P < 0.001, HR 4.48, 95% CI 2.62–7.65) and TERT overexpression  were significant prognostic factors for IHR-free survival in both univariate and multivariate analyses. TERT overexpression was the only significant prognostic factor for late IHR in HCC treated with curative resection. And, the statistical significance of TERT overexpression on late IHR was limited to HBsAg-positive patients.This study was designed to investigate the associations between TERT overexpression and the clinicopathologic factors of hepatocellular carcinoma (HCC). A total of 291 patients with HCC were enrolled. The site of first recurrence (anywhere in the liver) was classified as intrahepatic recurrence (IHR). Recurrence was then sub classified as either early or late IHR according to whether it was discovered within 2 years of resection, or after, respectively. TERT overexpression was not significantly correlated with previously recognized prognostic factors. During follow-up, early IHR occurred in 126 (63.6%) patients, while late IHR was detected in 59 patients among 145 patients who remained free of HCC recurrence for ≥ 2 years after surgery. Multivariate analysis showed late IHR was significantly correlated with TERT overexpression . Intrahepatic metastasis ( Currently, HCC is the fifth most common cancer, and the second leading cause of cancer-related death worldwide2. One study found that the local control rate of HCC was comparable between surgical resection and RFA3. Surgical resection is generally preferred, if possible, however, because of the difference in overall recurrence after surgery compared to that after other treatment modalities6. Unfortunately, recurrence is still detected in approximately 50% of patients after surgical resection7. There is no standardized adjuvant treatment modality after HCC surgical resection. Therefore, it is very important to identify reliable biomarkers of recurrence, as well as the most probable sites of recurrence, in order to develop customized management plans.The Barcelona Clinic Liver Cancer (BCLC) system is the most widely used HCC staging and treatment guideline. According to BCLC, the curative treatments for HCC include liver transplantation (LT), surgical resection, and local ablative therapy, including radiofrequency ablation (RFA)8. In most somatic cells, TERT expression is suppressed. In contrast, TERT is actively expressed in self-renewing cells, such as stem cells9. Overexpression of TERT and telomere dysfunction is frequently detected in a variety of human cancer specimens, including thyroid cancer10, bladder cancer11, melanoma12, and brain tumors13. This TERT overexpression is detected in up to 90% of cancer cells, compared to in <20% of normal cells14. Evidence has suggested that TERT overexpression in HCC is associated with poor clinical outcomes; however, this hypothesis was based on a small patient population, without consideration of other clinicopathologic factors that might affect clinical outcomes15. Therefore, it is essential to more precisely evaluate the role of TERT overexpression in HCC, as well as that of other clinicopathologic factors in homogenously-treated patients.Telomerase reverse transcriptase (TERT) is a core catalytic component of telomerase that plays a crucial role in maintaining telomere lengthWe investigated the associations between TERT overexpression and the clinicopathologic factors of HCC in Korean patients mainly caused by HBV infection. We also studied the clinical significance of TERT overexpression with regard to HCC recurrence and survival durationThe patients and tumor characteristics of the 291 enrolled cases of HCC are displayed in Table P < 0.001, Fig. P-values 0.0006 for both cut-off points). Diagnostic accuracy of TERT mRNA of 0.3 to predict recurrence and survival was displayed in Supplementary Table Gene expression analysis using quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR) revealed that TERT messenger RNA (mRNA) expression was significantly higher in HCC tissues than it was in background, non-tumor liver tissue (at least 2 cm from the HCC) and normal liver tissues. Normal liver tissue was obtained from patients who underwent hepatectomy due to pathologically-confirmed metastatic colon cancer to the liver without a history of viral hepatitis or alcohol use P < 0.00, Fig. 1.P = 0.67), microvascular invasion (P = 0.91), intrahepatic metastasis (P = 0.40), and background liver status (P = 0.89).The correlations between elevated TERT mRNA expression and clinicopathologic variables are displayed in Supplementary Table During the follow-up period, overall recurrence was detected in 198 (68.0%) patients. Among them, 103 (35.4%) patients had isolated intrahepatic recurrence (IHR), 82 (28.2%) had both IHR and distant metastases (DM), and the remaining 13 (4.5%) had isolated DM. Among all of the patients with IHR (with or without DM), early IHR developed in 126 (63.6%) patients. In contrast, late IHR was detected in 59 of the 145 patients that were followed for over 2 years. The estimated recurrence rate (per month) after surgical resection is displayed in Fig. P = 0.02, hazard ratio [HR] 2.81, 95% confidence interval [CI] 1.20–6.60), and intrahepatic metastasis . Early IHR was somewhat related to TERT expression, although the trend was not statistically significant .The results of univariate and multivariate analyses of clinicopathologic variables and early/late IHR are displayed in Tables P = 0.72), intrahepatic metastasis (P = 0.75), and BCLC stage (P = 0.33). Multivariate analysis showed TERT expression  and HBsAg  were the only significant prognostic factors for late IHR. In the subgroup analysis by HBsAg status, late IHR was significantly associated with TERT overexpression in HBsAg-positive patients , but not in HBsAg-negative patients . The monthly IHR rate according to probable prognostic clinicopathologic factors is displayed in Fig. In contrast, late IHR was not significantly correlated with any variables, including those known to be significant prognostic factors of HCC, including microvascular invasion ; however, the effect of TERT expression  on RFS increased with time.Multivariate analysis Table  showed sP < 0.001), an even clearer stratification was identified for IHRFS and RFS. According to the risk grouping, the median IHRFS was not reached in group 1, was 50.3 months  in group 2, 10.3 months  in group 3, and 3.4 months in group 4 . Similarly, RFS was not reached in group 1, but was 42.5 months  in group 2, 9.4 months  in group 3, and 3.41 months in group 4 .We performed predictive grouping of IHRFS according to the significant or marginally significant factors  using the following criteria: group 1, no risk factor; group 2, one risk factor; group 3, two risk factors; and group 4, all three risk factors. The curves for IHRFS, DMFS, RFS, and overall survival (OS) according to the risk groupings are shown in Fig. In this study, we evaluated the correlation between clinicopathologic variables and the prognostic significance of TERT overexpression in HCC treated with curative surgical resection. Interestingly, TERT overexpression was not related to other clinicopathologic variables, including well-known prognostic factors for HCC. Instead, TERT overexpression was significantly correlated with late IHR . The previously identified prognostic factors for HCC were only related to early IHR in our study. Furthermore, the prognostic significance of TERT overexpression on overall IHRFS was maintained in multivariate analysis.16. In most normal somatic cells, TERT expression is suppressed and strictly controlled by transcriptional regulation; in some self-renewing cells such as stem cells and/or reproductive cells, however, TERT is expressed more ubiquitously9. There has been a lot of interest in understanding the pivotal role of increased TERT-mediated telomere maintenance in HCC carcinogenesis17. The mechanisms by which TERT expression is increased in HCC may include TERT gene amplification18, and TERT promoter mutations20.The TERT is the most important unit of the telomerase complex, which allows potentially unlimited cell proliferation through the addition of repeated TTAGGG sequences and subsequent telomere maintenance17, TERT abnormalities also have prognostic significance in thyroid cancer10, bladder cancer21, and brain tumors13. In HCC, however, there are few studies defining the role of TERT expression as a prognosticator for the disease. It also remains to be determined whether TERT expression during recurrence reflects metastasis or a new primary lesion. Therefore, understanding the crucial role of TERT expression in HCC carcinogenesis17, as well as its clinical significance on metastasis and/or de novo primary HCC is extremely important for optimizing HCC management.In addition to HCC carcinogenesisThis study reveals the prognostic significance of TERT expression on IHRFS. With the exception of other potential confounders during multivariate analysis, TERT expression demonstrated an HR of 1.78 on overall IHRFS. Although the HR of TERT expression on IHRFS was lower than that of intrahepatic metastasis HR 4.22), it still suggests that it is an independent prognostic factor for overall IHRFS. There was no statistically significant correlation between intrahepatic metastasis and TERT expression  between July 2000 and May 2006 was used in this present study. Patients were eligible if they underwent complete surgical resection with curative intent. Complete resection was defined as the removal of all hepatic tumor nodules with microscopic clear resection margins. Any patients who received neoadjuvant or adjuvant treatments were excluded. The Institutional Review Board at Samsung Medical Center approved this study, and waived informed consent.Prior to surgery, patients underwent a work-up of their liver function and HCC status. This examination included the following: comprehensive history, patient demographics, viral marker, chest X-ray, complete blood count, chemistry profile, liver function test, indocyanin green retention test, AFP level, and contrast enhanced liver CT and/or magnetic resonance imaging (MRI) of the abdomen and pelvis. Chest CT and whole body bone scan were performed to rule out distant metastasis. Positron emission tomography (PET)-CT was alternatively used for the same purpose at the physician’s discretion.Patients were generally followed every two to three months after surgical resection. During the follow-up period, evaluations included physical examinations, laboratory tests (including liver function tests and AFP), chest X-rays, and CT of the abdomen and pelvis. Patients with suspicious imaging findings and/or continuously elevated AFP levels were further evaluated with PET-CT and/or MRI.16. The resected HCC tissues were graded according to the Edmonson and Steiner grading system: grade I (well differentiated), grade II (moderately differentiated), or grade III (poorly differentiated)30.Based on the Liver Cancer Study Group of Japan guidelines, the following histological parameters were postoperatively assessed: largest tumor diameter, number of tumors, microvascular invasion, and intrahepatic metastasisTM FFPE Total RNA Miniprep System . RNA was reverse transcribed using a high-capacity, complementary deoxyrinuleic acid (cDNA) Reverse Transcription Kit  according to the manufacturer’s instructions. The expression of TERT was measured using a gene expression assay containing forward and reverse primers, and a FAM-labeled MGB TaqMan probe from Applied Biosystems  for the detection of overlap at the exon 3-exon 4 junctions. GAPDH was used as an endogenous control . The PCR reaction mixture contained TaqMan Universal PCR master mix with AmpErase UNG , 900-nM primers (forward and reverse), 250 nM TaqMan probe, and 5 μl of cDNA sample for a total reaction volume of 20 μl. The PCR conditions were as follows: 95 °C for 10 min followed by 40 cycles of amplification at 95 °C for 15 s, and 60 °C for 1 min using the ABI PRISM 7500HT Fast Real-time PCR system (Applied Biosystems). The threshold cycle (Ct), which is the fractional cycle number at which the amount of amplified target reached a fixed threshold, was determined. The relative changes in gene expression were measured using the 2−ΔΔCt (ΔΔCt = ΔCttarget gene − ΔCtGAPDH) method.The total RNA was isolated from fresh samples using the ReliaPrepet al.31. All other sites of recurrence were defined as DM.Recurrence was generally diagnosed using radiologic examinations without histologic confirmation, except in patients for whom further surgical resection or liver transplantation was indicated. The site of first recurrence was classified as IHR, which is defined as recurrence occurring anywhere inside the entire liver. The IHR was sub classified as either early recurrence or late recurrence according to whether it was detected <2 years or ≥2 years after the initial surgery, respectively. This cut-off point was based on the study by Imamura The duration of IHRFS, DMFS, RFS, and OS were calculated from the date of surgical resection to the date of each event, or the last day of follow-up.P-values < 0.05 were considered statistically significant in two-tailed tests. The correlation between high TERT expression or intrahepatic metastasis and other clinicopathologic variables was evaluated using the Chi-square or Fisher’s exact tests. Univariate analysis was performed using the Cox proportional hazard model or the time-dependent Cox model according to satisfaction of the proportional hazard assumption for each variable. The proportional hazard assumption was confirmed using the correlation between partial residuals from the estimated Cox proportional hazard model and the time to the event. Variables with P < 0.1 in univariate analysis were included in the multivariate analysis. Multivariate analysis was conducted using a time-dependent Cox model, because some variables included in the multivariate analysis were against the proportional hazard assumption. Multicollinearity was identified by a Variance Inflation Factor (VIF) > 4.0. Some of those variables were excluded from the multivariate analysis. Statistical analyses were performed using SAS version 9.4 . Variable risk was expressed as an HR with corresponding 95% CI.The cut-off value of TERT mRNA expression on overall recurrence was determined using the log-rank test and the minimum p-value approach. For internal validation of the estimated cut-off value of TERT expression level, the distribution of the cut-off values was examined using 1000 bootstrap samples with replacement from the study data. Supplementary file


C. striatus in chronic unpredictable mild stress (CUMS) model of depression in male rats and its mechanism of action. The animals were subjected to CUMS for six weeks by using variety of stressors. At the end of CUMS protocol, animals were subjected to forced swimming test (FST) and open field test followed by biochemical assay. The CUMS protocol produced depressive-like behavior in rats by decreasing the body weight, decreasing the sucrose preference, and increasing the duration of immobility in FST. The CUMS protocol increased plasma corticosterone and decreased hippocampal and prefrontal cortex levels of monoamines  and brain-derived neurotrophic factor. Further, the CUMS protocol increased interleukin-6  and nuclear factor-kappa B . The lipid extract of C. striatus  significantly (p < 0.05) reversed all the above parameters in rats subjected to CUMS, thus exhibiting antidepressant-like effect. The mechanism was found to be mediated through decrease in plasma corticosterone, increase in serotonin levels in prefrontal cortex, increase in dopamine and noradrenaline levels in hippocampus and prefrontal cortex, increase in BDNF in hippocampus and prefrontal cortex, and decrease in IL-6 and NF-κB in prefrontal cortex.This study evaluated the antidepressant-like effect of lipid extract of Acute stress and chronic stress have different effects on human health. Acute stress prepares body for “fight or flight” situation and is beneficial for the survival, while chronic stress may have opposite and deleterious effects , 2. ChroChronic stress has an influence on neuroendocrine responses in men . An animChronic uncontrolled stress increases the allostatic load of an organism and impairs homeostasis, thus causing overactivity of some systems and underactivity of some systems in the body . Major dChanna striatus  is a freshwater snakehead fish indigenous to Malaysia [ C. striatus belongs to the family Channidae. Its flesh is included in postparturition diet as a rejuvenating diet and to aid wound healing in local Malay population [ C. striatus showed significant antidepressant-like effect in our previous experiments [ C. striatus may have antidepressant-like effect in CUMS model of depression. Hence, this study aimed to evaluate the antidepressant-like effect of C. striatus extract in CUMS model of depression and the possible mechanism of action.Malaysia . C. stripulation . Aqueouseriments , 31. TheMale Sprague-Dawley rats, approximately 6–9 weeks old, weighing between 150 and 190 g were used. The animals were sourced from Takrif Bistari Enterprise, Seri Kembangan, Selangor, Malaysia. All the animals used in this study were cared for and treated humanely in accordance with the protocols specified by the Institutional Animal Care and Use Committee, UPM, and also with the “Principles of Laboratory Animal Care” . The animals were housed for 2 weeks under controlled conditions for acclimatization before the experiments. These conditions were as follows unless otherwise specified: light: 12 h light/dark cycle, lights on at 7:00 am; temperature 25 ± 1°C; free access to food and water. The animals were randomly assigned to different groups for the experiments, namely, no stress , CUMS control , CUMS + fluoxetine 10 mg/kg , CUMS + lipid ext 125 mg/kg , CUMS + lipid ext 250 mg/kg , CUMS + lipid ext 500 mg/kg , and no stress + lipid ext 250 mg/kg . All efforts were done to minimize the number of animals used in the experiment. All the experimental protocols were approved by Institutional Animal Care and Use Committee, UPM (UPM/IACUC/AUP-R042/2013).Chronic unpredictable mild stress was applied in rats for a total duration of 6 weeks based on the previously established protocols , 14 withThe noise and cage tilting stressors were applied randomly at any time of the day. The overnight illumination was carried out from 7.00 pm to 7.00 am the next day. The order of the stressors was randomized in order to avoid any habituation effect. The sucrose preference test was conducted on every Sunday morning between 9 and 10 am. The sucrose preference test requires food and water deprivation 24 hr before the test to avoid any nonspecific influence of diet on sucrose consumption . Hence,  Channa striatus (2 kg) was procured from local wet market in Selangor, Malaysia. The fish were identified by Dr. Mohd Shafiq Bin Zakeyuddin, Research Assistant, Department of Environmental Management, Faculty of Environmental Studies, Universiti Putra Malaysia, Malaysia, by following previously published data [Thehed data , 36. A vhed data . The flehed data . One groThe sucrose preference test was conducted based on previously published protocols , 34 withThe forced swimming test was conducted based on the original method  with sliThe spontaneous locomotor activity was evaluated by following previously described methods . The appThe OFT was conducted at the end of CUMS protocol period on the day of decapitation, before FST. The animals were transferred to OFT test room and acclimatized for 1 hr. The animals were placed onto the center of the box and allowed to explore the box freely for 5 min. After conducting the test on each animal, the box was cleaned with dry tissue paper first and later with 70% alcohol solution and allowed to dry in air to avoid the influence of urine and feces of the previous animal on the next animal. The top view of the box was recorded by a video camera mounted on the ceiling. The video recordings were later scored by an observer blind to the treatment regimen for number of squares crossed and number of rearings. After the OFT, the same animals were tested in FST.At the end of CUMS protocol, 20 min after the FST , the aniThe brain was dissected out quickly and carefully and placed on an ice-cold plate. The prefrontal cortex and hippocampus of both sides of the brain were carefully removed , 45. TheDuring the homogenization, the Eppendorf tube containing tissue sample was maintained in an ice-cold environment. The homogenates were stored at −20°C overnight and thawed. After two freeze/thaw cycles to break the cell membranes, the homogenates were centrifuged for 5 min at 5000 ×g at 2–8°C. The supernatants were collected and used immediately for ELISA analysis and protein determination or stored at −80°C until analysis. For analysis, either the right or left hippocampus or prefrontal cortex was analyzed. The sample consisted of equal number of right and left hemisphere parts to counterbalance the lateral effects of brain. This procedure was adapted in protein assay as well as in biochemical assays.The protein determination was carried out to standardize the expression of results of marker proteins per g of wet brain tissue. Protein concentration was determined in hippocampus and prefrontal cortex tissues of rats by using a protein assay kit from Bio-Rad, CA, USA, with dye reagent concentrate  and bovine serum albumin as standard . Microassay standard procedure was used. The principle of protein assay is based on Bradford's method . The homκB), and brain-derived neurotrophic factor (BDNF) by using separate enzyme-linked immunosorbent assay (ELISA) kits as per the manufacturer's instructions . Briefly, a calibration curve was constructed by using the given standards. Each standard and sample were analyzed in triplicate and mean ± SEM were calculated and used for analysis. The unknown sample concentrations were interpolated from the standard curve and expressed with respect to per gram of protein for IL-6, NFκB, and BDNF.The plasma was analyzed for corticosterone and oxytocin levels by using ELISA kits as per the manufacturer's instructions . The homogenates from hippocampus and prefrontal cortex were analyzed for serotonin, dopamine, noradrenaline, interleukin-6 (IL-6), nuclear factor-kappa B  in no stress group, no stress group treated with 250 mg/kg of lipid extract, and groups subjected to CUMS protocol and treated with fluoxetine, 125, 250, and 500 mg/kg doses of lipid extract, when compared to CUMS control group  when compared to CUMS control group  in their sucrose preference when compared to no stress group (p < 0.01) decrease in sucrose preference until week 6  increased the sucrose preference at week 6 when compared to CUMS control group (p < 0.05) increased the sucrose preference at week 5 and week 6 when compared to CUMS control group . Further post hoc analysis revealed that the CUMS control group had significant (p < 0.05) increase in duration of immobility in FST when compared with no stress group (p < 0.001) decrease in the duration of immobility when compared with CUMS control group (p < 0.001) when compared to CUMS control group (p < 0.05) in immobility when compared with no stress group.In FST, one-way ANOVA indicated significant difference between the treated groups  and in number of rearing instances . Further post hoc analysis indicated that the CUMS control group had significant (p < 0.05) decrease in number of squares crossed . Further post hoc analysis indicated that the CUMS control group had significant (p < 0.01) increase in plasma corticosterone level when compared to no stress group (p < 0.01) decreased plasma corticosterone level when compared to CUMS control group (p < 0.05) decreased the plasma corticosterone level in stressed rats when compared to CUMS control group  between all the groups  in hippocampal serotonin levels and significant difference  in prefrontal cortex serotonin levels between the treated groups. In further post hoc analysis, the CUMS control group showed significant (p < 0.01) decline in hippocampal (p < 0.05) increased serotonin level in hippocampus (p < 0.001) increased serotonin level at 250 and 500 mg/kg when compared to CUMS control group  in hippocampal noradrenaline levels and significant difference  in prefrontal cortex noradrenaline levels between the treated groups. In further post hoc analysis, the CUMS control group showed significant (p < 0.05) decline in hippocampal (p < 0.001) increase in noradrenaline level in hippocampus when compared to CUMS control group (p < 0.05) increased noradrenaline level at 250 mg/kg dose only when compared to CUMS control group (p < 0.05) increased noradrenaline level in hippocampus  in hippocampal dopamine levels and significant difference  in prefrontal cortex dopamine levels between the treated groups. In further post hoc analysis, the CUMS control group showed significant (p < 0.05) decline in hippocampal (p < 0.01) increase in dopamine level in hippocampus when compared to CUMS control group (p < 0.05) increased dopamine level in hippocampus  in hippocampal BDNF levels and significant difference  in prefrontal cortex BDNF levels between the treated groups. In further post hoc analysis, the CUMS control group showed significant (p < 0.05) decline in hippocampal (p < 0.05) increase in BDNF level in hippocampus (p < 0.001) increased BDNF level in hippocampus  in hippocampal IL-6 levels and significant difference  in prefrontal cortex IL-6 levels between the treated groups. In further post hoc analysis, the CUMS control group showed significant (p < 0.001) increase in hippocampal (p < 0.05) decrease in IL-6 levels in hippocampus (p < 0.001) reduced it in prefrontal cortex  and in prefrontal cortex NF-κB levels  between the treated groups. In further post hoc analysis, the CUMS control group showed significant (p < 0.01) increase in NF-κB levels in prefrontal cortex (κB levels in hippocampus (p < 0.001) decrease in NF-κB levels in prefrontal cortex at 125, 250, and 500 mg/kg  reduced it in prefrontal cortex  increased the decreased monoamines caused by CUMS protocol in both hippocampus and prefrontal cortex of rats, similar to a previously reported finding . The lip C. striatus had significant effect on the brain monoamines and this influence on monoamines may be attributed to its antidepressant-like effect in FST and sucrose preference test consistent with the previously reported findings on fish oils [ C. striatus.These results suggest that the lipid extract ofish oils , 76. Furish oils , our finα-linolenic acid (1.10% w/w) (data not shown). Considering the impact of unsaturated fatty acids on the monoaminergic neurotransmission [The lipid extract was found to contain oleic acid (23.38% w/w) and smission , 76, theBDNF is considered as a critical marker and mediator of mood disorders, particularly major depression and assumed to be involved in the etiology, pathogenesis, and treatment response to antidepressants , 78. Chrω-3 fatty acids), upregulation of BDNF was observed in hippocampus [ω-3 fatty acids and supporting the antidepressant-like effect observed in sucrose preference test and FST.Fish oil (12% EPA and 18% DHA) supplementation at 3 g/day was reported to produce increased BDNF expression in hippocampus and prefrontal cortex of rats . In ratspocampus . Dietarypocampus . Similar C. striatus, especially at 500 mg/kg in both hippocampus and prefrontal cortex, suggesting a potential anti-inflammatory effect of the lipid extract of C. striatus. The lipid extract at 125 mg/kg was ineffective in reducing the IL-6 level both in hippocampus and prefrontal cortex. The anti-inflammatory effect of lipid extract appears to be dose-dependent with maximum effect that occurred at maximum dose.Elevated plasma proinflammatory IL-6 levels were reported in patients with major depression , 86. EviκB is a critical mediator of inflammatory processes [κB activity has been observed in chronic stress [β activate NF-κB signaling to constitute an inflammatory response [κB was found to be involved in the activation of the IL-6 gene [κB in the frontal cortex of rats [κB level and subsequently IL-6 level. Similar results were obtained in our study. The CUMS protocol increased the NF-κB level in both hippocampus and prefrontal cortex. This suggests that the CUMS protocol induced an inflammatory response in hippocampus and prefrontal cortex of rats. Fluoxetine decreased NF-κB level in hippocampus and prefrontal cortex of rats consistent with previous findings [κB activation in prefrontal cortex but not significantly in hippocampus. The decrease in the levels of NF-κB in the prefrontal cortex is very striking. Previous studies showed that ω-3 fatty acids suppressed the NF-κB activation [ C. striatus. Furthermore, our results indicated that similar pattern of response was observed in IL-6 and NF-κB protein levels in prefrontal cortex of rats, suggesting a link between IL-6 and NF-κB, as reported previously [κB in rat prefrontal cortex in chronic stress and depression.NF-rocesses  and uprec stress , 90. Cytresponse . NF-κB wL-6 gene . Munhoz findings , 94. Thetivation , 96. Heneviously . Further C. striatus to inhibit the activation of NF-κB. Inhibition of NFκB activity might have reduced IL-6 synthesis [κB in rat prefrontal cortex and subsequently reduced IL-6 in prefrontal cortex and increased serotonin level in prefrontal cortex, thus suggesting that the action of lipid extract may stem from its ability to inhibit NF-κB and IL-6. However, this cannot be concluded from this study. Further studies are required to explore the effect of unsaturated fatty acids in the lipid extract of C. striatus on the molecular pathways interconnecting serotonin, NF-κB, and IL-6 in prefrontal cortex of rats.Collectively, the mechanisms of action appear to stem from the ability of lipid extract ofynthesis , subsequynthesis . Cytokinynthesis , 99. A rynthesis . Therefo C. striatus in chronic unpredictable mild stress model of depression in male rats through sucrose preference test and forced swimming test. Furthermore, the mechanism of the observed antidepressant-like effect was found to be mediated through decrease in plasma corticosterone, increase in serotonin levels in prefrontal cortex, increase in dopamine and noradrenaline levels in hippocampus and prefrontal cortex, increase in BDNF in hippocampus and prefrontal cortex, and decrease in IL-6 and NF-κB in prefrontal cortex.This study demonstrated the antidepressant-like effect of lipid extract of


Despite the recent increasing worldwide attention towards pulmonary hypertension (PH), its epidemiology remains poorly described in Africa. Accordingly, we performed a systematic review and meta-analysis of PH prevalence, incidence and etiologies in Africa.χ2 test on Cochrane’s Q statistic which is quantified by I2 values. Using Newcastle-Ottawa Scale, we considered a score of 0–4, 5–7, and 8–10 as indicative of high, moderate, and low risk of bias in included studies, respectively.We searched PubMed, EMBASE, African Journals Online, and Africa Index Medicus. Published observational studies until September 20, 2017, including adult participants residing in Africa were considered. Two review authors independently selected studies, assessed included studies for methodological quality, and extracted data. A random-effects model was used for meta-analysis. Heterogeneity was evaluated by the 2 = 99.4%; 6 studies) in 11,163 people presenting with cardiac complaints; 10.6%  in 937 HIV-infected people; 32.9%  in 2077 patients with heart failure; 23.2%  in 248 patients on hemodialysis; 12.9%  in 3750 patients with rheumatic heart disease; 36.9%  in 79 patients with sickle cell disease; 62.7%  in 51 patients with chronic obstructive pulmonary disease; 25.4%  in 63 patients with systemic lupus erythematous; 68.7%  in 259 patients with cardiac surgery; and 7.4%  in 202 patients with systemic sclerosis. No study reported PH incidence. From one international study (n = 209), PH etiologies were: left heart disease (68.9%), pulmonary arterial hypertension (15.8%), lung disease and/or hypoxia (12.0%), chronic thromboembolic PH (1.9%) and unclear/multifactorial PH (15.8%).Of 1611 entries, 25 studies were retained. Twelve (48%), seven (28%), and six (24%) papers had respectively a low, moderate and high risk of bias. The prevalence of PH widely varied across different populations: 9.8%  contains supplementary material, which is available to authorized users. Pulmonary hypertension (PH) is a hemodynamic and pathophysiological condition characterized by abnormally elevated pressures in the pulmonary vasculature. It is defined by a mean pulmonary arterial pressure ≥ 25 mmHg at rest by right heart catheterization . PH can Irrespective of the cause, PH is associated with debilitating symptoms and reduced life expectancy. Late diagnosis and ineffective treatment are the main drivers of its poor survival , 4. For Despite the point made above and the potential risk of highly prevalent PH, the epidemiology of PH remains poorly described in Africa. In fact, there is a lack of studies on PH prevalence from this continent . High prHerein, we present the first systematic review and meta-analysis which synthesizes the current knowledge on the epidemiology of PH in Africa. Our objective was to determine the prevalence, the incidence and the etiologies of PH among people residing in Africa. The ultimate goal of this review is to provide evidence which could guide contextualized policies for the prevention and treatment of PH in Africa, and underpin further research.The MOOSE guidelines served as the template for reporting the present review . This rePublished observational studies until September 20, 2017, including adult (18 years or older) participants residing in Africa were considered. Studies were also considered if the diagnosis of PH was based on right heart catheterization with a mean pulmonary arterial hypertension ≥25 mmHg or Doppler echocardiography examination with pulmonary arterial systolic pressure > 35 mmHg [An expert librarian performed a search of PubMed/MEDLINE, Excerpta Medica Database, African Journals Online and African Index Medicus without any language restriction. The search strategy included the following terms: ‘Africa’, ‘pulmonary hypertension’, and ‘pulmonary arterial hypertension’. Individual country names for the 54 African countries were also used as additional key search terms to identify more abstracts on the subject. References of all relevant original and review articles were scrutinized for additional potential data sources. The main search strategy conducted in PubMed is available in the review protocol .Two review authors (JJB and JJN) independently screened abstracts and then full texts. They consensually retained all studies to be included in the review, and disagreements were solved by arbitration of a third review author (JRN).The Newcastle-Ottawa Scale was used to evaluate the methodological quality of studies included in this review . There iTwo review authors (JJB and JJN) independently extracted data including: first author name, year of publication, year of participants’ inclusion, country, study design, setting, sample size, age distribution, proportion of males, diagnostic criteria for PH, number of cases of PH, and etiologies of PH as classified by international guidelines . Authorsχ2 test on Cochrane’s Q statistic which is quantified by I2 values [2 values of 25%, 50% and 75% respectively represent low, medium and high heterogeneity [p value <0.1 was considered indicative of statistically significant publication bias. Data were analyzed using Stata software . We conducted a narrative synthesis in the case of limited data for a meta-analysis.A meta-analysis was conducted for data obtained from studies in the same population . Standard errors for the study-specific estimates were determined from the point estimate and the appropriate denominators. Then, the study-specific estimates were pooled through a random-effects meta-analysis model, to obtain an overall summary estimate of the prevalence across studies, after stabilizing the variance of individual studies using the Freeman-Tukey double arc-sine transformation . Heterog2 values , assuminogeneity . Inter-rogeneity . Egger’sInitially, a total of 1611 records were identified. After elimination of duplicates, 1543 records were retained. Titles and abstracts were screened and 1470 records were found irrelevant and excluded. Agreement between review authors on abstract selection was moderate (κ = 0.69). Full-texts of the remaining 73 papers were scrutinized for eligibility, among which 48 were excluded  [2 = 99.6%) in a pooled sample of 937 patients from four studies conducted in different countries including Cameroon, Mozambique, South Africa and Tanzania (p Egger = 0.351) [2 = 97.2%) was found in a pooled sample of 2077 patients with heart failure from three studies conducted in Morocco and Nigeria (p Egger = 0.445) [2 = 59.4%) was found in a pooled sample of 248 patients undergoing hemodialysis from three studies conducted in Egypt and Morocco (p Egger = 0.376) [2 = 96.8%) was found in a pooled sample of 3750 patients with rheumatic heart disease from two studies, one from Uganda and one international  [2 = 79.7) was found in a pooled sample of 171 people with sickle cell disease from two studies conducted in Congo Brazzaville and Nigeria [Figure = 0.478) , 46, 49.= 0.351) , 40, 43.= 0.445) , 31, 35.= 0.376) , 32, 47.nd Togo) , 41. A p Nigeria , 48.Fig.In one study conducted in Egypt, the prevalence of PH was 62.7% (95%CI: 49.0–74.7) among 51 patients with chronic obstructive pulmonary disease . One othOnly one study, the Pan African Pulmonary hypertension Cohort (PAPUCO) study, reported the etiologies of PH as classified by Simmoneau and colleagues . This stAlthough, we have neither found studies reporting the prevalence of PH in the general population nor those reporting the incidence of PH in Africa, available studies showed that the prevalence of PH may vary widely across specific populations: from 7.4% in patients with systemic sclerosis to 68.7% in patients with cardiac surgery. The main driver of PH among patients presenting with cardiac complaints may be left-sided heart disease, which seems to represent two-thirds of PH etiologies in Africa.Among patients presenting with cardiac complaints, the prevalence of PH was 9.8% which is in the 10–20% range reported in the US general population . In moreGroup 2 causes included: rheumatic heart disease (12.9%), heart failure (32.9%), and cardiac surgery (68.7%). Similarly, a recent global systematic review by Dzudie and colleagues reported a prevalence of PH varying from 22 to 83.3% in adults with left heart disease , 59. IndFor group 3 causes of PH, we found a single study from Egypt, which reported a prevalence of 62.7% among those with chronic obstructive pulmonary disease. This is higher than findings from developed countries among patients with end-stage chronic obstructive pulmonary disease (48.7%) . For groOverarchingly, one can note that the prevalence of PH in different specific populations in Africa is either similar to, or in the majority of situations higher than that reported in other non-African settings. This is potentially explained by the presence of many known endemic risk factors for PH in Africa (some not fully explored) –16. We fAbout half of studies included in this review had a low risk of bias in their methodological quality. We observed no publication bias, suggesting that future research endeavors are likely to have no important impact on the confidence in our estimates. A key exclusion criterion for studies during the review process was wrong diagnostic criteria for PH . Indeed, some studies used a cut-off between 20 and 30 mmHg –79 and oFurther studies are needed to investigate PH etiologies in Africa. Clinicians and researchers are invited to comply with updated guidelines for accurate diagnosis and classification of PH, as recommended by international guidelines . While tThis review had some limitations. First, although we searched several databases, this review is based on a limited number of original studies published in specific populations. Second, the limited number of studies included in the review did not permit us to perform meaningful subgroup analyses to determine sources of heterogeneity. Third, not all African sub-regions were represented in all the specific populations, making the results of this study particular in terms of generalizability to the entire continent. Nonetheless, this is the first comprehensive review on the prevalence, incidence and etiologies of PH in Africa; it gives a clear overview of the burden of PH in Africa and crucial gaps to fill in future research.This review suggests that the prevalence of PH is relatively high in some populations in the African context. These high prevalence estimates seem to be driven by left heart diseases. To tackle this high disease burden, policy makers and healthcare providers must be aware of this reality and invest in interventions towards improving prevention, detection and management of PH in Africa. Community-based studies are required to better characterize the epidemiology and natural history of PH in Africa.Additional file 1:Individual characteristics of included studies. (PDF 634 kb)Additional file 2:Risk of bias in individual studies. (PDF 287 kb)


Cognitive dysfunction affects 40–65% of multiple sclerosis (MS) patients and can occur in the early stages of the disease. This study aimed to explore cognitive functions by means of the Italian version of the minimal assessment of cognitive function in MS (MACFIMS) in relapsing-remitting MS (RRMS) patients with very mild clinical disability to identify the primarily involved cognitive functions. Ninety-two consecutive RRMS patients with Expanded Disability Status Scale (EDSS) scores ≤ 2.5 and forty-two healthy controls (HC) were investigated. Our results show that 51.1% of MS patients have cognitive dysfunction compared to HC. An impairment of verbal and visual memory, working memory, and executive functions was found in the RRMS group. After subgrouping RRMS by EDSS, group 1 (EDSS ≤ 1.5) showed involvement of verbal memory and executive functions; moreover, group 2 (2 ≤ EDSS ≤ 2.5) patients were also impaired in information processing speed and visual memory. Our results show that utilizing a comprehensive neuropsychological assessment, approximately half of MS patients with very mild physical disability exhibit cognitive impairment with a primary involvement of prefrontal cognitive functions. Detecting impairment of executive functions at an early clinical stage of disease could be useful to promptly enroll MS patients in targeted rehabilitation. Cognitive impairment (CI) is a common deficit of multiple sclerosis (MS), with prevalence rates ranging from 40 to 65% . It can Cognitive dysfunction can be detected even at the earliest stages of the disease , 7; neveMany other clinical variables have been explored to determine which one mostly influences cognition in MS patients, and results have been controversial. In particular, CI is scarcely correlated with disease duration , 13. TheOur aim was to explore cognitive function in RRMS patients with very mild clinical disability by means of an ad hoc comprehensive neuropsychological assessment ) , to idenOur study included ninety-two patients with a diagnosis of RRMS   and fortInclusion criteria were as follows: age 18 or older, fluent in Italian, able to provide informed consent to all procedures, and EDSS ≤ 2.5 (for patients only).Exclusion criteria were as follows: neurological disorders other than MS; psychiatric disorder other than mood, personality, or behavior change following the onset of MS; medical condition that might influence cognition; history of developmental disorder ; history of substance or alcohol dependence or current abuse; motor or sensory deficits that might interfere with cognitive test performance; and relapse and/or corticosteroid pulse within four weeks of assessment (for patients only).We decided not to include or exclude patients on the basis of the medication they were taking. However, none of the participants were under treatment that has a significant impact on their cognitive performance; all patients were under immunomodulant therapy (interferon or glatiramer acetate). A detailed clinical interview was performed to verify the inclusion and exclusion criteria, and each patient underwent complete neurological examinations including EDSS rating. According to Kurtzke's criteria , patientBoth MS patients and HC underwent an Italian version of MACFIMS  and neurMoreover, the participants completed the Beck Depression Inventory (BDI)  and StatPatients were diagnosed as having CI when at least two tests were found to have more than 1.5 standard deviations (SD) below the control mean, according to the proposal of Amato et al. . MoreoveOn the basis of the number of test in the CI range, patients were classified as mildly (two tests impaired), moderately (three tests impaired), or severely affected (four or more tests impaired) , 12. ThiIn addition to the number of tests failed, we aimed to consider the number of cognitive domains impaired, according to Migliore et al. . More spt-test or univariate ANOVA). Correlations among neuropsychological tests and disease duration, BDI and STAI-Y, were evaluated using Spearman's correlation coefficients. Analyses of covariance (ANCOVA) were applied to examine differences in test performance considering diagnosis and sex as factors and BDI as a covariate. To obtain neuropsychological profiles shown in Figures z-scores using the mean and the standard deviation (SD) of healthy controls of the present study. Repeated-measures ANOVA were performed on these z-score variables to compare the performance of each patient group to each neuropsychological test, considering the test type as the within-subjects factor and the group as the between-subjects factor. If the assumption of sphericity was violated, Greenhouse-Geisser correction of degrees of freedom was considered.Group differences regarding demographic and clinical data were assessed using parametric tests , the group X test type interaction  = 1.963; p = 0.02) was also significant, indicating that the difference between the two groups changed across tests , 51.1% of patients were classified as impaired . Moreover, even when considering the 5th percentile (1.645 SD below the control mean), 40.2% of patients had impaired results . Detailed data about each NP test are reported in Regarding the number of impaired cognitive domains, according to the cut-off of 1.5 SD below the control mean, 43.5% of MS patients showed at least two domains compromised . Moreover, if we consider the 5th percentile, 33.7% of patients were found compromised .p = 0.006). ANCOVA with diagnosis and sex as factors and BDI  as a covariate was applied to compare test scores in MS patients and HC. In general, the differences between MS patients and controls still remained significant, with the exception of PASAT 2″, where the effect of depression  = 3.69, p = 0.058) tempered down the difference between the two groups . Moreover, a significant effect of sex was observed on the COWAT score  = 5.15, p = 0.026). STAI-Y did not differ between MS patients and controls. Also, disease duration did not significantly influence NP scores .A significant difference was also found on BDI scores comparing MS patients and HC (p < 0.001). According to pairwise Tukey's comparisons, the controls' overall estimated mean was not significantly different from that of the ND group (p = 0.085) but was significantly higher than that of the VMD group (p < 0.001) as expected. Also ND's overall estimated mean was significantly higher than that of the VMD group (p = 0.015).Repeated-measures ANOVA revealed a significant main effect of the groups  = 8.632; Greenhouse-Geisser = 12.25 and df2Greenhouse-Geisser = 802.59) = 1.771; p = 0.047) was also found, indicating again that the differences among groups were dependent on the type of test  = 8.597, p < 0.001), visuospatial long-term memory  = 3.977, p = 0.021), executive functions  = 6.441, p = 0.002; DKEFS DS: F = 8.144, p < 0.001; and COWAT: F = 11.195, p < 0.001), and information processing speed  = 3.220, p = 0.043).A significant effect of the group X test type interaction (F(df1 of test . Separatp = 0.007) and executive functions  , visuospatial long-term memory , information processing speed , and executive functions . No significant differences were observed between the ND and VMD subgroups  . HC and ubgroups .It should be noted that with such an unbalanced distribution of cases in the two subgroups (73 ND versus 19 VMDP), only large standardized effect sizes (>0.8) have enough probability (power > 80%) to be recognized as statistically significant at the defined significance threshold (0.05).In Our results demonstrate that 51.1% of our MS patients with EDSS ≤ 2.5 have cognitive dysfunction (at least two tests ≤ 1.5 SD); in particular, 16.3% had a mild (two tests failed), 15.2% had a moderate (three tests failed), and 19.6% had a severe (≥four tests failed) level of impairment. Even when we applied a more restrictive cut-off (5th percentile), 40.2% of patients were cognitively impaired. Moreover, considering the number of impaired cognitive domains, we found that 43.5% of MS patients showed at least two compromised domains. Also, by applying the 5th percentile cut-off to the number of impaired domains, we observed that 33.7% of MS patients were significantly altered. Verbal memory , visuospatial long-term memory, working memory, and executive functions (DKEFS and COWAT) were the cognitive functions mostly impaired in MS patients compared to HC. As shown in In ND (EDSS ≤ 1.5), an unsatisfactory cognitive performance was limited to verbal memory and executive functions, while VMD patients (2 ≤ EDSS ≤ 2.5) also performed badly in information processing speed and visuospatial long-term memory. Overall, the VMD patients received lower scores than the ND patients in almost every test considered . FinallyThese findings highlight and confirm that even considering very mildly clinically disabled MS patients, almost half of them experience some degree of cognitive impairment, suggesting that cognitive dysfunction can occur early in the disease. Particularly, executive functions and verbal memory can be impaired even before the onset of significant disability and can remain stable in VMD patients. In addition, information processing speed and visual memory are relatively preserved in ND patients and tend to deteriorate in the VMD group  . Memory n = 19). Future studies should therefore take into account the possibility of increasing the sample to evaluate possible differences. Another limitation of this study was the lack of a measure to assess fatigue and nutritional life style, two factors possibly influencing cognitive performance. Moreover, comparing different groups of patients, cognitive reserve is another important variable to be considered in future studies. Finally, another limitation could be the effect of interferon and glatiramer acetate on cognitive functioning; it has been demonstrated that some aspects of cognitive functioning may improve in patients with MS [The high percentage (51.1%) of cognitive impairment in our sample of MS patients with very mild clinical disability could be due to employment of a complete and comprehensive neuropsychological analysis. It would be useful in clinical practice to use reliable and sensitive tools in order to early detect executive function impairment and to suggest an adequate cognitive training. The lack of a significant difference in the cognitive performance of the two patient groups can be attributed to the small VMD sample  by ANCOVA model.


Hantavirus in bats. Historically, it was thought that these viruses were harbored primarily by rodent and insectivore small mammals. Recently, new species of hantaviruses have been identified in bats from Africa and Asia continents expanding the potential reservoirs and range of these viruses. To assess the potential of Neotropical bats as hosts for hantaviruses and its transmission dynamics in nature, we tested 53 bats for active hantaviral infection from specimens collected in Southeastern Brazil. Part of the hantaviral S segment was amplified from the frugivorous Carollia perspicillata and the common vampire bat Desmodus rotundus. DNA sequencing showed high similarity with the genome of Araraquara orthohantavirus (ARQV), which belongs to one of the more lethal hantavirus clades (Andesorthohantavirus). ARQV-like infection was detected in the blood, urine, and organs of D. rotundus. Therefore, we describe a systemic infection in Neotropical bats by a human pathogenic Hantavirus. We also propose here a schematic transmission dynamics of hantavirus in the study region. Our results give insights to new, under-appreciated questions that need to be addressed in future studies to clarify hantavirus transmission in nature and avoid hantavirus outbreaks.Bats (Order: Chiroptera) harbor a high diversity of emerging pathogens presumably because their ability to fly and social behavior favor the maintenance, evolution, and dissemination of these pathogens. Until 2012, there was only one report of the presence of  Hantaviridae) are membrane-enveloped viruses. Their genomes are small and consist of three segmented negative single-stranded RNAs designated large (L), medium (M), and small (S). These segments encode the virus polymerase, glycoproteins (Gn and Gc) and the nucleocapsid (N) protein, respectively2. Within natural hosts, hantaviruses do not cause apparent pathogenic effects3. However, transmission to humans can lead to severe diseases and death6; especially in South America, where hantavirus lethality rate reaches more than 40%7.Hantaviruses  from two broadly distributed insectivorous species: Eptesicus serotinus and Rhinolophus ferrumequinum9. Recently, evidence of a lethal genotype of Andes orthohantavirus (ANDV), Araraquara orthohantavirus (ARQV), was documented by de Araujo et al.10 and Sabino-Santos Jr et al.11 among several Neotropical bats in Brazil. All the remaining reports of Hantavirus infection in bats involved genotypes not known to be pathogenic to humans.Hantaviruses are predominantly rodent-borne pathogens, although insectivore-bat-associated hantaviruses have been reported in the last decade13. Bats are ranked second when considering top reservoirs of zoonotic viruses, and they host more zoonotic viruses per species than the first place order, rodents15. Therefore, the understanding of how these pathogens are transmitted and cause disease in humans is important in public health. Here, we show a systemic infection of hantavirus in Neotropical bats for the first time.Bats (order: Chiroptera) have highly social behavior that favors the maintenance, evolution, and dissemination of pathogenic viruses2h, in southeastern Brazil , A. obscurus (n = 1), A. planirostris (n = 1), Carollia perspicillata (n = 1), Chiroderma villosum (n = 1), Chrotopterus auritus (n = 1), and Desmodus rotundus (n = 3) 11. Blood, excreta (feces and urine), saliva and organs  of all seropositive animals had RNA extracted and screened. Hantaviral RNA was detected only in the blood of two phyllostomids species: the frugivorous Carollia perspicillata and the sanguivorous common vampire bat Desmodus rotundus  detecting the viral nucleocapsid protein. Active infection was suggested in the organs of only D. rotundus. The monoclonal antibody to N protein of HTNV showed an intense cytoplasmic staining in myocardial cells and hepatocytes . Hantavirus nucleocapsid protein, the most synthesized viral protein, was found in all tested organs (except kidneys and liver) from the infected vampire bat, which indicates viral replication   Fig. . In the 19. Viruses from bats can become zoonoses that are transmitted to domestic animals and humans presumably due to their ability to fly vast distances disseminating these viruses15. In our study, we found hantaviral RNA closely related to ARQV in urine of the common vampire bat Desmodus rotundus. This vampire bat could be transmitting ARQV-like through contaminated aerosol of its urine that could be inhaled thus infecting other animals20. However, this transmission mechanism should be confirmed by isolation of the hantavirus from urine to investigate if the pathogen shedding by the vampire bat is viable and infectious21.Viruses are the most abundant biological entities on the planet Earth, and those originating in animal world cause most of emerging diseases of humansApodemus agrarius) and bats22; although, hantaviruses can also be found in other organs24. In the present study, we detected the N protein in almost all organs of D. rotundus, particularly in heart and liver, indicating virus replication . Guo et al.18 suggested that hantaviruses originated in bats and then dispersed among other mammalian reservoirs. However, due to the apparently common evolutionary history of bats with insectivores (shrews and moles), it is more likely that insectivores were the original reservoirs, and from these the virus has spread to bats and rodents28. There are reports of Sigmondontinae rodents infected in this same trap site region as the bats in the current study30. It is thought that small rodents may feed in the monocultures, but not necessarily nest in these monocultures. These rodents are more likely to live in the fragments where the vegetation is most preserved, thus sharing the same environment as bats, which depend on natural landscapes to survive environmental degradation33. D. rotundus feeds on blood of large and medium-sized mammals, including livestock, feral pigs, tapirs and red brocket deer. The bats potentially “take a ride” with the animals and disperse over long distances35. D. rotundus is broadly distributed over Latin America and in Brazil in particular, and considered commensal with humans and animals . It has also been approved by the Ethics Committee for Animal Research of the University of São Paulo (020/2011) and the Federal University of Minas Gerais (333/2013). The research was approved by state laws (licenses IF-SP/COTEC 260108-007.043 and IEF-MG 012/2014). All laboratory procedures, until viral inactivation, were conducted in our BSL-3 + facility.Captured bats were handled following the guidelines of the American Society of Mammalogists for the use of wild animals in research and biosafety recommendations for working with animals potentially infected with hantaviruscerrado vegetation (a Brazilian savanna-like vegetation) in addition to small enclaves of semideciduous forest patches. Site 2, located in Cajuru county, 21°16′ 31″S and 47°18′ 15″W, with most of the original vegetation (cerrado) converted into mono-specific cultivars. Site 3, located in Batatais county, 20°53′ 28″S and 47°35′ 06″W, is characterized by large sugar cane plantations with small patches of other types of vegetation. Site 4 was located at Sapucaia Ecological Park, which has an area of 37.66 ha and karst topography. Site 5 was located at Lapa Grande Ecological Station with an area of 9,600 ha, where a high concentration of caves and shelters can be found. Both sites 4 and 5 are located in Montes Claros county, 16°43′ 41″S and 43°51′ 54″W, and have a predominant vegetation of cerrado with patches of deciduous seasonal forest (dry forest), and stretches of transition to caatinga (a Brazilian desert-like vegetation). Field trips were conducted from February 2012 to April 2014. Places were sampled twice: in dry season (April to September) and in rainy season (October to March). We used 12 mist-nets in sites 1–3 and 6 mist-nets in sites 4 and 5 . Bats were identified following standard ecological taxonomic keys40 and supplemented when needed by molecular data 42. Vouchers specimens positive for hantaviral RNA, D. rotundus and C. perspicillata, were deposited in the Collection of Mammals from the Center for Taxonomic Collections from the Federal University of Minas Gerais, under numbers: 6948 and 6949, respectively. Saliva and excreta (feces and urine) were collected from all captured bats. According to ethical statements, only one specimen per species per trap-night could be anesthetized and euthanized. Thus, using Halothane TM  selected bats were anesthetized, and had blood collected by cardiac puncture. Next, bats were euthanized by deepening anesthesia and had organs collected: spleen, liver, kidneys, lungs, and heart. Organs were collected using sterilized tweezers, one for each specific organ to avoid cross-tissue contamination, washed with sterilized nuclease-free water. One-half of each sample was stored in cryovials with cell culture medium  for viral molecular screening, RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The other half sample was placed into cryovials containing paraformaldehyde 4% , for immuno-histochemistry assays. All samples, except those in 4% paraformaldehyde, were flash-frozen in liquid nitrogen.Bats were captured at five ecologically distinct sites in the northeast region of Sao Paulo State and northern region of Minas Gerais State, in southeastern Brazil Fig. . Site 1:43, for further purification and sequencing. PCR products were purified with ExoSap-TI  and used for nucleotide sequencing in an Applied Biosystems® - 3500 Genetic Analyzer .The RNA was extracted from plasma, feces, urine, and tissues of bats with the QIAamp Viral RNA Mini Kit , according to the manufacturer’s instructions. Tissues were homogenized in TissueLyser II  as recommended by manufacturer. RNA extracts were quantified in NanoDrop ND1000 (USA) spectrophotometer. For cDNA synthesis, we used the High-Capacity cDNA Reverse Transcription Kit  following the manufacturer’s instructions. PCRs were performed according to Moreli and others43. These PCR products  were purified and cloned into pGEM-T Easy Vector , according to manufacturers’ guidelines, and used to transform DH10β E. coli cells. Colonies of bacteria were selected in LB medium supplemented with ampicillin 100 ng/ul and confirmed by PCR using same primers of product amplification. Plasmid preparation, from positive clone colonies, was isolated with Plasmid Mini Kits  and used as positive controls for PCR reactions.Rio Mamore orthohantavirus (strain HTN-0007-TPV 4645) were amplified as previously described44. Tissues from bat organs fixed in 4% paraformaldehyde  were dehydrated in increasing concentration ethanol solutions . Then, followed by xylene (1:1 xylene: ethanol and 100% xylene), embedded in paraffin, and 4 μm sections of tissue were obtained in a Leica microtome . Slides were mounted with Entellan . Antigen retrieval in tissue slides was conducted by immuno-histochemistry. First, we used a monoclonal antibody (1:300 dilution in PBS/BSA 1%) against the N protein of HTNV . The tissue in the slide had endogenous peroxidase blocked by 10% hydrogen peroxide and was incubated in humidified chamber at room temperature, for 60 min, washed with PBS, and incubated for 30 minutes with a biotinylated secondary horse antibody (1:300 dilution in PBS/BSA 1%) (BA-2000)  at room temperature (RT). The signal amplification was obtained by incubation with a polymer conjugated with streptavidin-peroxidase , at RT. Samples were counterstained with Harris hematoxylin (Vector). Positive staining was visualized by Nova Red peroxidase system with Vector Nova Red Substrate kit . Based on comparison with infected HeLa cells as positive control and uninfected heart tissues of Desmodus rotundus used as negative control, tissues showing red-brown cytoplasmic staining were considered as positive for hantaviral antigen.To determine the presence of the structural nucleocapsid protein of ARQV in tissue samples from PCR positive bats a protocol previously standardized was performedTissues were incubated in a lysis buffer  supplemented with a protease inhibitor mixture . Simultaneous disruption and homogenization were achieved through a high speed shaking of samples in micro-centrifuge tubes with stainless steel beads using TissueLyser II equipment . Each of ten cycles of high-speed shaking, samples were followed by ice incubation for 1 min. The supernatant was collected after the lysates were centrifuged at 16.000 g for 20 min at 4 °C to remove tissue debris. Samples were mixed with loading buffer  and boiled at 95 °C for 5 min. Proteins were resolved by SDS-PAGE and stained with Coomassie blue; and densitometry analyses of protein bands were calculated to be equalized to same amounts using Image Lab™ Software . Proteins were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane , and blocked with PBS-T  and non-fat dry milk, for one h. Primary antibody (1:500 dilution in PBS/BSA 1%), a hyperimmune murine anti-hantavirus ascitic fluid against the recombinant N protein of ARQV, was incubated overnight at 4 °C. After three washes with PBS-T, membranes were incubated with HRP-conjugated antibody anti-mouse (1:2000 dilution in PBS/BSA 1%) for one h at RT. After washing out the antibodies, proteins were detected by enhanced chemiluminescence ECL .Desmodus rotundus  and Carollia perspicillata (blood) and an additional 47 nucleotides of S segment from representative hantaviruses. Multiple sequence alignment (MSA) was conducted by using combinations of Mafft v.745 and Muscle v.3.746. Phylogenies were inferred with IQ-TREE version 1.4.3 software that automatically obtained the best-fit model based on Bayesian Information Criterion. All segments were analyzed under the GTR + I + G4 model substitution model with 1,000 replicates47. Statistical supports for individual nodes were estimated using bootstrap value. The phylogenetic tree was visualized using the FigTree software v.1.4.2. The identity distance for the partial S segment was calculated using Genious 9.1.6 . All sequences generated in this study can be retrieved from GenBank under accession numbers MF094267, for D. rotundus and MF094269 for C. perspicillata.Maximum likelihood (ML) phylogenetic tree was constructed using partial S sequences obtained from Supplementary file


Acetylcholine is the main transmitter of the parasympathetic vagus nerve. According to the cholinergic anti-inflammatory pathway (CAP) concept, acetylcholine has been shown to be important for signal transmission within the immune system and also for a variety of other functions throughout the organism. The spleen is thought to play an important role in regulating the CAP. In contrast, the existence of a “non-neuronal cardiac cholinergic system” that influences cardiac innervation during inflammation has been hypothesized, with recent publications introducing the heart instead of the spleen as a possible interface between the immune and nervous systems. To prove this hypothesis, we investigated whether selectively disrupting vagal stimulation of the right ventricle plays an important role in rat CAP regulation during endotoxemia. We performed a selective resection of the right cardiac branch of the Nervus vagus (VGX) with a corresponding sham resection in vehicle-injected and endotoxemic rats. Rats were injected with lipopolysaccharide  and observed for 4 h. Intraoperative blood gas analysis was performed, and hemodynamic parameters were assessed using a left ventricular pressure-volume catheter. Rat hearts and blood were collected, and the expression and concentration of proinflammatory cytokines using quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were measured, respectively. Four hours after injection, LPS induced a marked deterioration in rat blood gas parameters such as pH value, potassium, base excess, glucose, and lactate. The mean arterial blood pressure and the end-diastolic volume had decreased significantly. Further, significant increases in blood cholinesterases and in proinflammatory  cytokine concentration and gene expression were obtained. Right cardiac vagus nerve resection (VGX) led to a marked decrease in heart acetylcholine concentration and an increase in cardiac acetylcholinesterase activity. Without LPS, VGX changed rat hemodynamic parameters, including heart frequency, cardiac output, and end-diastolic volume. In contrast, VGX during endotoxemia did not significantly change the concentration and expression of proinflammatory cytokines in the heart. In conclusion we demonstrate that right cardiac vagal innervation regulates cardiac acetylcholine content but neither improves nor worsens systemic inflammation. Despite intensive research, sepsis and septic shock still represent the major causes of death in intensive care units (ICU) ,2,3. In Otherwise, as is well known, sepsis is mainly characterized by the occurrence of strong inflammatory processes .The cholinergic anti-inflammatory pathway (CAP) concept can be defined as the missing link between regulation of the inflammatory response and the neural circuit. According to the CAP concept, acetylcholine—the principal vagal neurotransmitter—was able to reduce in vitro proinflammatory cytokine levels in response to endotoxin in human macrophages and in vivo through direct electrical stimulation of the efferent vagal nerve in rats . These e2, pO2, bicarbonate (HCO3−), base excess (BE), hematocrit (Hk), hemoglobin (Hb), potassium (K+), glucose (Glu), and lactate (Lac) concentrations. Thus, all these measured parameters were in the physiological range.Before administering LPS , no significant differences between the groups were determined for the following parameters: pH value, pCO+, BE, bicarbonate, glucose, and lactate concentration in endotoxemic rats compared to saline-injected rats , however, marked, significant changes were found in pH value, KResection of the right cardiac vagus nerve (VGX), however, did not show significant changes in blood gas parameters  under sa§: p ≤ 0.05, p > 0.05, In rat blood, acetylcholinesterase (ACHE) and butyrylcholinesterase (BuCHE) activities were measured four hours after injecting LPS (T5). The activity of both enzymes was significantly higher after LPS stimulation, irrespective of the selective vagus intervention, than in the corresponding saline groups (§: p < 0.05).LPS significantly increased acetylcholine in rat myocardium after a selective vagotomy of the right branch of the cardiac vagus nerve  without LPS significantly reduced the ACH concentration in myocardial tissue, .ACHE activity in heart tissue increased after selective VGX  was not significantly different between the four groups (I–IV) at the end of the experimental period: (I) 278.4 ± 10.9, (II) 283.0 ± 9.7, (III) 326.4 ± 14.2, and (IV) 303.8 ± 14.9 ng/mL.§, p < 0.05), both in gene expression in the heart and protein levels in plasma (p > 0.05).We measured the proinflammatory cytokines IL-1β, IL-6, and TNF-α in rat plasma and myocardial tissue. All three proinflammatory cytokines had significantly increased after LPS treatment (n plasma A–C and mn plasma A–C. A sep > 0.05).At baseline .After administering LPS, and irrespective of a vagal intervention, animals exhibited a significant decrease in mean arterial blood pressure . The end-diastolic volume (EDV) was significantly decreased in VGX LPS rats compared to VGX saline §: p < 0.05). In saline-injected rats, the effect of VGX was obtained in heart frequency (HF), cardiac output (CO), and EDV (#: p < 0.05). Under LPS, the effect of VGX was only significantly pronounced in rat HF .After administering LPS and irrespective of vagal intervention, animals exhibited a significant decrease in mean arterial blood pressure  reduced (i) HF under saline and LPS conditions, and (ii) CO under saline injection in rats. EDV, however, was significantly increased in VGX rats under the condition of saline injection (#: p2 = 0.05).A right cardiac vagus nerve resection (VGX) significantly (p < 0.05) in sham-operated rats, whereas VGX seemed to attenuate this effect , the MAP was significantly decreased and the CO was significantly increased in all animal groups A,C. HF ws effect B. A signs effect D.The main result of the present study showed that vagal outflow is related to cardiac acetylcholine content, but neither improves nor worsens systemic inflammation.Apart from the LPS models, the other main animal model of sepsis is the surgical model  model), which is also clinically relevant as this model induces a polymicrobial sepsis that mimics human sepsis ,16. HoweIn accordance with the literature ,18, endoSummarizing the results after LPS injection, we demonstrated that a single LPS dose (1 mg/kg body weight administered intravenously) was able to mimic some of the most important signs that are observed in septic patients, and therefore it is an appropriate animal model to use for studying open questions in inflammation-induced, cardiovascular alterations. Nevertheless, with only 4 h of observation time, we can only assess the very early changes using our acute LPS model. To assess compensatory mechanisms occurring delayed, further models must be utilized.Early studies considered that fine, silver-stained fibers coursing among ventricular myocardial cells were most likely cardiac parasympathetic postganglionic fibers . To our It is known that inflammation can be influenced by stress response through surgical intervention. Therefore, we measured the plasma corticosterone concentration. No changes in corticosterone plasma levels were observed between the groups; thus, possible differences between sham-operated and the right cardiac vagus nerve resected rats were not directly associated with surgical stress-related deterioration in the hypothalamic-pituitary-adrenal (HPA) axis and related peripheral corticosterone concentration.Selective VGX depressed cardiac function at the end of the experimental period: the VGX effect was mainly pronounced for HF, CO, and EDV in the present study. Alternative pathways partially compensating for the detrimental impact of a systemic loss of vagal tone were described in the studies of Horn and Friedman and Martelli et al. ,26. For 2 and significantly lower glucose levels in animals after LPS and VGX. Thus, because organ perfusion is compromised as a result of endotoxemia, increased lactate levels were observed. However, whether the observed influence of the vagus nerve is due to changes in cardiac metabolism or a result of hemodynamic alterations should be the subject of further research.Under LPS-induced inflammation, the MAP was reduced, irrespective of whether the vagus nerve was intact or resected (at the end of the experiments). The persistent drop in HF after VGX appears somehow paradoxical, but might be based on an (over)compensatory loss of sympathetic tone. As a consequence, CO is also reduced, which might reduce the supply of substrates to the tissue, as indicated by unchanged pOIt was shown in previous experiments that myocardial cells are capable of secreting pro-inflammatory cytokines upon sensing of circulating inflammatory mediators or pathogen-associated molecular pattern, e.g., LPS .Contrary to our primary hypothesis about a significant role of the heart in CAP, our present results showed an unchanged inflammatory response after VGX, as assessed by proinflammatory cytokine levels of IL-1β, IL-6, and TNF-α in the plasma and ventricular tissues. Analyses of myocardial expression of these genes are in concordance with the protein levels.In contrast to nerve resection, vagus nerve stimulation represents a common therapy option under some clinical circumstances, for instance, epilepsy and depression . ElectriSummarizing the main results: our present study demonstrates significant changes in heart ACH and ACHE after ablation of the right cardiac vagus nerve. However, we did not find evidence for a significant role of the heart in the cholinergic anti-inflammatory pathway.The experimental protocol was approved by the review committee of the Medical Faculty of the University of Heidelberg (Germany) and complied with the guidelines of the responsible national government agency  and with international standards.The studies were performed on adult male Wistar rats , weighing 350–390 g. They were housed in individual cages in a temperature-controlled room at 22 ± 0.5 °C with a reversed day–night cycle . Free access to food  and water was allowed throughout the experimental period.(I)n = 7) = Sham nerve resection group with 1 mL i.v. saline ((II)n = 7) = Nerve resection group: with 1 mL saline i.v. ((III)n = 9) (sham LPS)Sham resection group + LPS (1 mg/kg body weight i.v.) ((IV)n = 9) (VGX LPS)Nerve resection group + LPS (1 mg/kg body weight i.v.) 2:N2O = 30:70), anesthesia was maintained over the entire experimental period with 3.5 vol. % sevoflurane and oxygen/nitrous oxide (O2:N2O = 30:70).After induction of anesthesia with 4 vol. % sevoflurane  via a vaporizer  and oxygen/nitrous oxide . After subcutaneous electrode placement, the electrocardiogram was recorded. Tympanal temperature was monitored and maintained at around 37 °C by using a heating pad and a heater.In all animals, catheters were placed in the A. and V. femoralis sinistra (5.0 nylon thread) for administering drugs, for drawing blood samples, or for blood gas analysis. We have a very long experience in preparing femoral vein instead of tail vein. Under the conditions of prolonged anesthesia, the femoral vein is easy and reproducible to prepare, especially if an agent/medication etc. had to be infused.Thereafter, the A. carotis communis dextra was prepared and a microtip pressure-volume catheter  was inserted through the A. communis dextra and advanced into the left ventricle for continuous hemodynamic measurement.All inserted catheters were fixed (6.0 nylon threads) accurately before the rat’s chest was opened. A 3-cm incision was made to divide the skin and muscle layers. An electrocautery device  was used to avoid or stop bleeding from intercostal arteries. The thorax was opened between the first rib to above the diaphragm using scissors. To prepare, loop, and cut the right cardiac vagus nerve (VGX), a small part of the right thymus was resected. Thereafter, the right cardiac nerve branch of each rat was exposed , which wA respective sham operation was performed in rats of groups I and III without cutting the nerve. After these manipulations, a 30-min steady state interval was maintained without any additional manipulations. To make a gap between surgical procedure and LPS administration is justified by the idea to give the animal defined time interval to recover from surgical procedures for 30 min.Thereafter, 1 mL of NaCl or LPS was given via the V. femoralis sinistra. After 4 h of LPS or vehicle injection, the animals were sacrificed under general anesthesia by bleeding out. Blood samples were taken, and cardiac tissue was sampled. The rat’s heart was separated into two sections: one half was immediately transferred into RNAlater  for RNA isolation; the other half was immediately transferred to dry ice. All tissue was stored at −80 °C before performing the biochemical analysis.Blood samples (0.1 mL per sampling point) were taken via the A. femoralis at 5 time points: before the surgical procedure was finished (T1), at the end of the steady state phases (T2), 1.5 h (T3) and 3 h (T4) after LPS administration, and immediately before sacrifice (T5).2, pO2, bicarbonate (HCO3−), base excess (BE), hematocrit (Hk), hemoglobin (Hb), potassium (K+), glucose (Glu), and lactate (Lac) concentrations using a blood gas analyzing system ]. Fifty microliters were separated and subsequently frozen at −80 °C to determine CHE [acetylcholinesterase (ACHE) and butyrylcholinesterase (BuCHE)] activities. The remaining blood was rapidly centrifuged at 7000 rpm at 20 °C for 15 min and the supernatant was separated and frozen at −80 °C to assess the biochemical parameters.Blood taken with a glass capillary (10 µL) was immediately used to analyze blood gas ).Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using TaqMan Gene Expression Master Mix . Assay numbers were as follows: Rn00580432_m1 (IL-1β), Rn01410330_m1 (IL-6) and Rn01525859_g1 , and Rn1775763_g1  . For data analysis, the Delta CT (cycles of thresholds) method was used , 100 mM potassium phosphate buffer, and 18 mM sodium hydrogen carbonate. After 3 min of preincubation at room temperature, 6.25 µL of a substrate solution were added (86 mM acetylthiocholine chloride to measure ACHE activity or 10 mM butyrylthiocholine chloride for BuCHE activity). Formation of free thiocholine and the resulting release of 5-thio-2-nitrobenzoate from DTNB were assayed at 405 nm for 4 min. Specific enzyme activity was expressed as U/g Hb for ACHE and U/L for BuCHE.Blood ACHE and BuCHE were measured spectrophotometrically by applying the method of Ellman et al.  with som® Red Acetylcholine Assay kit . Protein was measured according to Lowry et al. [To determine the acetylcholine (ACH) and acetylcholine esterase (ACHE) concentrations in the heart, tissue samples of the right ventricle were homogenized in 0.5 mL of 50 mM sodium phosphate buffer (pH 7.7) including a protease inhibitor cocktail in an ultrasonic homogenizer. Thereafter, the homogenates were centrifuged at 13,000 rpm at 4 °C for 5 min. ACH concentration was measured in the supernatant after dilution with a specific reaction buffer to prevent ACH hydrolysis using the Amplexy et al. . Serum bCorticosterone concentration was assessed in homogenates of the hippocampus by radioimmunoassay  after extraction with diethyl ether and ethanol as described in the manufacturer’s instructions. Intra-assay and interassay variations were under 10%.All measurements were performed by an independent investigator blinded to the experimental conditions. If the data were distributed normally, we presented data as mean; if the data were distributed not normally after Kolmogorov Smirnoff analysis, the data were presented as median. Thus, results are presented as mean with standard deviation (SD) in the tables and in the text. Data in the figures are presented as median (IQR = Inter Quartile Range) or as mean values ± SD.t-test.Differences within or between normally distributed data were subjected to an analysis of variance (ANOVA) using SPSS v.22.0  followed by post-hoc Fisher’s LSD test. Two time points were compared using Student’s U test. Results were considered significant at p ≤ 0.05.Nonparametrically distributed data were analyzed with Kruskel-Wallis test followed by Mann-Whitney The beneficial effect of cholinergic stimulation during inflammation is indisputable and has been shown in a variety of conditions. Assuming that there is no splenic involvement in this “reflex” and reconsidering the early experiments of Otto Loewi, we put the heart in the center of interest. Contrary to the assumption that ventricular myocardium is only sparsely innervated by the N. vagus, we showed significant changes in heart ACH and ACHE after ablation of the right cardiac vagus nerve. However, we did not demonstrate a significant role of the heart in the CAP.


Polycystic ovary syndrome (PCOS) affects 5–10% of women in reproductive age and is characterized by oligo/amenorrhea, androgen excess, insulin resistance, and typical polycystic ovarian morphology. It is the most common cause of infertility secondary to ovulatory dysfunction. The underlying etiology is still unknown but is believed to be multifactorial. Insulin-sensitizing compounds such as inositol, a B-complex vitamin, and its stereoisomers (myo-inositol and D-chiro-inositol) have been studied as an effective treatment of PCOS. Administration of inositol in PCOS has been shown to improve not only the metabolic and hormonal parameters but also ovarian function and the response to assisted-reproductive technology (ART). Accumulating evidence suggests that it is also capable of improving folliculogenesis and embryo quality and increasing the mature oocyte yield following ovarian stimulation for ART in women with PCOS. In the current review, we collate the evidence and summarize our current knowledge on ovarian stimulation and ART outcomes following inositol treatment in women with PCOS undergoing in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI).  Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder of women in reproductive age with a prevalence ranging from 5 to 10% . There iA comprehensive literature search was performed using the electronic databases PubMed, Medline, and Google Scholar for relevant articles in the English language until May 2016. The following combinations of search terms were used: “polycystic ovarian syndrome”, “PCOS”, “inositol”, “ovulatory dysfunction”, “hyperandrogenism”, “insulin resistance”, “ovulation induction”, “in vitro fertilization”, “IVF”, “Assisted reproductive technology”, “ART”, “metformin”, and “treatment outcome”. The abstracts identified were reviewed and evaluated by both authors. Any disagreement between the two researchers was resolved with discussion. The reference lists of the selected papers were manually searched in order to identify additional potentially relevant studies.Inositols and their derivatives are sugar alcohols which belong to the vitamin B family, representing nine cyclohexane-1,2,3,4,5,6-hexol stereoisomers. They are chemically stable molecules which can be ingested from the diet . Dietary2+ release during oocyte maturation and fertilization [Inositols and their phosphates function as secondary messenger molecules for various cellular signaling pathways including insulin signal transduction, calcium trafficking, lipid metabolism, cytoskeletal protein assembly, cell growth and differentiation, modulation of serotoninergic pathways, oocyte maturation, and fertility . Uptake lization , 35. Myolization , 36. Morlization , 38. In lization , 39. Furlization , 40, 41.It is now recognized that insulin resistance has a critical role in the pathogenesis of PCOS. Hyperinsulinemia secondary to insulin resistance is common in a high proportion of PCOS patients and is associated with metabolic morbidities as well as reproductive dysfunction . InsulinInositol and its stereoisomers are classified as insulin sensitizers and act as a second messenger of insulin signaling pathways . Indeed,Women with PCOS have reduced serum level and increased urinary loss of DCI , 49. It As mentioned earlier, MYO is converted to DCI via an insulin-induced epimerase enzyme in different tissues based on their requirement for each of these molecules , 56. DCIIn contrast, the benefit of MYO supplementation in PCOS has been demonstrated by several studies. Administration of MYO combined with folic acid 2 g twice a day for 6 months in PCOS patients showed maintenance of normal ovulatory activity in 72%, with singleton pregnancy rate of 40% during the 6-month observation period . In a rer = −0.56, P = 0.025), suggesting that obesity plays a role in abnormal inositol metabolism independently of PCOS. In a randomized trial of inositol treatment in PCOS women, Gerli et al. have shown that metabolic risk factor benefits of inositol treatment were not observed in the morbidly obese subgroup of patients, noting an inverse relationship between BMI and treatment efficacy [Another important consideration when evaluating the effects of inositols in PCOS women is the interaction with obesity. Baillrgeon et al. showed that obese PCOS women have diminished release of DCI-IPG in response to insulin elevation compared to normal weight women . Since tefficacy . Recentlefficacy . For theefficacy . It was Inositol plays an important role in the follicular microenvironment and affects oocyte maturation and embryo development . ElevateP = 0.01), significantly lower peak E2 levels at hCG administration , and reduction in degenerated oocytes  without compromising oocyte yield in the myo-inositol group in comparison to folic acid alone group [Papaleo et al. investigated the effect of MYO supplementation of 2 g twice a day on ART outcomes in sixty patients with PCOS undergoing ovarian stimulation for intracytoplasmic sperm injection (ICSI) cycles. They found significant reduction in the total number of days of stimulation  2 times a day for 3 months, while group B received only folic acid (200 μg). Their results showed lower peak E2 levels at hCG administration, less cycle cancellation, higher number of oocytes retrieved, significantly lower number of immature oocytes, and better quality of embryos with higher number of transferred embryos in group A in comparison to group B [P < 0.04), with significant reduction in grade I embryos (P = 0.004) in DCI supplementation group suggesting the negative effect of DCI on oocyte and embryo quality and worsening ovarian response with increasing DCI dosage in PCOS patients [Similar findings were reported by Ciotta et al. in a randomized study in which they evaluated the effects of myo-inositol on oocyte and embryo quality in 34 PCOS patients undergoing IVF/ICSI. Patients in this study were divided into two groups: group A was given myo-inositol (2 g) and folic acid , number of transferred embryos , and improved embryo quality  in MYO-DCI treated group in comparison to DCI only treated group [Colazingari et al. studied the role of combined MYO and DCI in comparison to DCI alone in PCOS patients undergoing IVF. They included PCOS patients with BMI less than 28 and FSH less than 10 IU/L undergoing IVF-ET and treated them with MYO combined with DCI in a physiological ratio (1.1 g myo-inositol plus 27.6 mg of D-chiro-inositol) or with DCI alone (500 mg) for 12 weeks. They found reduced number of degenerated oocytes (1.04 ± 1.15 versus 1.82 ± 1.55), better fertilization rate (0.75 ± 0 versus 0.58 ± 0.29) (n = 195), group A , and group B . All patients received their treatment from first day of the menstrual cycle until 14 days after embryo transfer. Patients in group A required decreased dose of gonadotropins  and had enhanced quality of oocytes (group A: 48.2% versus group B 35.0% versus control group 38.2%) and embryos (45.7% in group A versus 30.4% in group B and 25.6% in the control group), suggesting the synergistic effect of MYO and melatonin on improving oocyte and embryo quality [In the largest study to date evaluating the effects of MYO supplementation in PCOS women undergoing IVF, Pacchiarotti et al. randomized 526 PCOS patients into three groups: control  and α-lipoic acid (800 mg) per day for 3 months. In MYO and α-lipoic acid group, significant reduction was noted in immature oocytes , with improvement in mature oocytes  and increase in grade 1 embryos  and higher number of pregnancies achieved  in comparison to MYO alone group [α-lipoic acid may enhance the beneficial effects of MYO in PCOS women. In another study by Rago et al. the combined effect of MYO and ne group . While tIn summary, current data on inositol supplementation and ART outcomes in women with PCOS is limited to small randomized clinical trials which suggest beneficial effects of MYO on folliculogenesis with improved oocyte maturation and embryo quality. However, data on the effects of MYO supplementation on implantation, pregnancy, and live birth rates following ART in these women is scarce. Thus, large clinical trials aimed at assessing these important ART outcomes are needed. In addition, further studies are needed to determine the optimal ratio of administered MYO : DCI resulting in maximal beneficial effect.In conclusion, myo-inositol is an insulin sensitizer which appears to have beneficial effects on ovarian function and response to ART in women with PCOS. It induces nuclear and cytoplasmic oocyte maturation and promotes embryo development. In contrast, D-chiro-inositol appears to exert opposite and detrimental effects on the ovary. While accumulating evidence suggests that myo-inositol improves the number of mature oocytes retrieved, oocyte quality, and embryo quality in women with PCOS undergoing ART, data on its effects on pregnancy and live birth rates in these women is much more limited. Further research on larger patient populations is needed to determine whether inositol supplementation, possibly in combination with other drugs, could improve clinical pregnancy and live birth rates in PCOS women undergoing ART. It is an affordable, widely available, and easy to administer agent which has the potential of improving the outcomes of fertility treatments in women with PCOS.