time of each testing (26 d difference between the first and last enrolled cow). For this reason, we controlled the analysis of milk components by DIM. A summary of milk components by study group is presented in Table P = 0.13). No interaction was observed between treatment group and parity (P = 0.52).Finally, daily milk yield (kg/d) for the overall 305 d period was compared between treatment groups. A tendency was observed in favor of the ORG with extra 0.8 kg/d compared to CON cows . Fluctuations of BCS across the study period are presented in Table P = 0.002). On the other hand, no differences in BCS were observed during grazing or at the end of the supplementation.Metabolic status was assessed through the variation of body condition scores during transition and grazing season, and through serum glucose and NEFA determination. Body condition scores before the start of the supplementation (1 DIM) did not show differences between ORG and CON . Serum concentration dynamics throughout the first 21 DIM are presented in in Fig. Serum concentrations of glucose (mg/dL) and NEFA (mEq/L) were measured at 1, 3, 7 and 21 DIM. One-hundred and forty-seven animals were screened . Accordingly, the time-to-event analysis showed no differences between treatment groups (P = 0.85), where the Kaplan-Meier median time of resumption of ovarian cyclicity was 38 d for ORG (37–48 d) and CON (37–49 d). No differences between treatment groups were established for the time of AI (P = 0.5); ORG cows had a Kaplan-Meier median AI time of 67 d (63–76 d) whereas CON cows had a time of 72 d (64–83 d). The number of cows that resulted pregnant from the first AI did not differ between treatment groups (P = 0.43). Seventy-seven animals resulted pregnant at 150 DIM. However, there were no differences between treatment groups and parity according to the logistic regression models .There was no association between the resumption of ovarian cyclicity after calving and the supplementation of RPF  were diagnosed with health disorders at 150 DIM. There was no association between treatment and disease occurrence (P = 0.8). No effect in disease occurrence was observed for the interaction between the variables treatment and parity (P = 0.5). Additionally, the occurrence of disease until 305 DIM was evaluated to assess a carry-over effect of the ORG supplementation. Sixty-four animals  were determined sick throughout this period. No effect was found for treatment , parity (P = 0.8), or their interaction (P = 0.5). The time-to-disease diagnosis for ORG and CON cows was not different within 150 DIM (P = 0.6) and 305 DIM (P = 0.6).Fifty-six cows  left the herd within 150 DIM. No association was observed between treatment groups and culling (P = 0.25). After 305 DIM of follow-up, a total of 44 animals  were culled. No association was found between treatment groups and culling (P = 0.16) during this period. On the other hand, the main effect of parity was associated with culling, where multiparous (MP) cows had 3.7  times greater odds of being culled compared to primiparous (PP) cows.Voluntary culling was compared during the supplementation period and until 305 DIM. Twenty-three animals  and the interaction between treatment group and parity (P = 0.17) were not significantly associated with eating time within the time of pellet delivery and therefore were removed from the model. Thus, the final model considered treatment effect, week of supplementation, and their interactions. There was a significant overall effect of treatment on eating time (LSM) by week during the supplementation period . However, the multiple comparison test did not indicate significant differences on eating time between treatment groups for specific time points during the supplementation period. Similarly, a significant overall treatment effect was found on daily eating time , but no differences were established when specific days were compared.Parity  that conditions the level of milk production in dairy cows . Some stBody condition changes during transition have been associated with milk yield, post-partum health, and decreased fertility –37. Few After 30 d in grazing, the group difference in BCS observed at 80 DIM was lost because ORG cows decreased their BCS. Despite this reduction, ORG group sustained higher milk production during the grazing season and the BCS was not different during grazing and by the end of the supplementation Figs.  and 3.N = 18) in mid lactation cows (98 DIM) for 4 weeks. Under those study settings, the researchers did not find differences in daily and weekly milk yield, with both groups producing approximately 32.7 kg/day. These results contrast with those found in our study during the mid-lactation stage. Although, the management differs due to grazing and organic production, our study determined that ORG cows produced more milk after 90 DIM  with RPF tuna oil during the grazing season  and multiparous [MP] ≥2 lactations), and randomly assigned into the two study groups. Each study group was conformed by 30% of PP cows. The ORG group was supplemented with 1.5 kg/d of a treatment pellet formulated to contain 0.45 kg of the organic RPF , whereas the CON group was supplemented with 1.05 Kg of a control pellet formulated to match all feed components except the organic RPF  and management except when the treatment pellets were fed. For pellet delivery, the ORG and CON pellets were individually fed once a day during the trial, after the 07:00 h milking. As individual feeding was required, the study cows were separated in two groups when exiting the milking parlor, based on color marks in both id-ear tags and collars in the control group. After the group separation, the treatment pellets were delivered on top of the TMR. Once the study cows consumed all pellets they were released, and the two groups were allowed to mix in the research pen. More details about sorting methods and efficiency can be found in Manriquez et al. .During the grazing season, the study cows obtained at least 30% of their DMI from pasture. The TMR was based on corn silage (5 to 7%), wheat silage (17 to 19%), grain mix containing soy bean, soy hulls, corn, wheat, and minerals and vitamins (38 to 41%), sorghum silage (5 to 7%), alfalfa hay (2%), grass hay (0 to 1.5%), and pasture grazing (estimates as 30 to 38%). Grazing management considered rotational grazing in pastures based on perennial forages, alfalfa, Italian rye grass, oat rye grass, and teff grass.n = 72; CON; n = 76). Vein puncture was performed using vacutainer system tubes without anticoagulant . After collection, blood was allowed to clot for 1 h at 4 °C, and then centrifuged at 2800 rpm for 15 min. Supernatant was recovered and stored at -20 °C until lab analysis. Glucose (mg/dL) was measured using a handheld meter  as referenced by Voyvoda and Erdogan in 2010 [Blood samples were collected from the coccygeal vein within 24 h after calving and at 3, 7, and 21 DIM for determination of glucose, and NEFA concentrations. Blood sample collection was performed after the morning milking (0700 h). One-hundred forty-eight serum samples were subject to laboratory analysis , milk components, resumption of ovarian cyclicity at 49 DIM, time to the first AI, pregnancy at the first AI, pregnancy within 150 DIM, and time to pregnancy. All these variables were longitudinally measured from ORG and CON groups from calving until the end of the supplementation.Farm personnel performed daily health monitoring according to farm protocols during the supplementation period and until 305 DIM. The assessed health outcomes included metritis, endometritis, pyometra, subclinical ketosis, digestive disorders , respiratory disease, and lameness. Clinical disease information was retrieved from farm records as well as culling and mortality.Blind body condition scoring was performed at 1, 3, 7, 21, 80 and 150 DIM using the standard scoring chart of 5 point with a 0.25-point scale . AdditioMilk was evaluated by daily milk yield up to 150 DIM. Individual milk yield (kg/d) was available from the farm’s milking machine software . Milk components were analyzed every 2 weeks by an independent laboratory . Components included fat, protein and lactose. Fat corrected milk (FCM = 0.4324*milk in lb. + 16.216*fat content) was calculated at every test day. Additionally, fluctuations in milk yield were evaluated before and during the grazing season. This evaluation was standardized by DIM, and the weekly milk yield averages were compared 1 week before grazing and for up to 5 weeks after grazing started.During the supplementation trial, only AI was performed as breeding procedure based on heat detection. Cyclicity at 49 DIM was assessed through transrectal ultrasonography evaluating the presence of a corpus luteum at 35 DIM and 49 DIM. The number and date of AI were obtained from farm records. Cyclicity and pregnancy were recorded as binary variables, whereas DIM at AI were analyzed as time-to- event data.Individual and group eating time (min/d) were estimated using accelerometers  tagged in the left ear . The accData analysis was performed using SAS software . Descriptive statistics and univariate analysis for parity, DIM, and disease frequency were performed using Chi square test in SAS (PROC FREQ). The effect of RPF on health outcomes was evaluated using logistic regression (PROC LOGISTIC) and survival analysis (PROC LIFETEST). The occurrence of health disorders, culling and mortality were analyzed as a binary response  until 150 and 305 DIM. The logistic models included the effect of RPF, parity and their interaction. Time-to-disease diagnosis, culling and mortality were compared between ORG and CON groups until 150 and 305 DIM. Differences between treatment groups was determined by the Wilcoxon test. Serum concentrations of glucose, and NEFA were analyzed using PROC MIXED for repeated measures. The model included the effect of the RPF, sample point and their interaction.Analyses of milk yield, milk components, and BCS were performed using PROC MIXED for repeated measures. This model included treatment effect, parity, evaluation times and interaction between treatment effect and parity , and evaluation times. The evaluation of daily milk yield considered the sum of the three-daily milking. The model included the fixed effects of treatment (ORG and CON), parity, DIM, and the interaction between treatment effect and DIM. Milk yield analyses during grazing included interaction terms between treatment group, parity and week of evaluation, and a triple interaction term between treatment group, parity, and week of evaluation. Milk fat and protein and FCM were compared by treatment group, parity and evaluation date, including the interaction between treatment group and evaluation date. For BCS at 1 DIM treatment effects, parity and their interaction term were included. To compare BCS between ORG and CON cows, the main effect of treatment, type and evaluation time, and their interactions were included in the mixed model.P-values were used to test equality of strata (ORG and CON) of the survival curves.Reproductive performance outcomes were analyzed through logistic regression (PROC LOGISTIC), including treatment group, parity, and their interaction terms in the model. Additionally, to explore treatment effect on time-to-first AI and pregnancy, survival analysis was performed (PROC LIFETEST). Wilcoxon Differences in weekly eating time between treatment groups were compared using PROC GLIMMIX, by treatment groups, parity, and their interaction. The analysis was stratified between 0700 and 0800 h to compare eating time during the delivery of the treatment pellets. In addition, overall daily eating time was analyzed in weekly period.P-values < 0.05. Multiple comparison P-values were adjusted through the Tukey-Kramer test. Variables with P-values ≤0.15 were kept in the models for confounding control.Statistical significance was determined at


Grape leaves provide the biochemical substrates for berry development. Thus, understanding the regulation of grapevine leaf metabolism can aid in discerning processes fundamental to fruit development and berry quality. Here, the temporal alterations in leaf metabolism in Merlot grapevine grown under sufficient irrigation and water deficit were monitored from veraison until harvest.2+ and sucrose to the osmotic adjustment concomitant with the initiation of leaf shedding. Last, towards harvest under progressive stress conditions following leaf shedding, incremental changes in leaf water potential were measured, while the magnitude of perturbation in leaf metabolism lessened.The vines mediated water stress gradually and involving multiple strategies: osmotic adjustment, transcript-metabolite alteration and leaf shedding. Initially stomatal conductance and leaf water potential showed a steep decrease together with the induction of stress related metabolism, e.g. up-regulation of proline and GABA metabolism and stress related sugars, and the down-regulation of developmental processes. Later, progressive soil drying was associated with an incremental contribution of CaThe data present evidence that over time grapevine acclimation to water stress diversifies in temporal responses encompassing the alteration of central metabolism and gene expression, osmotic adjustments and reduction in leaf area. Together these processes mitigate leaf water stress and aid in maintaining the berry-ripening program.The online version of this article (10.1186/s12870-019-1652-y) contains supplementary material, which is available to authorized users. Vitis vinifera L.) is considered suited to Mediterranean like climates s were vedings of  that shoVitis leaves [. Calcium ion plays multiple roles in plant stress response such as stomatal closure [+ might reflect its involvement in maintaining the aforementioned cellular processes in water-stressed leaves.One of the interesting result in this research was that leaf shedding mediated the recovery of the vine at the physiological and molecular levels. For instance, following the marked leaf shedding event, the vines regained leaf water potential and ease the extent of metabolite perturbation. As a likely stress relaxer, this mechanism will directly reduce the evaporative surface of the vines and can also aid in redirecting photoassimilates translocation from older leaves to younger ones . Coincids leaves  and parts leaves  probably closure , osmotic closure , 59, sig closure –63, impr closure  and deto closure . The oveChanges in metabolite level during the stress depended upon the length of imposed drought. Both the transcriptional and metabolomic data suggest the increase in stress related amino acids of the Glu family, GABA and proline Fig. a. The fiMetabolic adjustments in response to stress conditions often occurs in a very short time, for example GABA metabolism can react within seconds from the beginning of the stress . HoweverThe role of ABA in plant responses associated with stress signaling and transduction pathways is well described in the plant literature –82. In lIn general, the parallel transcriptional and metabolomic analysis showed a coordinated modulation of leaf metabolism under water deficit. Earlier comparative study of the effect of water deficit and salinity on grapevine leaf have shown the major stress effects in relation to ROS and energy metabolism, transcription factors, ABA and osmolite concentration . The autaba responsive transcription factors, at the early stage was followed by stress related responses such as increased osmolite concentration, , upregulation of ROS metabolism and stress associated gene transcripts and corresponding metabolites such as GABA, Proline, myo-inositol, galactinol and raffinose.The integrative leaf physiology, transcript and metabolite analysis under partially controlled field condition enabled us to examine stress temporal effect on grapevine leaf metabolism. Progressive water stress caused a sequential stress response from gene transcript-metabolite alteration, osmotic adjustment to leaf shedding and metabolic relaxation. The progressive leaf shedding and the consequential reduction in transpiration rates led to a rise in leaf water potential as the season progressed. The activation of stress associated responses, e.G. LYS, [The experiment was conducted on 4-years old Merlot vines grafted onto SO4 rootstock at the Udine University experimental farm  during 2014 and 2015 growing seasons exactly as describe in , 34. To LYS, ). The siLYS, .LYS, WW) and water deficit  with four replicates applied in randomized manner along the row of pots as described in [Two irrigation treatments were applied starting from veraison (50% color change)– well watered (120% of ETribed in . Three dribed in ). ResultThe leaf area of experimental vines was evaluated five times during the experiment by a linear model relating the leaf length  and the leaf area as described in .l; MPa) was periodically measured at midday during clear sunny days using a pressure bomb chamber  as previously described [− 2 s− 1) and CO2 concentration (400 μmol.mol− 1) at ambient humidity and leaf temperature. The same leaf used for gas exchange measurements was then sampled for the extraction of metabolites and transcripts as described below.Leaf water potential (Ψescribed . Pressurescribed  on 6 aduv/v/v). The extraction mixture contains internal standards ribitol (i.e. 0.2 mg/ml in water), ampicillin (1 mg/ml in water) and corticosterone (1 mg/ml in methanol) to give a concentration of 0.86 mg ml− 1, 5.6 mg ml− 1, 7.16 mg ml− 1, respectively in the final injected volume. The sample-extraction mix was then briefly vortexed, centrifuged for 2 min at 14,000 RPM (microcentrifuge 5417R) and the supernatant was decanted into the new tubes. The supernatant was mixed with 300 ml of chloroform and 300 ml of UPLC-grade water, briefly vortexed and then centrifuged at 14,000 RPM for 2 min. For GC-MS analysis, 100 μl of extract from the water/methanol phase was dried in a vacuum concentrator (Eppendorf Concentrator Plus) for derivatization [Samples for metabolic analysis were taken at nine time points in 2014 (− 1 (one day before deficit irrigation imposition), 1, 4, 8, 13, 18, 28, 35 and 53 days following deficit irrigation imposition) and six time points in 2015 . At each sampling time point, young, sun exposed and fully expanded leaf samples were taken, divided into two portions and used for metabolite and transcript analysis at the respective sampling stages. Each portion was immediately wrapped in aluminum foil and snap frozen with liquid nitrogen, and then kept at − 80 °C until further analysis. Sample preparation, tissue grinding and metabolite extraction for parallel GC-MS and LC-MS analysis were performed exactly as previously described . Samplestization . The remArabidopsis thaliana from a bulked extraction of Arabidopsis thaliana Columbia-0 plants and a mixture of authentic metabolite standards (0.05 mg/ml). A volume of 1 μl was then injected into the GC column in a splitless mode or with a split ratio of 50:1. Spectral searching, based on the National Institute of Standards and Technology  algorithm incorporated in the Xcalibur® data system (version 2.0.7), was done against retention index (RI) libraries downloadable from the Max-Planck Institute for Plant Physiology in Golm, Germany [Samples for GC–MS analysis were derivatized exactly as described previously . The sam Germany  and norm− 1, in 50/50 of acetonitrile/H2O with 0.1% v/v formic acid. The MS conditions were set essentially as described in [http://www.chemspider.com/) and further confirmed with previous metabolite annotations [For LC-MS analysis, 2 μl of extracted sample was injected onto a UPLC-QTOF-MS system equipped with an ESI interface  operating in negative and positive ion modes. The chromatographic column conditions, solvent composition and gradient program were maintained exactly as described in . All anaribed in . UPLC daribed in . The rawribed in . Metabolotations –99.Transcriptomics data was analyzed only for the early time points  during the 2014 season, while one time point from the 2015 season (i.e. 12 DAT) was used as confirmatory data.Vitis vinifera genome (12x) [Vitis vinifera annotation (GTF format) file. Differential expression (DE) analysis was conducted with DESeq2 [Frozen leaf was ground in liquid nitrogen and total RNA was extracted from 100 mg tissue using Sectrum™ plant total RNA kit . The quality and concentration of extracted RNA was determined using Bioanalyzer Chip RNA 7500 series II  and a Nanodrop 2000 spectrophotometer . Following quality assessment, library construction was conducted using TruSeq RNA Sample Prep Kit v2.0 (Illumina). Sequencing was performed on an Illumina HiSeq 2000 sequencer (Illumina) at IGA Technology Services . Quality check, filtering, and trimming of raw sequence reads  was performed using trimmomatic v0.36 [me (12x)  using bome (12x) . Read sume (12x)  using thh DESeq2 . A falseGene ontology (GO) enrichment analysis of DE genes were performed with BinGO  using thInorganic ions were extracted from leaf samples at  using 20 mg of ground sample with 15 ml distilled water, boiled at 100 °C for 5 min, kept at orbital shaker for about 1 min, and filtered through a cellulose nitrate filter . The ion10 transformed data using TMEV software package [For metabolite analysis, the normalized data set  was subjected to Student’s t-test to compare between treatments at each sampling data point. Principal component analysis (PCA) was carried out on log package . ResultsAdditional file 1:Table S1. Stomatal conductance measurements of 2014 and 2015 presented as mean (N = 4) and standard error. The respective time points are indicated with days from treatment application. Table S2. Fold change (Treatment/Control) of leaf secondary metabolites during the time course of the experiment in 2014 and 2015. Table S3. Fold change (Treatment/Control) of leaf central metabolites during the time course of the experiment in 2014 and 2015. Table S4. Summary of RNA sequencing analysis metrics. The table show the number of filtered reads and aligned reads (filtered) to the 12X grapevine reference genome that are counted. Table S5. Transcript abundance of the all 29,970 genes, reported as Reads Per Kilobase of transcript, per Million mapped reads (RPKM), in leaves of well-watered and water deficit treated grapevines at day 4, 8 (2014) and day 12 (2015). Tables S6–S8. Summary of differentially expressed genes  in grapevine leaves comparing well-watered and water deficit treatments. DESeq2 statistical outputs of day 4, 8 (2014) and day 12 (2015) treatments are listed in Table S6, S7, and S8, respectively.. Annotation of genes were referred from Vitisnet  . Table S9. List of differentially expressed genes in grapevine leaves that are common and unique leaves in each intersection of the Venn diagram described in Fig. Table S10. Summary of enriched (FDR < 0.05) plant GO slim categories in grapevine leaves comparing well-watered and water deficit treatments at day 4, 8 (2014) and day 12 (2015). Other statistical outputs of BiNGO  are reported. Table S11. Summary of cis-regulatory elements (CRE) enriched at a threshold of FDR < 0.01 in promoters of drought-regulated genes at day 4, 8 (2014) and day 12 (2015). For each treatment group, the number of genes containing the CRE, group size, total number of genes in the genome containing the CRE, promoter size, enrichment P-value, FDR, and lists of genes containing the CRE with their CRE position along the gene promoter are reported. (XLSX 3596 kb)Additional file 2:Figure S1. Number of significantly altered metabolites under water stress identified using GC-MS (primary metabolites) and LC-MS (secondary metabolites) during the course of the experiment. (PPTX 38 kb)


Gut microbiota has the capacity to impact the regular function of the brain, which can in turn affect the composition of microbiota. Autism spectrum disorder (ASD) patients suffer from gastrointestinal problems and experience changes in gut microbiota; however, it is not yet clear whether the change in the microbiota associated with ASD is a cause or a consequence of the disease.We have investigated the species richness and microbial composition in a valproic acid (VPA)-induced rat model autism. Fecal samples from the rectum were collected at necropsy, microbial total DNA was extracted, 16 rRNA genes sequenced using Illumina, and the global microbial co-occurrence network was constructed using a random matrix theory-based pipeline. Collected rat microbiome data were compared to available data derived from cases of autism.We found that VPA administration during pregnancy reduced fecal microbial richness, changed the gut microbial composition, and altered the metabolite potential of the fecal microbial community in a pattern similar to that seen in patients with ASD. However, the global network property and network composition as well as microbial co-occurrence patterns were largely preserved in the offspring of rats exposed to prenatal administration of VPA.Our data on the microbiota of the VPA rat model of autism indicate that this model, in addition to behaviorally and anatomically mimicking the autistic brain as previously shown, also mimics the microbiome features of autism, making it one of the best-suited rodent models for the study of autism and ASD.The online version of this article (10.1186/s13229-018-0251-3) contains supplementary material, which is available to authorized users. The gut and brain form the gut-brain axis through bidirectional nervous, endocrine, and immune communication. A change in one of these systems will most certainly have effects on the other systems. Disorders in the composition and quantity of gut microbiota can affect both the enteric nervous system and the central nervous system . SpecifiValproic acid (VPA) is a medication used for epilepsy and mood swings. Children prenatally exposed to VPA have an increased chance of being diagnosed with autism –7. In adFirmicutes, Bacteroidetes, and Proteobacteria with the abundance of Lactobacillares and Clostridia [Bifidobacterium, Lactobacillus, Prevotella, and Ruminococcus [Bacteroidetes in VPA exposed offspring [ASD patients suffer from gastrointestinal problems and experience changes in the gut microbiota, including shifts in levels of ostridia , 13. Othnococcus . Microbiffspring . It is nP < 0.05, the Welch t test). In the control rats, Chao1 value was 1005.62 ± 120.00 (N = 11). VPA injection significantly reduced Chao1 to 925.98 ± 76.62 . However, other microbial diversity indicators, such as Pielou’s evenness, PD whole tree, and Shannon and Simpson indices, remained unchanged by VPA.A single IP injection of VPA during pregnancy in rats had a significant effect on fecal microbial richness in their offspring (P = 1.78 × 10−12). A cluster analysis using the group average approach of the resemblance values suggested individual microbial communities from the control and VPA groups were able to form two distinct clusters, respectively  level, mean Bray-Curtis similarity values (%) within either the control or VPA groups were 63.57 ± 4.04, a significantly higher than mean similarity between the control and VPA groups , including one class . The abundance of three families, Eubacteriaceae . On the other hand, the abundance of Enterobacteriaceae . At the genus level, a significantly higher abundance level of the genus Anaerotruncus  scores , representing approximately 10% of all OTU in a given gut microbial community  while the abundance of Actinobacteria was significantly increased by VPA in females only , as compared to controls of the same sex. VPA significantly repressed the abundance of the class Coriobacteriia while it increased the two classes Bacteroidia and α-Proteobacteria in males only (LDA > 2.0 and P < 0.05). The abundance of several genera was significantly increased by VPA only in females, including Allobaculum, Bifidobacterium, Odoribacter, and Staphylococcus (LDA > 2.6 and P < 0.05). Intriguingly, the abundance of the genus Candidatus Arthromitus, a group of the segmented filamentous bacteria (SFB), was also significantly increased by VPA in female rats (LDA = 3.774 and P = 0.015) but not males. There is strong evidence demonstrating that these gut epithelium-associated bacteria possess strong abilities to modulate host immune responses.Differences in microbial composition between the sexes were investigated by comparing male and female rats prenatally exposed to VPA with same-sex control rats. While uneven sample size in the male and female comparison may be a concern, the drastic sex-dependent changes induced by VPA were evident Fig. a, b. At Ruminococcus flavefaciens was significantly higher in both male and female rats with prenatal VPA exposure  while 7 other OTU were significantly reduced in fecal microbial communities of both male and female rats with VPA exposure (LDA > 2.0 and P < 0.05).At the species (OTU) level, VPA prenatal exposure induced significant changes in the relative abundance of 66 and 72 OTU in male and female rats, respectively. Among them, the abundance of 61 OTU was also significantly impacted by VPA exposure regardless of gender. A total of 9 OTU displayed significant directional changes by VPA in both male and female rats  and ATP-binding cassette, subfamily B, bacterial (K06147), and RNA polymerase sigma-70 factor, ECF subfamily (K03088) were among the most abundant. Compared to the control, VPA injection repressed the abundance of 11 KEGG proteins, including putative ABC transport system ATP-binding protein (K02003), multiple sugar transport system substrate-binding protein (K02027), LacI family transcriptional regulator (K02529), methyl-accepting chemotaxis protein (K03406), two proteins related to two-component system, K07718 and K07720, and four proteins in the peptide/nickel transport system .P < 0.05), resulting in a significantly elevated hit count for 21 pathways while repressing 8 pathways . Pregnant control dams of the same age were injected with sterile saline also at E12.5 (n = 5). The pups of these dams were the subjects of this study. We collected stool and tissue samples from 10 VPA offspring and 11 control offspring equally distributed among groups.Intraperitoneal administration of VPA  was delivered to pregnant Sprague Dawley rats (8 weeks old) at E12.5 . DNA integrity and concentration were quantified using a BioAnalyzer 2100 .2SO4, 2.0 mM MgSO4, 1% glycerol, and 100 ng/uL bovine serum albumin . PCR was performed using the following cycling profile: initial denaturing at 95 °C for 2 min followed by 20 cycles of 95 °C 30 s, 60 °C 30 s, and 72 °C 60 s. Amplicons were purified using Agencourt AMPure XP bead kits  and quantified using a BioAnalyzer DNA 7500 chip kit and a QuantiFluor fluorometer. The purified amplicons from individual samples were pooled in equal molar ratios. The purified amplicon pool was further spiked with approximately 25% of whole-genome shotgun libraries prepared using an Illumina TruSeq DNA sample prep kit with a compatible adaptor barcode to enhance sequence diversity during the first few cycles of sequencing for better cluster differentiation. The concentration of the pooled final library pool was quantified using a BioAnalyzer high-sensitivity DNA chip kit (Agilent). The library pool was sequenced using an Illumina MiSeq Reagent Kit v3 on an Illumina MiSeq sequencer as described previously. The mean number of 2 × 250 bp pair-end sequences obtained was 347,849.14  per sample.The 16S rRNA gene sequencing was performed as previously described , 18. TheThe sequence data were preprocessed using MiSeq Control Software (MCS) v2.4.1. Raw sequences were first analyzed using FastQC version 0.11.2 to check basic statistics, such as GC%, per base quality score distribution, and sequences flagged as poor quality. The four maximally degenerate bases (NNNN) at the most 5′ end of the read pair, which were designed to maximize the diversity during the first four bases of the sequencing run for better identification of unique clusters and improve base-calling accuracy, were then removed. The presence of forward and reverse PCR primers at the 5′ and 3′ ends of each sequence read was scanned; the reads without primers were discarded. Chimeric reads were also removed. The processed pair-end reads were then merged using PandaSeq v2.8 to generate representative complete nucleotide sequences (contigs) using default parameters. The overlapping regions of the pair-end read were first aligned and scored, and reads with low score alignments and high rate of mismatches were discarded. After these quality control steps and filtering procedures, greater than 91% of the input raw sequences  retained for subsequent analysis.lbl.gov). PyNAST (v1.2.2) was used for sequence alignment. PICRUSt (v1.0.0), a software package designed to predict metagenome functional contents from marker gene surveys , was used with default parameters to predict gene contents and metagenomic functional information based on the OTU table generated using the closed-reference protocol in QIIME. Briefly, the OTU table was first normalized by dividing each OTU by the known/predicted 16S copy number by using the PICRUSt workflow: normalize_by_copy_number.py. The gene contents or the abundance of KEGG Orthology (KO) were predicted from the normalized OTU table using the workflow: predict_metagenomes.py. The predicted metagenome function was further analyzed by collapsing thousands of KEGG Orthologs into higher functional categories (pathways) (categorize_by_function.py). In addition, specific OTU contributing to a given function or pathway was identified by using the workflow: metagenome_contributions.py, as described previously [10 LDA score or LDA > 2.0 and P values < 0.05 based on the Kruskal-Wallis test by ranks).The QIIME pipeline (v.1.9.1) with the default reference v. 0.1.3 was used to analyze the 16S rRNA gene sequences. Both “closed reference” and “open reference” protocols in the pipeline were used for OTU picking as previously described . The rareviously . The lineviously  with a dThe global microbial co-occurrence network was constructed using a random matrix theory (RMT)-based pipeline , 21. TheThe gut and brain form the gut-brain axis through bidirectional nervous, endocrine, and immune communications. Mammalian species often contain similar microbiome richness at the level of phylum, but diversity and richness of species are highly variable among individuals . This vaPrevotella and other fermenters [Humans with autism and mice models of autism have shown significant alterations in their microbiota composition. Children with autism present with more GI symptoms than typically developing children, and the severity of their GI symptoms is correlated to the severity of their behavioral symptoms , 27. Thermenters –35.Akkermansia and a decrease in abundance of Bifidobacterium and Clostridiales [. In addition, BTBR mice have impaired intestinal integrity and a deficit in the intestinal tight junction proteins Ocln and Tjp1 [Lactobacillus, Parabacteroides, Helicobacter, and B. uniformis. In this study, we demonstrated that species richness in the fecal microbial community in the autistic-like rat model, the 400-E12 VPA rat, was significantly reduced. Using next-generation sequencing technology in a murine autism model, it was reported that the microbiome composition in mice in utero exposed to VPA presented with a decreased of Bacteroids [Deltaproteobacteris and Erysipelotrichales. These changes in VPA mouse microbiota composition were coincident with changes in behaviors linked to autism [Studies in mice have allowed to better understand the role of the microbiota in autism . The lacridiales –45. In aand Tjp1 . Environand Tjp1 . In the cteroids . Other go autism .α-Proteobacteria, Eubateriaceae, Rikenellaceae, and Staphylococcaceae. On the other hand, Enterobacteriaceae was significantly decreased by VPA exposure in utero. At the genus level, we found a significantly higher abundance of the genus Anaerotruncus in the control group and a significantly increased abundance of the genera Allobaculum, Anaerofustis, Proteus, and Staphylococcus in the VPA group.Our 400-E12 VPA rats showed a decrease in microbial diversity (species richness). Specifically, significant increases in the abundance of +Itpr3tf/J mouse model of autism [This is the first time the microbial species richness and microbiome composition have been studied in a rat model for autism, the 400-E12 VPA rat. The decrease in microbial diversity in this rat model was consistent with the observations in human autism and most of the mouse models of autism studied to date. The gut microbial composition was largely similar to that of humans with autism and murine autism-like models. The enteric bacteria, especially the class Clostridia, are known to play an important role in children with autism . In our study, Clostridia is the most dominant class in the rat fecal microbial community, accounting for more than 60% of all sequence reads, followed by the class Bacteroidia with more than 30% of the sequences. Among the 100 OTU significantly impacted by prenatal VPA administration, the vast majority of them, 94, belonged to Clostridia, suggesting that ecological manipulation via antibiotics or pre- or pro-biotic approaches targeting this class of gut bacteria may prove effective in alleviating autism symptoms. A significant reduction in microbial species richness, such as Chao1, in the 400-E12 VPA rats was consistent with the observation in BTBR Tf autism . Howeverf autism . While aH17 immune responses as well as innate immunity, SFB may play a role in the pathogenesis of autism. Indeed, a recent study shows that pregnant mice colonized with SFB were more likely to produce offspring with maternal immune activation (MIA)-associated abnormalities [Moreover, our findings provide further evidence of sex-specific alterations of gut microbiome by prenatal VPA administration in rodents . For examalities .Bifidobacterium and Lactobacillus, that inhabit in the gut, have the capacity to produce anti-inflammatory cytokines, while other, such as Clostridium and Ruminococcus [. Metabolic products of the gut microbiota, such as short-chain fatty acids, have also been implicated in autism. Gut microbiota has been suggested to regulate many nervous functions including neurogenesis, differentiation, myelination, formation and integrity of the blood-brain barrier, neurotrophin and neurotransmitter release, apoptosis, gap junction modification, and synaptic pruning [The composition of the microbiota is of great importance to the function of the brain. Bacteria can regulate brain function through several mechanisms. Some bacteria, such as nococcus , can pro pruning . Moreove pruning . In addi pruning –56. HereIn conclusion, our data on the gut microbial community of the 400-E12 rats in response to prenatal VPA exposure indicate that this model, in addition to demonstrating behavioral and anatomical similarities to autism, also mimics the microbiota features of autism, making it one of the best-suited rodent models for the study of autism.Additional file 1:All OTU in a given gut microbial community. (XLSX 180 kb)