Hyperuricemia is common after renal transplantation, especially in those receiving calcineurin inhibitors. Little, however, is known about the relationship between uric acid (UA) levels and allograft outcome.N = 368) in order to assess UA blood levels post-transplant association with allograft outcome. For this study, a median serum UA level of all measured UA levels from 1 month to 1 year post renal transplantation was calculated.We conducted a retrospective single-center analysis (N = 164) versus median UA level < 7 and < 6 mg/dL for men and women respectively (N = 204) had lower GFR values at 1, 3 and 5 years posttransplant . In multivariate models, UA levels were no longer significantly associated with renal allograft function. In a multivariate cox proportional hazard model, UA level was found to be independently associated with increased risk for death-censored graft loss .Patients were divided into 2 groups based on the median UA level measured between 1 and 12 months post-transplant. Those with median UA level ≥ 7 and ≥ 6 mg/dL (Hyperuricemia was found to be associated with increased death- censored graft loss but not with allograft function. Increased UA levels were not found to be an independent predictor of long-term allograft function despite the known association of hyperuricemia with the progression of cardiovascular and renal disease. Post-renal-transplant hyperuricemia is common with a prevalence of 15.5% to 84% . ExperimThe introduction of Cyclosporine in the 1980s led to dramatic reductions in the rate of early acute rejection and greatly improved 1-year graft survival ,21. DespThis is a retrospective analysis of data gathered from the Research Patient Data Registry (RPDR) system at Brigham and Women’s Hospital (BWH), in addition to data we collected from the clinical records. Biochemical data were retrieved in an automated fashion from the laboratory database and clinical data from electronic medical records. This study was approved by the local ethics committee.n = 449) including those whose graft failed in the first year post-transplant (n = 39).All patients, who underwent kidney transplantation at BWH between January 2000 and September 2013 were evaluated for inclusion in the study and constituted a population of 856 transplants. Exclusion criteria were: patients without serum UA levels from 1 month–1 year post-transplant , or Thymoglobulin . All patients received corticosteroids, beginning with a single preoperative bolus of 500 mg methylprednisolone intravenously, with gradual tapering to 20 mg oral prednisone at postoperative day 5. If the patient was a candidate for early steroid withdrawal (ESW), the prednisone was continued at 20 mg PO daily until tacrolimus levels have reached the therapeutic goal (>8 ng/ml) and then stopped. If the patient did not achieve therapeutic tacrolimus trough concentrations by postoperative day (POD) 12 the prednisone dose was tapered down below 20 mg/day. In patients who were not candidates for ESW, prednisone was tapered down to 5 mg/day within 2 months of transplant.All patients were started orally on 1 gm mycophenolate mofetil  or 720 mg of mycophenolate sodium  preoperatively; followed by 2 × 1 gm/day or 2 × 720 mg/day, respectively, with tapering of the dosage depending on individual needs and/or side effects.CNI therapy was started post-transplantation, using mostly tacrolimus, aiming for trough levels of 8–10 ng/mL in the first 3 months, and 5–8 ng/mL thereafter with reduction of trough goals if patients had side effects.Biopsy proven acute rejection (BPAR) was treated with three doses of intravenous methylprednisolone (500 mg daily) on consecutive days. All patients received pneumocystis jiroveci pneumonia prophylaxis (sulfamethoprimcotrimoxazole or atovoquone). Prophylaxis for cytomegalovirus (CMV) disease using gancyclovir, (Cytovene Roche Pharma) up to 2003 and then valganciclovir  was started in all patients.The following biochemical post-transplantation variables were retrieved from the RPDR system: serum creatinine, mean serum LDL and HDL from 1 month to 1 year post-transplant and Tacrolimus 12 h trough blood levels. Median serum UA values from 1 month to 1 year post-transplant were calculated using all samples available from this period. High median serum UA level was defined in the present study as a median serum UA level ≥7 mg/dL in men and ≥6 mg/dL in women, consistent with previous epidemiologic studies –27. We cThe presence or absence of SGF was determined based on whether serum creatinine on postoperative day 5 was above 3 mg/dL (presence of SGF) or ≤3 (absence of SGF).Median tacrolimus 12 h trough blood levels were calculated based on all trough levels available from 1 month to 1-year post-transplant in order to associate tacrolimus trough with serum UA within the same period. Both the median and mean trough levels of tacrolimus from 1 month to year post-transplant were recorded. As those gave similar results, only the results based on the median are reported here.α * max −1.209 * 0.993Age * 1.018 [if female] * 1.159 [if black]. .Serum creatinine levels were retrieved at different time points: 3 months post-transplant (3 months ± 2 weeks post-transplant), 1 year post-transplant (1 year ± 3 months post-transplant), 3 years post-transplant (3 years ±3 months post-transplant) and 5 years post-transplant (± 6 months). Glomerular filtration rate was calculated according to the following CKD Epi formula: GFR= 141* min Based on the electronic patient medical records, details on the following relevant items were collected: age, gender, race, transplant type, type of induction treatment, medical history {specifically the history of hypertension (HTN), heart failure (HF), cardiovascular disease (CVD) or pretransplant diabetes}, new-onset diabetes after transplant (NODAT), rejection episodes from 1 month to 1-year post-transplant, use of any loop diuretics, thiazides, Allopurinol, prednisone, angiotensin-converting enzyme inhibitors (Ace inh.), angiotensin receptor blockers (ARB’s), beta-blockers (BB’s) and statins from 1 month to 1-year post-transplant. The nature of the original kidney disease was noted as either ‘diabetic nephropathy’, or ‘other’.Descriptive statistics were expressed as percentages for categorical data or mean (±SD) for continuous variables. Differences in baseline characteristics between the groups were tested using Chi-square for the categorical variables or t-test for the continuous variables.We analyzed subgroups based on the availability of serum creatinine at 1, 3 and 5 years post-transplant.A multivariable linear regression analysis was constructed to analyze the association between median UA levels and allograft outcome while adjusting for covariates and potential confounders. The variables used in the multivariate analysis were chosen based on their clinical relevance, i.e., those with possible interaction with UA blood levels and/or influence on allograft function.p-values are provided via Satterthwaite’s degrees of freedom method. Mixed-effects model analysis was performed using the R packages lme4 and lmerTest.In order to account for patient-level variability, a repeated measures analysis was performed using a linear mixed-effects model with a random intercept calculated for each patient ID number. Median UA level, years post-transplant, and the confounders were used as the fixed effects. We assessed the risk of graft loss and patient’s death based on UA level using Kaplan-Meier curves and the log-rank test, followed by multivariable-adjusted Cox proportional hazards models adjusted for covariates and potential confounders.http://www.r-project.org).A p-value of less than 0.05 was considered statistically significant. Data were analyzed using R version 3.5.2. (Three hundred sixty-eight transplants (208 men and 160 women) of the original population of 856 transplants fulfilled the inclusion criteria. Patients were divided into 2 groups based on the median UA level measured between 1 and 12 months post-transplant. 164 (45%) had median UA level from 1 month to 1-year post-transplant ≥7 and ≥6 (high UA group) and 204 (55%) had median UA level <7 and <6 in men and women respectively (low UA group).Pre and post-transplantation clinical and biochemical parameters comparing the high and low UA groups are presented in p < 0.001). Medical history of HTN and CVD was more prevalent in the high compared to the low UA group (p = 0.005 and p < 0.001 respectively). 9.7% of KTR in the high compared to 3.9% of patients in the low UA group had NODAT (p = 0.044). Due to the association of diuretics with hyperuricemia, we looked at the use of loop diuretics as well as disothiazides from 1 month to 1-year post-transplant. The use of loop diuretics was higher in the high compared to the low UA group (p < 0.001 for PO loop diuretics and p = 0.002 for the use of intravenous loop diuretics). More KTR used disothiazides in the high compared to the low UA group (p = 0.016) as well as other medications including prednisone for maintenance immunosuppression, Ace inh/ARB’s and BB’s. Dyslipidemia with low mean HDL cholesterol levels was more prevalent in the high compared to the low UA group . No statistically significant differences were observed for the mean LDL cholesterol level between the groups. 52.5% of the patients in the high UA group had SGF compared to 29.9% in the low UA group (p < 0.001). Baseline allograft function derived from GFR at 3 months post-transplant was lower in the high compared to the low UA group . For all other non-significant differences between variables in the two groups see 138 patients (67.6%) in the low UA group had a living transplant compared to only 70 KTR (42.7%) in the high UA group (p < 0.001), 3 years  and 5 years post-transplant   see .p < 0.01).We performed a multivariable linear regression analysis to establish the change in GFR in relation to UA blood level. UA level was not found to be an independent predictor of GFR at 1, 3 and 5 years post-transplant. For every increase in baseline allograft function of 1 mL/min GFR increased by 0.8 [0.7–0.9], 0.54 [0.37–0.71] and 0.7 [0.4–1.00] ml/min at 1, 3 and 5 years post-transplant respectively (p < 0.05) but not at 1 or 5 years post-transplant. An increase in median CNI trough level was associated with an increased GFR at 1-year post-transplant but not at 3 or 5 years post-transplant. All other variables including age, gender, race, transplant type, induction therapy, medical history of HTN or heart failure (HF), tacrolimus coefficient of variation, pre-transplant diabetes and the presence of SGF were not significantly associated with differences in GFR at 1, 3 and 5 years post-transplant , medical history of CVD , black race  and with baseline allograft function . All other variables including UA blood level, age, gender, transplant type, induction therapy, medical history of HTN, HF and pre-transplant diabetes, median and coefficient of variance for tacrolimus 12 h trough levels, use of loop diuretics and the presence of SGF were not significantly associated with GFR over time . 14 (9.7%) and 13 (7.3%) patients died in the high and low UA groups respectively (p = 0.59).Patients were followed up for up to 120 months post-transplant with a mean follow-up time of 50.1 months. During this period, 21 cases in the high UA group (14.5%) and 8 in the low UA group (4.5%) lost their grafts (p = 0.024). When death with a functioning graft was excluded from the analysis, we observed a greater variance between the two groups (p = 0.006) (see p = 0.931)  see . Patient931) see .p < 0.05 for every increase of 1 mg/dL in UA level). Among the other variables included in the analysis black race, coefficient of variance CNI trough level and baseline graft function were found to be independently associated with death-censored graft loss  or with good allograft function at 1-year post-transplant (GFR> 60 mL/min). In addition, we could not show an independent association to allograft function when looking at UA level at 3 months post-transplant or median UA level from 1 to 6 months post-transplant (results not shown). Median UA level from 1 month to 1-year post-transplant was found to be independently associated with increased risk for death-censored graft loss over time, despite the absence of association to allograft function.2, no association was observed between UA and CKD progression [In CKD, renal excretion of UA is decreased, resulting in hyperuricemia. Interstitial accumulation of sodium urate may induce deterioration of the disease. A summary of results from 24 studies in which most reports identified hyperuricemia as an independent risk factor for CKD progression concluded that decreasing UA levels in hyperuricemic CKD patients may attenuate CKD progression . Moreovegression .Studies have raised the hypothesis that elevated UA is associated with progressive renal allograft dysfunction. A correlation of hyperuricemia with the rate of decrease in renal allograft function over time, independent of initial eGFR has been observed. Hyperuricemia was also found predictive of cardiac events in this population . In a reSeveral mechanisms for the effect of UA on native renal and renal graft outcome have been proposed. The association between hyperuricemia and cardiovascular risk, HTN, microalbuminuria, diabetes mellitus, obesity and CKD has been well known for many years . UA was The role of hyperuricemia in CKD progression as well as in renal allograft function and outcome remains controversial. This is partly related to the difficulty in isolating the relation between UA and GFR, because of the multiplicity of confounding effects of factors, such as a change in GFR over time, use of diuretics, antihypertensive medications and immunosuppressive therapy in the KTR population. The association of hyperuricemia to CVD, HTN and other risk factors make it even more complex as multiple closely related factors which play a role cannot be easily taken into account simply by multivariate analysis. Although many of these associations were detected in our study by univariate analysis, adjustment for covariates rendered some associations nonsignificant. In fact, only the presence of CVD was found to be an independent predictor of long-term renal allograft function both in multivariate analysis and in a mixed effect model. This finding may be due to the awareness of immunosuppressive exposure reduction post-transplantation as well as addressing cardiovascular risk factors in the modern era. Consistent with previous reports, the present study also shows that increased UA level is associated with an increased risk of long-term graft failure . Interesvia its association with cardiovascular risk factors and with CVD and is largely a marker of reduced allograft function. Our observation of increased UA level post-renal transplantation with lower quality kidney donors (deceased donors and DCD/ECD as opposed to living donors) with possibly reduced tubular function and impaired ability to excrete UA supports this hypothesis. However, our finding of an independent association of increased UA to death-censored graft loss may indicate that UA level by itself is a risk factor for adverse allograft outcomes. It is possible we could show UA level association to allograft survival since the follow-up time in survival analysis is longer (up to 10 years post-transplant). Survival analysis takes into consideration overall follow-up time and so patients that died during the follow-up period are included in the survival analysis but are excluded from the multivariate analysis of the years following the death. In addition, in the multivariable analysis, we are comparing mean GFR, and since the number of patients with graft loss is fairly small it is possible that their effect on the overall mean is not significant enough.Certain limitations should be considered when interpreting the results of this study. The study is an observational study performed retrospectively showing the association (but not the causative relationships) between the primary variables of interest and outcome. Even though we adjusted for numerous variables, residual confounding parameters may still exist. The long follow-up period and standardization of treatment practices limit confounding. This is a retrospective study, which often deliberates a choice of immunosuppressive agents, possibly leading to inclusion bias. It does not however abolish the relationship between UA level to graft function and outcome. We believe that increased UA level exerts its negative effect on renal allograft function Until larger prospective randomized controlled studies that allow the adjustment of confounding variables are accomplished the role of UA in progressive renal disease, as well as CVD in the transplant population, will stay debatable. In the meantime, hyperuricemia remains a marker for progressive renal allograft dysfunction and CVD after renal transplantation. Considering the link between UA level, traditional CVS risk factors and CVD, lowering UA level to minimize these risk factors may be beneficial for improving graft outcomes. Minimizing the use of diuretics and cyclosporine and avoiding purine-rich foods and alcohol may also be effective strategies to decrease the serum UA level in KTR.


Results confirmed that aging exerts a harmful effect on oocyte quality measured by using different parameters and gene expression levels of cumulus cells. Moreover, UA supplementation was an effective way to prevent oocyte aging, improving the subsequent bovine embryonic development.Post-ovulatory and maternal oocyte aging impair female reproductive capacity through several mechanisms that are not fully understood. Urolithin A (UA) is a natural compound previously identified to exert an anti-aging effects in several cells, which has never been used in bovine germinal cells. Our goal was to study UA effect on the developmental potential of the female gamete and the surround cumulus cells obtained from young and adult cows. A model for in vitro aged oocytes (30 h of IVM) obtained from prepubertal and adult females, either supplemented with UA or not were assessed. Additionally, the amount of mRNA of several genes  and the number of mt-ND5 DNA copies were quantified in cultured GCs from prepubertal and adult females, either supplemented with UA or not. Our study confirmed the harmful effect of oocyte aging on the nuclear maturation progression, MMP, developmental competence and gene expression levels. UA treatment during in vitro maturation enhanced (p < 0.05) the maturation rate and subsequent developmental capacity of aged oocytes. A positive effect (p < 0.05) of UA on physiological maturation, MMP and embryonic development was also identified. UA also interfered on the expression profile of NFE2L2 and NQO1 genes in GCs cultures. Our findings demonstrate that UA supplementation is an effective way to prevent oocyte aging and improves the subsequent bovine embryonic development.Oxidative stress and mitochondrial dysfunction have been associated with the age-related decline of oocyte quality and strategies for their prevention are currently quested. Urolithin A (UA) is a natural metabolite with pro-apoptotic and antioxidant effects, capable of preventing the accumulation of dysfunctional mitochondria in different aged cells. UA has never been tested in bovine oocytes. Our aim was to study the effect of UA on the developmental potential of cumulus-oocyte-complexes (COCs) and granulosa cells’ (GCs) expression of important genes related to reproductive competence. Nuclear maturation progression, mitochondrial membrane potential (MMP) and developmental competence of physiologically mature (22 h) and  In ad periods . The imp periods ,8. Ovula periods ,9,10. ThNQO1), heme oxygenase-1 (HMOX1), and glutamate-cysteine ligase catalytic subunit (GCLC) [One of the major causes of impaired developmental competence in aged oocytes is the increase in oxidative stress, which induces mitochondrial dysfunction, DNA damage and spindle formation errors, influencing the oocyte quality . It is wt (GCLC) ,16. Prevt (GCLC) ,18. Howet (GCLC) ,10.Conversely, mitochondria are involved in several critical cellular functions and are fundamental for meeting the demand of energy production required during oocyte maturation and subsequent embryonic development . CompeteSOD, NQO1, GCLC and HMOX1), indicating a promising anti-aging effect [Mitochondria-targeted therapeutic approaches prompted a huge interest for several pathologies associated with aging due to their great potential in enhancing mitochondrial function . Within g effect .Several studies have been performed with the goal of delaying ovarian aging, consequently improving oocytes quality and the fertility outcomes. Due to the contribution of oxidative stress to the ovarian aging process, as well as to mitochondrial dysfunction, supplementation with antioxidants has appeared as a promising therapy ,37. Howein vitro aging (30 h of maturation) or to the physiological maturation (22 h) processes were applied.This study was approved by the Animal Care Committee of the National Veterinary Authority (N°08965DGAV), following European Union guidelines (no. 86/609/EEC). To investigate the effect of aging on the alteration of oocyte quality and the potential anti-aging effect of UA, a model using COCs collected from prepubertal and adult cows submitted to in vitro maturation. After the maturation period, some oocytes (n = 154) were stained to determine the chromosomal configuration and maturation stages. The remaining matured oocytes were submitted to in vitro insemination with frozen/thawed semen. Presumptive zygotes were cultured, and cleavage and blastocyst rates were determined at day 2 and day 7 of culture, respectively. Based on the obtained results, namely the absence of harmful effects and the promotion of maturation and blastocyst development, the concentration of 1 μM of UA was selected. A previous assay to determine the concentration of UA that should be used during the bovine COCs maturation process was performed based on a dose-response study in four sessions. Since UA has never been tested in bovine oocytes, previous doses successfully applied for prevention and mitigation of some cancers and to demonstrate the anti-aging effect of UA in different cell lines were used ,38,39. Cin vitro maturation (n = 149); (4) UA aged 30 h prepubertal group, COCs from prepubertal calves aged in vitro for 30 h in maturation medium supplemented with 1 μM of UA (n = 144); (5) control adult group, COCs from adult cows matured for 22 h (n = 155); (6) UA adult group, COCs from adult cows matured in medium supplemented with 1 μM of UA for 22 h (n = 129); (7) control aged 30 h adult group, COCs from adult cows aged through 30 h of in vitro maturation (n = 148); and (8) UA aged 30 h adult group, COCs from adult cows aged in vitro for 30 h in maturation medium supplemented with 1 μM of UA (n = 138). After the respective in vitro maturation periods, oocytes were inseminated with thawed capacitated bull semen. Subsequently, embryonic development was assessed, evaluating both the rate of cleaved and produced embryos, as well as their quality.In this experiment, carried out in six sessions, both COCs from prepubertal  and adult  cows were collected to assess the oocyte quality and developmental potential of aged and physiologically matured oocytes as well as UA effect to rescue female fertility. COCs were randomly divided into 8 groups: (1) control prepubertal group, COCs from prepubertal calves matured for 22 h (n = 148); (2) UA prepubertal group, COCs from prepubertal calves matured in medium supplemented with 1 μM of UA for 22 h (n = 155); (3) control aged 30 h prepubertal group, COCs from prepubertal calves aged through 30 h of Additionally, in this experiment, COCs from each group were retrieved to assess their nuclear maturation stage . The mitochondrial membrane potential (MMP) of COCs was also evaluated . NFE2L2, NQO1 and mt-ND5. The number of copies of mt-ND5 gene was also evaluated. GCs were obtained after centrifugation of the follicular fluid aspirated from ovaries of prepubertal (mean age = 10 months) and adult cows (mean age = 62 months). These cells were cultured in the following conditions: (1) prebubertal control, culture of GCs of prepubertal calves; (2) prepubertal UA, culture of GCs of prepubertal calves supplemented with 1 μM UA; (3) adult control, culture of GCs of adult cows; and (4) adult UA, culture of GCs of adult cows supplemented with 1 μM UA. After GCs’ confluence at the 5th day of culture, they were snap frozen in liquid nitrogen and later the DNA and RNA were extracted, allowing the subsequent quantification of NFE2L2, NQO1 and mt-ND5 mRNA transcripts and also mt-ND5 copies number.As the GCs play an essential role in follicular growth and oocyte development, a second experiment was performed in five sessions to further study the effect of age and UA on the expression of w/v, BSA) supplemented with 0.05 mg mL−1 of kanamycin. At the laboratory, ovarian follicles with 2–8 mm in diameter were aspirated with a 19-gauge needle. Only COCs with at least three layers of compact cumulus cells and a homogeneous ooplasm were washed and selected for maturation according to the experimental design. Maturation was accomplished in an incubator at 38.8 °C, 5% CO2 in humidified air for 22 or 30 h in a maturation medium composed of tissue culture medium 199 (TCM) with 10% of fetal bovine serum, 0.2 mM sodium pyruvate, 10 ng mL−1 of epidermal growth factor, and 10 μL mL−1 of gentamicin [Ovaries from adult and prepubertal cows  were collected at a local slaughterhouse, and kept at 35–37 °C, in a phosphate-buffered saline (PBS) supplemented with 0.15% of bovine serum albumin (ntamicin .g [w/v), cells were seeded at a concentration of 2 × 105 viable cells mL−1 and cultured for five days at 38.8 °C, 5% CO2 in a humidified atmosphere until confluence. At every 48 h, the culture medium was discharged and refreshed with a new one. For DNA and RNA extraction, GCs were collected and washed by centrifugation at 200× g for 10 min. Cell pellets were resuspended in 1 mL of PBS, immediately snap frozen in liquid nitrogen, and stored at −80 °C.Granulosa cells were obtained from the recovered follicular fluid after centrifugation for 10 min at 200× g . The pelin vitro maturation. Denuded oocytes were fixed in an acetic acid/ethanol  solution, and maintained at 4 °C for 48 h. Then oocytes were stained with 1% aceto-lacmoid solution, mounted in a Neubauer chamber and observed under a phase contrast microscope (Olympus BX41). Oocytes were classified as follows: Germinal Vesicle (GV), Condensing Chromosomes I (CCI), Condensing Chromosomes II (CCII), Diakinesis, Anaphase-I/Telophase-I (AI/TI), and MII (Metaphase-II). Only oocytes with visible chromatin staining were taken into account [Nuclear maturation stages were assessed following the 22 h or 30 h period of  account .−1 of JC-1 [2 in humidified air in the dark. Oocytes were washed twice in PBS and immediately transferred to a pre-heated slide glass and observed under a fluorescence microscope (Olympus BX51) using the blue fluorescence filter . Mitochondrial membrane potential was then calculated as the ratio of the measured red/green fluorescence using the ImageJ software .To measure the mitochondrial membrane potential (MMP), an indicator of mitochondrial activity, mitochondria were stained with 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolcarbocyanine iodide . Denuded oocytes were incubated with 5 μg mL of JC-1  in maturIn vitro fertilization was performed with frozen-thawed sperm of a Holstein-Frisian bull, previously submitted to capacitation using the Percoll gradient (45 and 90) method, at a concentration of 2 × 106 spermatozoa mL−1. COCs and sperm were co-incubated for 20 h at 38.8 °C and 5% CO2 in humidified air. Presumptive zygotes were then transferred to droplets of synthetic oviductal fluid (SOF) medium supplemented with BME and MEM amino acids, glutamine, glutathione, and BSA [ and BSA . After 4 and BSA ,43 to as and BSA ,44.Total DNA and RNA were isolated from GCs using the High Pure PCR Template Preparation Kit  and PureLink™ RNA Mini Kit , respectively, according to the manufacturer’s instructions. Those protocols included the use of spin columns used to isolate high-quality total DNA and RNA, and DNase as treatment to remove genomic DNA from RNA . After eSynthesis of complementary DNA (cDNA) from RNA isolates were performed using the Xpert cDNA Synthesis Mastermix kit . The resultant cDNAs were stored at −20 °C until use for further assays.NFE2L2 and NQO1) and an endogenous control gene (β-actin) were designed using the Primer BLAST software of the National Center for Biotechnology Information (NCBI) . Sequences of primers for the reference genes and the target genes are depicted in mt-ND5 gene was used in this work and details about mt-ND5 primers were retrieved from a previous study [For this study, primers for the targeted  copies number of the ND5 gene was carried out by qPCR through the previously extracted DNA, using the same equipment as for the quantification of genes. Each optimized reaction was performed, consisting of Xpert Fast SYBR Green Mastermix 2X with ROX, primer (Forward and Reverse) of each target gene, sample (cDNA/DNA) and RNase free water making up a total volume of 10 μL. The samples were analyzed in duplicate and reactions containing water instead of template were included as negative controls. The samples were subjected to an amplification protocol that consisted of an initial cycle at 95 °C for 2 min of denaturation phase, followed by 40 denaturation cycles at 95 °C for 5 s, 40 annealing cycles for 30 s at 60 °C (depending on the melting temperature of primer sequences), and extension phase at 72 °C for 30 s and, lastly, a final extension period at 72 °C for 10 min. Real-time PCR analyses were performed using the Xpert Fast SYBR Green Mastermix 2X with ROX in a QuantStudio 3 thermocycler , using cDNA at a concentration of 25 ng uLFor the gene expression quantification, the relative quantification method was used. This method of relative quantification of gene expression was carried out with the expression levels of the target genes under study, which were normalized with the housekeeping genes, by the CT comparative method. As the amplification by RT-qPCR was performed in duplicate, the mean CT values for each gene were determined and the expression levels were calculated using the following formula:mt-ND5 copy number was assessed by qPCR and performed in duplicate, the mean CT values for the tests and calibrators samples were determined and the ratios were calculated using the following formula:For the quantification of the mt-DNA number of copies, the relative quantification normalized against unit mass method was used. This method was carried out with the CT values of GCs treated with UA (named as test), which were normalized with control samples . As the p < 0.05.Data from embryo production and quality sessions were analyzed by using Proc Glimmix from SAS , using the binary distribution and the logit as link function. The generalized linear mixed model included treatment  as fixed effect and replicates as random effect. In addition, the means for each treatment were calculated, and comparisons between groups were performed using the PDIFF test. The JC-1 data, mRNA transcript levels and mt-DNA number of copies were analyzed using the Proc Mixed of SAS with a model including treatment  as fixed effect. The session was considered a random effect. The data of chromosomal configurations were compared between groups using the exact Fisher test, in a 2 × 2 contingency table. The analysis of results was considered statistically significant when p = 0.0006) groups. In this phase, a higher rate in the 25 UA  group compared to the 10 UA group was also identified. Moreover, a trend was found between 50 UA group and 1 UA . The highest dose also showed a harmful effect, reducing the number of oocytes classified at the Metaphase-II (MII) stage  when compared to control , 1 UA , 10 UA  and 25 UA  groups. No differences were observed between the remaining stages of oocyte maturation. The GV and CCI maturation phases were not shown in the The oocyte maturation status observed during the dose-response study of UA supplemented to the maturation medium is represented in p = 0.004), 10 UA  and 25 UA  groups. Although no significant differences were found between the control and 50 UA groups, a trend (p = 0.06) was identified. Moreover, day 7 embryo rates tend (p = 0.07) to be higher after 1 UA dose supplementation compared to control. Moreover, the 1 UA group doubled the number of excellent/good embryo quality  when compared to control  and 10 UA . A low number of grade 1 embryos was obtained with the higher doses: 25 UA group  and 50 UA . Based on the above-mentioned results, the 1 μM UA was chosen to proceed to the following studies.The dose-response test showed no significant effect of UA concentrations on cleavage and hatched embryo rates. However, the rate of embryos produced at day 7 was influenced by the different concentrations of UA. The 50 UA group (7.2 ± 2.7%) had lower rates of embryos at day 7 compared to the 1 UA . Moreover, a delay on maturation progression was identified on aged gametes and prepubertal females with more oocytes at AI/TI phase . Moreover, a higher number of degenerated oocytes after 30 h of IVM was identified compared to after 22 h . The female age and UA supplementation did not interfere (p > 0.05) with the number of degenerated oocytes.We investigated the effect of UA supplementation on chromosomal maturation status of aged and physiologically matured oocytes collected from prepubertal and adult females. Independently of the UA effect, oocyte aging and female age induced a significant harmful effect on maturation. Higher rates of MII phase were identified in control oocytes compared to aged ones  and UA aged 30 h prepubertal , and both adult control  and control aged 30 h adult  groups. A trend was also identified between UA adult and UA aged 30 h adult (p = 0.0769).Both p = 0.05), UA aged 30 h prepubertal  and UA adult  and control aged 30 h adult  groups. Moreover, the UA adult group showed higher rates of matured oocytes (MII) compared to the UA aged 30 h adult , control aged 30 h adult  and control adult .Regarding the MII phase corresponding to a complete nuclear maturation, significant differences were also found between groups . Lower rNo differences were observed between the remaining stages of oocyte maturation.in vitro maturation progression in oocytes submitted to the aging process, especially in prepubertal females. An important anti-aging effect of UA was thus identified.In summary, UA supplementation improved In order to analyze the involvement of mitochondrial dysfunction in female aged oocytes and the effect of UA and maternal age, the mitochondrial membrane potential (MMP) was assessed in oocytes of different groups.p = 0.002).Independently of the female age and UA effects, oocyte aging had a significant harmful effect on MMP assessed as the ratio of the measured red and green fluorescence  influenced the MMP  and UA aged 30 h adult  groups. Moreover, a significant increase in JC-1 aggregate/monomers ratio, which indicates a significant increase in MMP, was observed in the UA adult group when compared with control adult , control prepubertal , control aged 30 h adult  and UA aged 30 h adult  groups. A trend was also identified between UA prepubertal, and UA aged 30 h prepubertal (p = 0.07).The combination of the female age, aging effect and the supplementation with UA during maturation significantly  and D7 embryo rates . The UA supplementation was also shown to improve the D7 embryo rates .The embryonic developmental potential was evaluated through cleavage, D7 and hatched embryo rates, obtained with the different treatments (aging and UA supplementation) applied to the prepubertal and adult females COCs during maturation. Independently of the female age and UA effects, oocyte aging had a significant harmful effect on cleavage  influenced the embryonic development, namely the cleavage and D7 embryo rates (p ≤ 0.01), control aged 30 h prepubertal  and UA aged 30 h prepubertal  groups (p = 0.09) was also identified between the control adult and the control aged 30 h adult groups.The combination of the female age, aging effect and the supplementation with UA during maturation significantly (yo rates . Higher ) groups . A trendp = 0.01) influenced by the combination of the female age, the supplementation with UA and COCs aging was the rate of embryos produced at day 7 (p ≤ 0.05). Moreover, lower D7 embryo rates were produced from aged oocytes from both adult and prepubertal females without the supplementation of UA  compared to those that were supplemented with UA, respectively . The supplementation of UA to the maturation medium did not significantly (p > 0.05) influence the quality rates of produced embryos of grade 1, 2 and 3, nor did the aging effect or all the studied effects together .In order to study the UA effect and maternal age influence in the Nrf2 signaling pathway, analysis of p > 0.05) influence the gene expression levels of NFE2L2, NQO1 and mt-ND5 genes. The supplementation of UA to the culture medium of GCs, both independently and considering female age, did not significantly (p > 0.05) influence the copy number of mt-ND5 gene. Analysis of mt-ND5 DNA content in GC culture from prepubertal and adult cows were assessed by qPCR. The supplementation of UA to the culture medium of GCs, both independently and considering female age, did not significantly  harmful effect of gamete aging on cleavage and day 7 embryo production rates were observed. Accordingly, several authors referred that old females have an age-associated decline in reproductive capacities, reflected in lower fertility rates and poor embryo quality [in vitro maturation on the embryo development of aged females from different species, such as cattle [Intrinsic quality of oocytes has been widely reported as the main determinant of subsequent embryonic development. Accumulated evidence revealed that several cellular and molecular abnormalities occur during extended ry aging . Furtherry aging . To demo quality ,55. On ts cattle , pig [38s cattle . For inss cattle . Our ress cattle ,35 is vaAdditionally, we also assessed the preventing role of UA in the age-related deterioration on embryo quality. Although in this study no significant differences were found on embryo quality rates, the oocytes supplemented with UA increased the number of transferrable embryos, compared to the untreated ones. A previous study reported that L-carnitine treatment could improve the quality of embryos developed from aged bovine oocytes through the reduction in ROS levels and higher levels of glutathione as well as of others antioxidant enzymes . The difNFE2L2 and its downstream antioxidant (NQO1). Our results showed a significant influence of female age on the level of NFE2L2 transcripts, which decreases with age. Indeed, greater mRNA expression levels were observed in prepubertal cows when compared to adults. This is in agreement with previous studies that reported a highly expressed level of Nrf2 protein and mRNA on ovarian tissues of childbearing young mice and women from 22 and 49 years old, whereas in aged mice and women a lower expression was found. It was suggested that decreased expression of Nrf2 may be involved in the decline of reproductive capacity of older women and its control may have important implications in delaying ovarian aging [Nrf2 targeted genes, such as SOD1, NQO1, GCLC and HMOX1 were reported. [NFE2L2 and NQO1 genes in GCs was not significantly affected by UA supplementation. However, further studies should be addressed to confirm the reduction in ROS and subsequent improvement of produced blastocyst, as well as the UA effect on mRNA expression of the aforementioned genes on aged oocytes.It is widely known that oxidative stress plays a crucial role in the age-associated decrease in fertility. Oxidative stress is one of the major contributors to low oocyte maturation efficiency, and oocyte quality deterioration, thereby impairing subsequent embryo development . Intercean aging ,61. Furtan aging . Similareported. . In our mt-ND5 mRNA expression, between the GCs retrieved from prepubertal and adult cows. Moreover, we also assessed the mt-DNA copy number, because the content of mt-DNA in GCs has been positively associated with oocyte quality and embryo development [In addition to the damage to mitochondrial DNA (mt-DNA) ,57, the elopment ,64. Althelopment . FurtherThe results obtained in this study confirmed the harmful effect of oocyte aging on its developmental competence. Moreover, our model for aging female gametes proved to be very efficient and useful to study different problems associated with reproductive aging and consequent fertility impairment. Additionally, UA supplementation during the maturation process of aged oocytes improved maturation rates and produced embryos. Therefore, an anti-aging effect of UA in rescuing aged gametes was identified for the first time, improving the blastocyst development, which leads to an increased number of embryos for transfer to recipient females. A positive effect of UA on physiological maturation, MMP and embryonic development was also identified. In conclusion, UA treatment provides a new therapeutic approach to prevent or delay gamete aging, and improve the subsequent blastocyst formation and fertility outcomes in ART.


The striatin-interacting phosphatases and kinases (STRIPAK) multi subunit complex is a highly conserved signaling complex that controls diverse developmental processes in higher and lower eukaryotes. In this perspective article, we summarize how STRIPAK controls diverse developmental processes in euascomycetes, such as fruiting body formation, cell fusion, sexual and vegetative development, pathogenicity, symbiosis, as well as secondary metabolism. Recent structural investigations revealed information about the assembly and stoichiometry of the complex enabling it to act as a signaling hub. Multiple organellar targeting of STRIPAK subunits suggests how this complex connects several signaling transduction pathways involved in diverse cellular developmental processes. Furthermore, recent phosphoproteomic analysis shows that STRIPAK controls the dephosphorylation of subunits from several signaling complexes. We also refer to recent findings in yeast, where the STRIPAK homologue connects conserved signaling pathways, and based on this we suggest how so far non-characterized proteins may functions as receptors connecting mitophagy with the STRIPAK signaling complex. Such lines of investigation should contribute to the overall mechanistic understanding of how STRIPAK controls development in euascomycetes and beyond. Posttranslational phosphorylation of eukaryotic proteins plays an essential role in modulating their function and is tightly regulated in time and space by the fine-tuned balance between protein kinases and phosphatases . The serHere, we briefly summarize our current knowledge about the function of the STRIPAK complex of ascomycetes, with an emphasis on the role of euascomycetous development. We further refer to recent structural investigations and cellular analyses, which help us understand how STRIPAK acts as a scaffolding complex that cross-links several signal transduction pathways. Finally, current molecular cellular investigations of the STRIPAK homologue in yeast provide insights into a mechanistical hypothesis of how STRIPAK interacts with other signaling pathways at the cellular level, and thus integrates various developmental programs.Striatin is the central subunit of STRIPAK, and the primary structure of this B‴ subunit is conserved in all eukaryotic systems studied so far ,4,5,6. SAspergillus nidulans [Although the association of STRIPAK into a heterotrimeric holoenzyme is validated, the overall structure of the complex and knowledge about the stoichiometric composition of the subunits are still cryptic. Assembly of the fungal STRIPAK complex was recently investigated in the euascomycete nidulans . Using wnidulans ,10. A funidulans . STRIPAKnidulans . The higSordaria macrospora, a self-fertile homothallic member of the Sordariaceae family, sterile strains were screened for mutant genes, many of which were found to encode STRIPAK subunits. The mutants only generated small immature fruiting bodies, called protoperithecia of about 50 µm in diameter, and contained no ascospores nor asci. Fertility was clearly restored when the wild type subunit gene was introduced into the mutant genome [Distinct genes encoding subunits of the STRIPAK complex have been analyzed in a variety of filamentous ascomycetes. Mutations in the corresponding genes lead to severe developmental defects as summarized in t genome . Such STNeurospora crassa strains with a defect in cell–cell fusion were investigated and the genes responsible were abbreviated as HAM for hyphal anastomosis. Of these, several showed a mutation in genes for STRIPAK subunits, such as HAM-2, -3, and -4 [N. crassa, female but not male fertility is affected by mutant genes for STRIPAK subunits. Furthermore, it has been hypothesized that the lack of hyphal fusions leads to impaired nuclear division during meiosis and to the formation of abnormal ascospores. These observations led to the hypothesis that subunits of STRIPAK are involved in regulating the cell cycle [In an analogous mutant screen, , and -4 ,15,16. Cll cycle . In thisll cycle ,19,20.A. nidulans when proper expression of the velvet complex [A remarkable effect was observed in  complex  was inve complex .Epichloë festucae forms a mutualistic symbiotic relationship with the grass Lolium perenne. The fungus produces several secondary metabolites thus protecting the host against herbivores. This interaction arises by fungal colonization of intercellular spaces and the leaf surface. Loss of mobC, which is homologous to mob3, completely abolishes the symbiotic interaction between the fungus and the grass. Instead, E. festucae shows extensive hyphal growth within the intercellular tissue of the grass, and in addition, hyphae are found within the vascular bundle tissue, which was never observed with the wild type. Infection with the mobC mutant leads to underdeveloped grasses, lacking a functional association with the fungal symbiont [Plant–fungal interactions are another developmental process that is controlled by STRIPAK. symbiont .Colletotrichum graminicola, Fusarium verticillioides, Fusarium virguliforme, and Magnaporthe oryzae. In the corn pathogen F. verticillioides, the N-terminal domains of the striatin homologue Fsr1, as well as the STRIP1/2 homologue FvStp1 are important in maize stalk rot virulence [C. graminicola, striatin null mutants have functional appressoria; however, the colonization leading to infection is diminished [F. virguliforme. Here, the striatin homologue FvStr1 controls the colonization of the vascular system, although phloem and xylem vessels of the roots are still infected by mutants [M. oryzae, strains lacking the catalytic subunit of PP2A (MoPPG1) were unable to form appressoria for penetrating the host plant [For a long time, plant pathogenic fungi were the subjects of genetic analysis in order to understand their pathogenic mechanisms. These investigations revealed that STRIPAK controls the virulence process in plant pathogens such as irulence ,24,25. Sminished . Virulen mutants . In the st plant . From thAspergillus fumigatus, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum will discover virulence mutants with a defect in genes for subunits of the STRIPAK complex. This assumption is supported by the fact that genes for STRIPAK subunits are very similar between pathogenic and non-pathogenic ascomycetes and basidiomycetes. At least, so far characterized protein domains in homologues of striatin, SLMAP and STRIP1/2 seem to be highly conserved  pathway [Genetic and molecular analysis in fungi has shown previously that STRIPAK is associated with signaling transduction pathways. In  pathway , which s pathway ,44.Saccharomyces cerevisiae broadens our mechanistic view of how multiple cellular localizations of STRIPAK subunits connect to signaling transduction pathways [A recent report from the protoascomycete pathways . In yeaspathways .So far, a homologue of Atg32p has neither been detected in mammals nor in filamentous euascomycetes ,48. HoweS. macrospora genes encoding proteins with sequence motifs that are characteristically found in Atg32p and/or Bcl2-L-13. Indeed, we detected 27 genes  was applied to analyze phosphorylation site occupancy in signaling components of the SIN signaling pathway . In conc


Potentiostatic polarization of the electrode at 2.0 V in H2SO4 solution resulted in a higher number of oxygen and nitrogen-containing groups on the electrode surface; lower charge transfer resistance; a 1.5 times increase in the effective surface area and a decrease in the UA oxidation potential by over 0.4 V, compared with the non-activated CVE, which was confirmed by energy dispersive X-ray spectroscopy, electrochemical impedance spectroscopy, chronoamperometry and linear sweep voltammetry. The developed sensor is characterized by a low detection limit of 0.05 µM and a wide linear range (0.09–700 µM). The results suggest that the sensor has perspective applications for quick determination of UA in artificial and human saliva. RSD does not exceed 3.9%, and recovery is 96–105%. UA makes a significant contribution to the antioxidant activity (AOA) of saliva (≈60%). In addition to its high analytical characteristics, the important advantages of the proposed CVEact are the simple, scalable, and cost-effective manufacturing technology and the absence of additional complex and time-consuming modification operations.The paper describes the development of a carbon veil-based electrode (CVE) for determining uric acid (UA) in saliva. The electrode was manufactured by lamination technology, electrochemically activated and used as a highly sensitive voltammetric sensor (CVE Uric acid (UA) is produced in the human body as a result of the enzymatic decomposition of purine bases and nucleic acids. The level of UA in blood serum, urine and saliva is a biomarker of some systemic diseases . High leSaliva is a very promising diagnostic matrix. As a biological material for analysis, saliva is more advantageous than plasma or blood serum. Its non-invasive sampling is characterized by cost-effectiveness, reduced complexity of the analysis and lower risk of patient’s infection, which is an obvious benefit. The UA level in saliva of patients with oral diseases is considered to be a biomarker of antioxidant defence against oxidative stress . MonitorA variety of methods can be used to measure UA in biological fluids, including fluorescent , chromatElectrochemical methods, characterized by simplicity, high sensitivity, and selectivity, serve as powerful analytical tools for monitoring UA levels. Among the advantages of electrochemical methods is miniaturization and the possibility for its use in on-site measurements. To date, numerous electrochemical sensors and biosensors have been developed to determine UA in biological  samples ,16,17,18Carbon veil (CV), or carbon paper (CP), is considered as an advanced material for creating new electrochemical sensors. Since this material has a well-developed three-dimensional (3D) surface, we think that the term “carbon veil” is preferable, as the term “carbon paper” is more associated with a two-dimensional (2D) material. CV is a thin non-woven fabric, with multiple fibers randomly intertwined and bonded by a light binder, which ensures a porous structure of the material with high surface area. CV has various properties: good electrical conductivity, electromagnetic interference shielding, static dissipation, chemical and thermoresistance, and low specific gravity. These properties have made CV a popular material in fuel cells, aerospace, and civil construction . Due to CV is an advanced material for manufacturing highly sensitive (bio)sensors with the use of amperometric or voltammetric methods of signal recording . CurrentThe present paper focuses on the development of a simple, highly sensitive voltammetric carbon veil-based sensor, fabricated by using the hot lamination technology, for determining salivary UA.2HPO4·12H2O , KH2PO4 , NaHCO3 , NaOH , KCl , NaCl , K3[Fe(CN)6] , K4[Fe(CN)6]·3H2O , Cementit universal , acetone , uric acid , ascorbic acid , creatinine , urea , glucose . All reagents were chemically pure and used without additional purification. Working solutions were prepared using deionized water .Chemicals Na2; surface electrical resistivity—8–10 Ω, manufactured by M-Carbo Ltd.  and polyethylene terephthalate sheets sized 303 × 216 × 0.125 mm .Electrodes were fabricated on the basis of polyacrylonitrile carbon veil (surface density—30 g/mCarbon veil electrodes (CVE) were fabricated utilizing an LM-260iD laminator . Scanning electron microscopy (SEM) images were taken by a Quattro S electron microscope . The analysis of the elemental composition of the CV surface was conducted by energy dispersive X-ray spectroscopy (EDS), utilizing a Quattro S scanning electron microscope and an EDAX Octane Elite detector  at an accelerating voltage of 2 kV and a sample current of 1 nA. Electrochemical impedance spectroscopy (EIS) was performed using a μAutolab Type III potentiostat/galvanostat . A semi-automatic computerized voltammetric analyzer IVA-5  with a PE-6100 magnetic stirrer and a three-electrode cell was used for cyclic, linear sweep voltammetric and chronoamperometric studies. A CVE was referred as the working electrode; a glassy carbon rod as the auxiliary electrode, and a silver–silver chloride electrode  EVL-1M3.1  as the reference electrode. Antioxidant activity (AOA) of saliva samples was measured utilizing a multifunctional potentiometric analyser MPA-1 . The planar platinum electrode  served as the working electrode and Ag/AgCl/KCl, 3.5 M served as the reference electrode in potentiometric measurements.2 (5 × 3 mm). Then the electrode was washed in the water–acetone mixture  by applying constant stirring for 15 min. The obtained electrode was labeled a carbon veil-based electrode (CVE). To activate the CVE electrochemically, it was placed in a solution of 0.05 M of sulfuric acid and kept at different potentials  for 5 min. Thus, CVEact was prepared.The working electrodes were fabricated by applying the hot lamination technology ,28. The 2PO4; 0.02 g of urea and 0.0631 g of NaHCO3 in 100 mL of deionized water. Prior to the analysis, salivary pH was changed to 7.0, applying NaOH.Artificial saliva and human saliva were used for sampling. Artificial saliva was prepared following  and contVolunteers, adults aged 25–28 years, provided saliva samples. The samples were collected in the morning following the recommendations ,32. Elig−1 and cyclic voltammograms, from 0.5 V to +1.1 V at 0.05 Vs−1. Chronoamperometric measurements were taken at the potential +0.5 V in 0.1 M KCl as background electrolyte. EIS measurements were performed at an ambient temperature, by applying the sine potential of 5 mV and polarization potential of 0.25 V. The frequency range was from 0.04 Hz to 1.0 kHz. EIS spectra were fitted to a Randles-type equivalent circuit.Phosphate buffer (PB) pH 5–8 was used as background electrolyte for recording UA signal with the linear sweep (LS) voltammetry. LS voltammograms were registered in the potential range from 0.1 V to 0.7 V at a potential scan rate of 0.025–0.300 Vsb is the slope of the dependences Ip vs CUA. The recovery of UA was determined in accordance with IUPAC guidelines 4−/3− Red/Ox pair on CVE and CVEact was studied. As can be seen in 6]4−/3− oxidation/reduction currents and a decrease in the potential difference between the anodic and cathodic peaks for CVEact as compared with the non-activated CVE. In addition, in comparison with the non-activated CVE, a better shape of the peaks for CVEact was observed. Cyclic voltammograms showed that the ratio of the cathodic and anodic currents was 1.01 for CVEact and 0.77 for CVE. The potential difference between the cathode and anode peaks was 0.34 V for CVEact and 1.05 V for CVE.The electrochemical behaviour of the [Fe(CN)6]4−/3− Red/Ox pair is irreversible on CVE and reversible on CVEact. In this regard, chronoamperometry and the Cottrell equation that is applicable for both reversible and irreversible electrochemical processes were used to measure the effective surface area of the electrodes. For that purpose the chronoamperograms of K4[Fe(CN)6] oxidation were registered at a potential of 0.5 V at CVE and CVEact (−1/2) (−1/2) for CVEact is approximately 1.5 times higher than for CVE.The obtained results demonstrate that electrochemical reaction of  concentration; D = 7.6 × 10−6 cm2 s−1 (diffusion coefficient); t (s)—time.The effective surface area of the electrode was computed following the Cottrell equation (Equation (1)):2, and the area of CVEact was 24.6 mm2. Due to electrochemical activation, the degree of coating of the carbon veil with various oxygen-containing functional groups increases, which contributes to a bigger active surface area  and 0.1 mM K4[Fe(CN)6]. The changes (decrease) in the potential were registered in the two-electrode cell containing the planar platinum electrode (working electrode) and silver/silver chloride electrode Ag/AgCl/KCl (reference electrode). When correlating salivary UA and AOA, UA concentration was expressed in μM-eq, taking into consideration that two electrons were involved in UA oxidation on CVEact  has demonstrated that the proposed sensor could be well applied for salivary UA determination. A good correlation (r = 0.97) has been observed between the levels of salivary UA and AOA. It has been found that UA provides about 60% of the total AOA of saliva.


Bacillus amyloliquefaciens (BA) and Bacillus subtilis (BS) are usually used as feed supplements directly or bacterial inoculants in biological feeds for animals. However, few research have reported the effects of BA and BS on fermentation characteristics and bacterial community successions of whole-plant corn silage during ensiling. If the BA and BS inoculants have positive effects on silages, then they could not only improve fermentation characteristics, but also deliver BA or BS viable cells to ruminants, which would play its probiotic effect. Therefore, the objectives of this study were to investigate the effects of BA and BS on the fermentation, chemical characteristics, bacterial community and their metabolic pathway of whole-plant corn silage.Acetobacter and Acinetobacter.Freshly chopped whole-plant corn was inoculated without or with BA and BS, respectively, and ensiled for 1, 3, 7, 14 and 60 d. Results showed that BA and BS inoculations increased lactic acid concentrations of whole-plant corn silages compared with control, and BA inoculation decreased acetic acid concentrations, whereas BS inoculation decreased fiber contents and increased crude protein (CP) content. Higher water-soluble carbohydrate contents and lower starch contents were observed in BA- and BS-inoculated silages compared with that in control. The decreased CP content and increased non-protein nitrogen content were observed in BA-inoculated silage, which was consistent with the higher amino acid metabolism abundances observed in BA-inoculated silage. In addition, it was noteworthy that BA and BS inoculations increased the metabolism of cofactors and vitamins, and decreased the relative abundances of drug resistance: antimicrobial pathways. We also found that the bacterial metabolism pathways were clearly separated into three clusters based on the ensiling times of whole-plant corn silage in the present study. There were no significant differences in bacterial community compositions among the three groups during ensiling. However, BA and BS inoculations decreased the relative abundances of undesirable bacteria such as Our findings suggested that the BS strain was more suitable as silage inoculants than the BA strain in whole-plant corn silage in this study. Lactobacillus plantarum [Pediococcus acidilactici with high-antioxidant activity [Saccharomyces cerevisiae, were defined as fourth generation silage inoculants where probiotic microbes delivered direct benefits to the animals through silages. Bacillus could also be classified as fourth generation silage inoculants because of its favorable ability to improve animal performance when orally administered [Whole-plant corn silage, which makes up over 40% of forage fed to dairy cows , has beclantarum  or Pedionistered  and imprnistered .Bacillus amyloliquefaciens and Bacillus subtilis are usually used as biological control agents to protect plants effectively against plant pathogens [B. amyloliquefaciens and B. subtilis, they are used as feed supplements directly or bacterial inoculants in biological feeds for monogastric animals, such as poultry and pigs [B. subtilis in silages. Lara et al. [B. subtilis used in silages improved fermentation quality and inhibited aerobic spoilage. However, few research have reported the effects of B. amyloliquefaciens on fermentation characteristics in silages. Sansinenea and Ortiz [B. amyloliquefaciens was related to B. subtilis and had the potential to produce many antimicrobial compounds. Thus, we speculated that B. amyloliquefaciens could also improve fermentation characteristics of silages and deliver B. amyloliquefaciens viable cells to ruminants.athogens , 8. In aand pigs –11. Thera et al.  and Bai a et al.  found thnd Ortiz  reportedB. amyloliquefaciens or B. subtilis on bacterial community succession and their functional shifts in whole-plant corn silage. Therefore, the objective of this study was to investigate the effects of B. amyloliquefaciens or B. subtilis on the fermentation characteristics, bacterial community succession and their functional shifts of whole-plant corn silage during ensiling.Understanding the succession of bacterial community could provide deep insight into the fermentation process underlying silage formation. The bacterial community succession at different phases of ensiling is a dynamic process that varies throughout the fermentation period, and single-molecule real-time (SMRT) sequencing technologies provide useful information on microbial shifts at the species level . In addiZea mays L. Dajingjiu 3876) was harvested at half milk-line from 4 randomly selected sites in a commercial farm located in Dingxi city, Gansu province, China. The whole-plant corn was chopped into 2 cm size by using a forage cuter  and then taken into the laboratory immediately. For each of the 4 randomly selected sites, there were 15 piles of forage . The corn forage piles were treated separately with distilled water ; Bacillus amyloliquefaciens HRH317 , which could inhibit fungus by producing antifungal proteins [Bacillus subtilis CP7 , which had the ability to inhibit gram-negative bacteria, such as Escherichia and Salmonella by producing antibacterial peptides [Whole-plant corn , and water-soluble carbohydrate (WSC) were referenced by the description of Licitra et al. [Twenty g of each fresh and ensiled sample was put in a juice extractor and squeezed with 180 mL distilled water for 30 s, and then filtrated through four layers of cheesecloth. The filtrate pH was measured using a glass electrode pH meter. Determination methods of lactic, acetic, and propionic acid concentrations were referenced by the description of Zhang et al. . Chemicaa et al.  and Ke ea et al. . The chea et al. . The deta et al.  and van a et al. . The staTotal genomic DNA of surface bacteria of fresh and ensiled whole-plant corn from the three groups fermented for 1, 3, 7, 14, and 60 d were extracted using a DNA isolation kit . Among the four replicates of each treatment, three replicated samples were randomly chosen for DNA extraction. The quality and quantity of extracted DNAs were measured according to the description of Guo et al. [Lactobacillus, Weissella, Leuconostoc and Pedicoccus, the blastn (blast-2.11.0+) program based on the best hit method was further used to assign the annotations to the species level by depositing the data in the SILVA 138 library [The rdp_classifier-2.2 software was used to annotate the species of OTU representative sequence basing the database of SILVA 138 firstly. For those OTU representative sequences that annotated to the genus levels but not to the species levels, such as  library , 24. Bef library .Yij represents the response variable, μ is the overall mean, Ti is the effect of inoculants, Dj is the effect of ensiling time, (T × D)ij is the effect of the interaction between the inoculants and ensiling time, and εij is the random residual error. Chemical compositions of 60-d silage samples were analyzed using one-way ANOVA. Tukey’s test was also used for pair-wise mean comparisons. Significance was considered at P < 0.05.The experimental protocol had a 3 × 5 factorial design with 3 inoculants and 5 ensiling times. The data for pH and organic acids were analyzed using the general linear model procedure of the Statistical Package for Social Science  according to the model:The chemical compositions of fresh whole-plant corn are presented in Table P < 0.001). The acetic acid concentration was higher in BA-inoculated silage compared with control after 1, 3, and 7 d of ensiling, while decreased after 14 and 60 d of ensiling period. The higher acetic acid concentration was in BS-inoculated silage compared with control at the ensiling stages of 1 to 14 d (P < 0.001), and no differences were observed after 60 d of ensiling period. Higher lactic acid/acetic acid (LA/AA) ratios were observed in BA- and BS-inoculated silages than that in control after 1, 3, 7, and 14 d of ensiling period, and the lowest LA/AA ratio was observed in BA-inoculated silage after 60 d of ensiling (P < 0.001). No propionic acid was detected in all whole-plant corn silages during the ensiling period.Fermentation characteristics and chemical compositions of whole-plant corn silages are listed in Tables P < 0.05, Table P < 0.001). On the other hand, the highest NPN content was in BA-inoculated silage, followed by control, and then in BS-inoculated silage (P < 0.001). However, no differences were observed in NH3-N contents among the three groups. The aNDF and ADF contents were lower in BS-inoculated silage compared with control and BA-inoculated silage (P < 0.05).The BA and BS inoculations increased WSC contents and decreased starch contents when compared with control  and Leuconostoc pseudomesenteroides (9.51%). After ensiling, the bacterial community compositions of the three silage groups were similar, and obvious regular changes were observed with the extension of ensiling. The relative abundance of Lactobacillus increased rapidly, and Weissella, Leuconostoc, and Gluconobacter decreased during the fermentation process. After 60 d of ensiling, the compositions of dominant bacteria at genus levels became simple that Lactobacillus was the main genus, and Lactococcus appeared in the three silage groups, and Lactococcus was higher in the BA inoculated silage. In addition, the species of Lactobacillus varied during the ensiling. After 60 d of ensiling, the bacterial community compositions at the species level were completely changed. The dominant Lactobacillus changed from L. brevis (> 30%) after 7 and 14 d of ensiling to L. buchneri (> 30%) after 60 d of ensiling. However, no differences were observed on the main bacterial community compositions between BA- and BS-inoculated silages and control.Bacterial diversity, bacterial community compositions and differences are shown in Fig. Bacillus, we only annotated Bacillus at the genus levels, but did not annotated B. amyloliquefaciens and B. subtilis at the species levels in this study. Therefore, we performed a statistical analysis on the relative abundance of Bacillus  among the three groups, Leuconostoc was in control, Lactobacillus was in BA-inoculated silage. Bacillus was higher in BS-inoculated silages after 7, 14, and 60 d of ensiling period. Acinetobacter and Acetobacter were higher in control than that in BA- and BS-inoculated silages after 14 and 60 d of ensiling, respectively. Lactobacillales was still higher in BA-inoculated silage than that in the other two groups after 60 d of ensiling period.Then we explored the differences in bacterial communities among the three groups using the LEfSe analysis Fig. . After 1The potential functions and enzyme of bacterial communities of the three groups at different ensiling stages were predicted by the PICRUSt2 software Fig.  and 4. TEC:3.2.1.1 (α-amylase), EC:3.2.1.4 (cellulase) and EC:3.4.21.107 (peptidase) that are closely related to silage fermentation were chosen in enzyme classification (EC) database , mid (7 to 14 d of ensiling), and late (60 d of ensiling) periods. We generally focused on the metabolism of carbohydrates, nucleotide, energy, amino acid and cofactors and vitamins (the main metabolic pathways in silages), which were related to the changes of fermentation and chemical characteristics , 17. In a silage . It mighSalmonella and Escherichia coli [The abuse of antibiotic results a global growth of multidrug-resistant bacteria, which becomes one of the greatest threats to human health. . In natuhia coli , 47. It In order to further confirm the effects of BA and BS inoculants on the chemical characteristics of whole-plant corn silages, EC numbers of α-amylase, peptidase and cellulase were chosen according to PICRUSt2 in this study. We found that the highest abundance of α-amylase was observed in BA-inoculated silage during the entire ensiling period, which was consistent with the lowest content of starch in BA-inoculated silage after 60 d of ensiling. The lowest abundance of α-amylase was in accordance with the highest content starch in control. The result indicated that the BA strain in this study produced α-amylase to degrade starch during ensiling of whole-plant corn silage. In addition, the BA inoculation increased the abundance of peptidase during the early ensiling periods, while the peptidase abundance became the lowest in BA-inoculated silage after 60 d of ensiling. The lowest CP content in BA-inoculated silage might be due to the stronger activity of the facultative aerobe BA strain in the early stage of ensiling, which produced more peptidase in the optimal growth environment. The lower abundance of peptidase was observed in BS-inoculated silage compared with BA-inoculated silage, which was consistent with the higher CP content and lower NPN content in BS-inoculated silage. The changes of cellulase abundance of the three silage groups could not explain the differences in aNDF and ADF contents. We found that the BS inoculation decreased aNDF and ADF contents of whole-plant corn silage compared with control and BA-inoculated silage. However, the cellulase abundance was higher in BA-inoculated silage than that in control and BS-inoculated silage after 1 and 3 d of ensiling, and no difference on the cellulase abundance was observed between BA- and BS-inoculated silages during the ensiling period from 7 to 60 d. Therefore, the decrease in fiber content in BS-inoculated silage might not be related to the abundance the effect of cellulase.Acetobacter and Acinetobacter according to LEfSe analysis. Higher abundances of Bacillus and Lactobacillales were observed in BS- and BA-inoculated silage, respectively. Additionally, the whole-plant corn ensiling process could be clustered into three stages through changes in metabolism pathways, which could be a new approach to silage research. We also found that BA inoculation increased the relative abundances of amino acid metabolism, which was consistent with the increased NPN content and the decreased CP content in BA-inoculated silage. Meanwhile, the decreased starch content and increased WSC content were observed in BA-inoculated silage after 60 d of ensiling period. Due to the special digestion and absorption mechanism of ruminants, the degradation of protein and starch by BA might not be suitable for whole-plant corn silage. However, the BS inoculation increased the CP content and reduced the contents of NPN, aNDF, and ADF when compare with control. It is noteworthy that the metabolism of cofactors and vitamins increased in BA- and BS-inoculated silages; meanwhile, the drug resistance: antimicrobial pathway decreased during the ensiling.The BA and BS inoculations had a little effect on bacterial community compositions. However, BA and BS inoculations significantly decreased the relative abundance of


IDH is a severe, chronic superinfected eczema generally associated with Staphylococcus aureus and/or Streptococcus beta haemolyticus infection that responds partially to antibiotic therapy but prompt recurrence develops upon treatment withdrawal. IDH could be a risk factor for progression toward both HAM/TSP and ATLL and, similarly to other diseases associated with HTLV-1, it is sub-diagnosed particularly in non-endemic areas. Here, we present a case of IDH in a young boy living in Buenos Aires with symptoms since 2010, at the age of 5. HTLV-1 infection was suspected and confirmed in 2016. The patient exhibited chronic dermatosis with exudative eruption involving mainly the scalp, retroauricular regions, neck and abdomen. Clinical evaluations, routine laboratory tests, full blood count, and HTLV-1 diagnosis for this case are included.Human T-lymphotropic virus type 1 (HTLV-1) is a neglected retrovirus distributed worldwide and the ethiological agent of several pathologies, such as adult T-cell leukemia/lymphoma (ATLL), a chronic myelopathy known as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and infective dermatitis associated with HTLV-1 (IDH). HTLV-1 presents tropism for CD4 Generally, IDH responds to antibiotic therapy but prompt recurrence develops upon treatment withdrawal. IDH is associated with high HTLV-1 proviral loads (PVL), and characterized by a predominantly Th1 immune response , with CD8+ T cell infiltrates in skin. CD4+/CD8+ T cell ratio is usually elevated. Tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-ɤ) levels are also frequently increased in IDH patients when compared to asymptomatics (Human T-lymphotropic virus type 1 (HTLV-1) is a neglected retrovirus distributed worldwide in endemic regions such as Japan, Rumania, Iran, Jamaica, Western Africa and South America, and mostly diagnosed elsewhere in immigrants from endemic areas. HTLV-1 causes adult T-cell leukemia/lymphoma (ATLL) and a chronic demyelinating neurologic disease known as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), but it is also associated with several inflammatory disorders like HTLV-1 related uveitis, chronic respiratory diseases, strongyloidiasis, and infective dermatitis associated with HTLV-1 (IDH)  and dyslipidemia. Parasitological analysis was negative. The patient was treated with topical corticosteroids and topical calcineurin inhibitors for skin lesions; topical salicylic acid, coal tar, and betamethasone lotions for the scalp. He also received antihistamines and antibiotics, such as cephalosporins, T/S. On antibiotic courses he experienced a good response, but relapsed after discontinuation.At the age of seven, he was hospitalized due to exacerbation of his symptoms, skin infection and blepharoconjunctivitis which improved with empiric antibiotics. Blood analysis was within normal values and both blood and urine cultures were negative for fungi and bacteria. Inverted psoriasis was suspected. Skin biopsy revealed psoriasiform dermatitis with epidermal hyperplasia, parakeratosis with few polymorphonuclear cells, acanthosis and hypergranulosis, elongations of vascular connective axes, edematous dermis with mononuclear inflammatory infiltrates and isolated melanophages. At the age of eight, due to the persistence of his psoriasiform-like dermatitis, narrow-band ultraviolet B (NB-UVB) therapy was initiated, and subsequently methotrexate. There was no favorable response to any of them so treatment was discontinued. Skin affection impacted not only on the child's school attendance but also on social interactions. The child's embarrassment and some discrimination episodes prevented him from participating in physical activities and social meetings, and he didn't like to be photographed.+ lymphocyte population, compatible with viral infection. Thus, HTLV-1 infection was suspected and confirmed at INBIRS Institute in Buenos Aires. Specific antibodies anti-HTLV-1 were detected by enzyme-linked immunoassay (ELISA) , and infection was confirmed by nested polymerase chain reaction  after DNA extraction . PVL was determined to be 11.5 copies/100 peripheral blood mononuclear cells (PBMC) by quantitative PCR (qPCR) . His mother was also found positive for HTLV-1, but none of his siblings. Human immunodeficiency virus (HIV) and other immunodeficiencies were excluded. IDH was confirmed based on La Grenade's modified criteria  were normal. Nonetheless, IgE levels increased . Cytokines in plasma were also analyzed. An increase in interleukin (IL)-6 and IL-9 concentrations was determined while no increase in plasma levels of Th1 , Th2  or Th17  cytokine profiles was observed by LEGENDplex immunoassay (BioLegend). Specific allergen radio allergo sorbent test (RAST) was requested, with a positive result for Dermatophagoides. A specific immunotherapy was indicated with slight improvement.After these events, at the age of 11 in 2016, he was re-evaluated at the dermatology unit. Infective dermatitis was suspected. A new skin biopsy revealed once again spongiosis and acanthosis, but with a perivascular lymphocytic infiltrate. Phenotypical analysis was performed by flow cytometry, which showed a decreased ratio of naive 49.6%) vs. memory effector (20.6%) CD89.6% vs. Staphylococcus aureus decontamination was periodically indicated with mupirocin 2% ointment and chlorhexidine. The clinical status evolved with recurrences but favorably. The patient continued to present lower intensity exudative and crusty eczema in folds, scalp, and retroauricular regions; generalized papular eruption involving the face and trunk.Once diagnosed, appropriate chronic low dose treatment  was indicated to avoid bacterial superinfection of the skin. T/S, minocycline and clindamycin were used according to local resistance patterns. On chronic antibiotic therapy, no further hospital admissions were required. Prophylaxis with T/S was started and the patient experienced partial remission although relapses of folliculitis and erythematous scaly crusty lesions were present. For acute skin infections, 10-day courses of these drugs or clindamycin were alternated. At the age of 14, in 2019, a complete urine test showed glomerular hematuria (>50% dysmorphic red blood cells) and significant proteinuria. Blood pressure was always within normal limits. He was then followed up in the nephrology department. A significant increase in the PVL values (22 copies/100 PBMC) was observed when compared to the first measurement in 2016.At the age of 15 (in 2020), prophylaxis with minocycline was prescribed, during which period less soft tissue infections were observed. However, the patient still suffered from relapsing chronic dermatitis, presenting recurrences when discontinuing or lowering the antibiotics dose, especially in hot and humid weather. Minocycline was interrupted 6 months later because of a photosensitivity reaction.Currently, the severity of outbreaks is lower  and the patient leads a normal social life. He started practicing physical activities and playing soccer. It should be noted that the patient's dyslipidemia improved with changes in lifestyle and diet. At present, the patient remains under multidisciplinary monitoring. New blood and urine controls, PVL determination, and a renal biopsy have been scheduled. A brief outline of the chronology of this case is provided in We present the first pediatric case of infective dermatitis associated with HTLV-1 in Argentina. A large number of articles on epidemiology and immuno-virology associated with ATLL and HAM/TSP have been published in the scientific literature; however, there are few reports about IDH. Besides, only 5% of HTLV-1 infected individuals develop associated diseases, suggesting host genetic risk factors, such as human leukocyte antigen (HLA) haplotypes –12. DiffMother to child transmission through breastfeeding has been associated with IDH in childhood and ATLL in adults . There iThis patient was breastfed until the age of five by his mother who was unawarely living with HTLV-1. This factor represents a great risk for both diseases, IDH and ATLL. High PVL values have been associated with an increased risk of progression to HAM/TSP –21. HoweRegarding the renal affection, it has been recently hypothesized that the chronic inflammatory state associated with HTLV-1 infection may be involved in its development . However+CD25+CCR4+ T cells. Tax and HTLV-1 bZIP factor (HBZ) are viral products associated with oncogenic transformation. They regulate transcription of viral proteins, host factors and cellular signaling pathways, resulting in the development of HTLV-1 associated diseases. HTLV-1 infection induces cytokine and chemokine deregulation. Increased concentrations of IL-1β, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), CXCL10 and IFN-γ have been reported in HAM/TSP cerebrospinal fluid, and recently, an increased concentrations of IFN-γ in plasma have been reported to be a biomarker of progression to the intermediate syndrome status and HAM/TSP  but Th9 cells have also been involved in inflammatory and neoplastic disorders of the skin such as psoriasis and cutaneous T cell lymphoma  motif in its proximal promoter  are pathognomonic of ATLL, so peripheral blood smear is also relevant .HTLV-1 is a neglected virus, recently recognized by the World Health Organization in the last report published in March 2021 . AntibodAn accurate diagnosis of IDH in infants is relevant to indicate the appropriate treatment and to monitor the possible development of other systemic related diseases . It is aThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.PB: data collection and analysis, writing, edition, and revision of the manuscript. ND: data analysis and revision of manuscript. LA, II, LP, ML, and MBe: data collection and revision of the manuscript. CB and MBi: conceptualization, supervision, writing, edition, and revision of the manuscript. All authors read and approved the final manuscript.This work was supported by the Argentinean Ministry of Science, Technology and Innovation (grant number PICT 2019-1633).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


More numbers of selective serotonin reuptake inhibitors (SSRIs) , were dispensed than other classes of antidepressants, with escitalopram (26%) at the top of the list. Preponderance of prescription of antidepressants for non-mental health diseases was observed. Population-level prescription trends, as we reported here, when combined with patient genetic variability and outcome data, will have the power to predict the potential for treatment failures and adverse effects of these medications in the population. We also recommend educating non-mental health prescribers about the adherence to evidence and guidelines to ensure patient safety while prescribing antidepressants.Studying the prescription pattern of medications will help in understanding potential unnecessary prescriptions, due to the trial-and-error method of prescribing, and the need for personalized medicine in a population. Therefore, in this study, our aim was to explore the prescribing pattern and off-label use of antidepressants in the Qatari population. We conducted a retrospective study of Qatari patients who received prescriptions for antidepressants from the major healthcare providers in Qatar, for a period of 24 months between June 2018 and May 2020. The number of patients, prescriptions, and diagnostic indications were analyzed. The chi-square test was used for identifying statistically significant association of the number of individuals prescribed with age category or gender. Of the 14,601 Qatari patients who were prescribed antidepressants, the majority were female (61%,  The burden of mental illness is increasing across the world; more than one in ten are estimated to live with a mental health disorder (MHD) . In the An increase in the usage of antidepressants over the years was observed. For example, the introduction of selective serotonin reuptake inhibitors (SSRIs) in the late 1980s resulted in increased prescriptions of antidepressants, which doubled in 10 years in western countries . An incrIt is a major concern that some patients may not respond as effectively to medications, but have adverse effects. In different types of MHDs, only 30% of patients reach full remission, and continue their medications ,14,15,16CYP2C19 and CYP2D6, which are important in the metabolism of several antidepressants [Our recent investigation of the pharmacogenomic profile of an apparently healthy adult Qatari population, from whole genome sequencing data, identified that a large proportion carry variants in genes, ressants ,21. Howeressants . TherefoWe conducted a retrospective database study of Qatari patients who received prescriptions for antidepressants from the major healthcare providers in Qatar, Hamad Medical Corporation (HMC), and Primary Healthcare Corporation (PHCC), for a period of 24 months between June 2018 and May 2020. The inclusion criteria for the study were (a) Qatari patients who were visiting outpatient clinics at HMC and PHCC between June 2018 and May 2020, and (b) prescribed antidepressants during the study period. Exclusion criteria included (a) non-Qatari patients, and (b) patients who were hospitalized (inpatients). Data points collected and the medications targeted are provided in the Anatomical Therapeutic Chemical classification (ATC) codes associated with the drug categories were obtained from the database of WHO Collaborating Centre for Drug Statistics Methodology  and CPICWe performed the chi-square test for the association between the drugs and the age category, and for the association between the drugs and gender. Prescription counts were summarized using balloon graphics, where the size of each balloon represented the magnitude of the count at a given point, while crossing the two variables. R statistical package was used for the analysis and plotting.p < 2.2 × 10−16). The majority of the antidepressant prescriptions were given to patients at or above 60 years of age , showing statistically significant differences among the age groups (chi-square p < 2.2 × 10−16) , and the gender difference was statistically significant (chi-square × 10−16) . Six non× 10−16) a.There were 48,031 antidepressant prescriptions dispensed for Qataris over the 24 months. SSRIs were the most highly prescribed , followed by ‘other antidepressants’ (ATC: N06AX) , and tricyclic antidepressants . Specifically, escitalopram , was the most commonly dispensed, followed by duloxetine  and amitriptyline  b.There were 8386 diagnostic indications recorded, which may be classified into mental health disorders (MHDs), and 6503 as non-MHDs. The prescription of duloxetine, amitriptyline, and imipramine were especially more for non-MHDs compared to MHDs, while several other antidepressants, including escitalopram, were also prescribed for non-MHDs .Major diagnostic indications for MHDs included depression  and anxiety , followed by insomnia , obsessive compulsive disorder  and schizophrenia  a. EscitaNon-mental health diagnoses observed for antidepressants included different types of chronic pain , chronic diseases , neuropathy , migraine , irritable bowel syndrome , and fibromyalgia , among others a. AmitriFor the on-label and off-label use of antidepressants, we found that 48% of prescriptions were following the United States Food and Drug Administration (FDA) approved indications while the majority of the indications of antidepressants (52%) were for FDA unlicensed use, such as for controlling chronic diseases, sleeping disorders, and schizophrenia.p = 3.45 × 10−6). Age category also showed statistically significant association with the number of patients prescribed (chi-square p < 2.2 × 10−16), with those ≥60 years receiving the greatest number of prescriptions  . Of the 3395 patients, there were more women than men who were prescribed antidepressants at MHH during the 24 month period  . When th96; 23%) b. The moIn our study of the prescription patterns in the Qatari population, we found that antidepressants were prescribed more for women and ages at or above 60 years. A Swedish study also reported that prescribing of antidepressants was high in women and aged 45–64 years . Indeed,SSRIs were the major class of antidepressants prescribed in our cohort. This finding is in parallel with studies from other countries . HoweverSome interesting patterns were also observed in the prescription numbers. There were distinctive dips in the month of August in both years, coinciding with the holiday season, characterized by the large outflow of residents due to the extreme hot temperatures in the country. A distinct dip was also observed in May 2020, especially in the number of prescriptions from the MHH, due to closure of clinics following the COVID-19 outbreak.For antidepressants, when FDA approved indications (on-label and off-label use) were considered, our study found variations in the most commonly prescribed medications between the Mental Health Hospital (MHH) and different facilities at the general hospitals in the HMC and PHCC. The differences are due to the use of some antidepressants, especially TCAs, for indications such as migraine, neuropathic pain, and different types of pain management. Both TCAs and serotonin-norepinephrine reuptake inhibitors (SNRIs) possess analgesic qualities, while the evidence for the effectiveness of SSRIs is weaker. Several studies have shown that antidepressants were effective in relieving neuropathic pain. Antidepressants might also provide effective pain relief for other painful conditions, such as chronic musculoskeletal pain and fibromyalgia. Since the pain relief effects of antidepressants seem to occur earlier, and at lower doses than for antidepressant effects, it was suggested that the mechanism of action to control the pain is separate from their antidepressant effects. However, none of the TCAs was approved by the FDA for any pain management indications since their mechanisms of action are unclear.Duloxetine is the only antidepressant that has FDA approval for the treatment of neuropathic pain and chronic musculoskeletal pain. In addition, duloxetine and milnacipran were approved for fibromyalgia. Depression and chronic pain are conditions having some overlap in their symptoms. A positive association between depression and chronic pain was also observed. An analysis of data from the National Health and Nutrition Examination showed that 90% of patients with depression were also having pain symptoms . The linIn Qataris, amitriptyline was the most highly prescribed antidepressant for different types of pain, followed by duloxetine and escitalopram. In the case of neuropathy, 60% of the patients were prescribed duloxetine followed by amitriptyline (34%). An off-label use of amitriptyline was for treating migraine, followed by escitalopram. According to the American Academy of Neurology and the American Headache Society guidelines, amitriptyline was considered for migraine prophylaxis, the only antidepressant with consistent evidence supporting its effectiveness for this use. In addition, studies found that amitriptyline was more beneficial for patients with mixed migraine and tension features .Antidepressants were also used in Qatar to treat some patients with irritable bowel syndrome (IBS). Meta-analyses of cumulative controlled experience have confirmed the efficacy of antidepressants in IBS and other functional gastrointestinal disorders . AccordiAnother common off-label use highlighted in our study was the use of antidepressants, especially amitriptyline and mirtazapine, for sleeping disorders. The use of antidepressants for treating sleeping disorders, particularly insomnia, is widespread. Nevertheless, none of the antidepressants is licensed for treating sleeping disorders, except doxepin, which is the only TCA that was approved by the FDA for the treatment of insomnia. This type of use for antidepressant medications may be driven by concern over longer-term use of hypnotics and the limited availability of psychological treatments for insomnia .Interindividual variability in response to antidepressants can affect the treatment outcome and lead to potential adverse effects. Pharmacogenomics testing of variants known to be associated with such variability can help in the personalized prescription or dosage adjustments for increased efficacy and reduced toxicity . ConsortCYP2C19 and CYP2D6 [Furthermore, our recent investigation of the pharmacogenomic landscape of the Qatari population identified the high prevalence of actionable variation in the genes, d CYP2D6 ,21. SincIt is also worth noting that, with the emergence and proliferation of direct-to-consumer genomic testing in some parts of the world, more patients are testing for genetic markers, for response to psychotropic medication, at a very high cost, with (currently) little clinical impact. In particular, the younger generation, who are more aware, often ask for such tests or enquire about them, preferring to have a confirmed/evidence-based personalized medication selection rather than trial and error.With the accumulation of big datasets generated from multiple technologies, and advancements in artificial intelligence and machine learning methodologies, the field of psychiatry is also progressing towards precision and personalized medicine approaches . Populat