Avian reovirus (ARV) is a prevalent infectious agent that has the potential to cause respiratory and gastrointestinal illnesses in poultry, leading to substantial financial losses in the poultry sector. Until now, there have been no investigations conducted to examine the epidemiological status of ARV infections in Morocco. The aim of this study was to investigate the seroprevalence of ARV infections with respect to area, types of chickens , vaccination status, and age of chickens. A total of 826 serum samples were collected from 36 broiler and broiler breeder flocks, with 14 of them being unvaccinated, fromsix different regions of Morocco, namely Casablanca-Settat, Rabat-Salé-Kénitra, Tanger-Tétouan-Al Hoceïma, Oriental, Marrakech-Safi, and Fez-Meknès between 2021 and 2022.These serum samples were screened using a commercial indirect ELISA ARV antibody test kit (IDEXX REO). The study found that all tested flocks were positive for ARV-specific antibodies, indicating that the virus was present in these flocks. Out of the 826 serum samples tested, 782 were positive for ARV-specific antibodies. The overall prevalence of ARV infections in breeder and broiler flocks was calculated to be 94.6% ± 0.78. To summarize, the current study provides evidence of the widespread distribution of ARV infections in Morocco, suggesting that the poultry industry in the country is highly infected with ARV. Avian reovirus is a virus that belongs to the Reoviridae family and the Orthoreovirus genus. It can infect various species of birds, including chickens, turkeys, ducks, and quails. The virus can cause several diseases in birds, including viral arthritis/tenosynovitis, respiratory tract infections, and malabsorption syndrome ,2,3. AviThe seroprevalence of avian reovirus varies depending on several factors, including the age and breed of the birds, management practices, and geographical location. In general, seroprevalence is higher in older birds than in younger birds, as older birds have had more opportunities for exposure to the virus ,8. SeropIn Morocco, avian reovirus presents a significant concern for the poultry industry, and ongoing research is necessary to develop new control strategies and to improve our understanding of the virus and its impact on bird health and production. This study, for the first time, investigates the seroprevalence of Reovirus in broiler breeder and broiler chickens in different regions in Morocco.In this study, a total of 826 sera were collected from 36 different broiler breeder and broiler flocks in 6 different regions of Morocco . In total, 379 serums were collected in 2021 and 447 in 2022, according to the distribution of collection dates. Several parameters, such as age, type of bird , and vaccination status were considered during the sample collection . The ageThe stratified random sampling strategy used in this study provided statistical representativity with a confidence level of 95% and a diagnostic test sensitivity of 86%. For the 6 Moroccan regions included in the study, the criteria “total poultry farms for each region” helped to divide the flocks into 3 subgroups called strata, each of which represented a density . For a given theoretical seropositivity rate of 50%, and according to the size of flocks in each region , the minimum required sample size is determined to be 6 per flock for each age group but can be increased to 12 to maximize the chances of having reliable results. For each bird, a sample random selection is applied to ensure an equal chance of being selected. Nevertheless, in some circumstances, the sample size was reduced due to potential losses during collection or storage.g for 5 min and then transferred into a clean Eppendorf tube. Each sample was labeled with an appropriate alphanumeric code. The collected sera were stored at −20 °C in preparation for performing the ELISA.Blood samples were obtained from the wing vein, using a 3 mL syringe, of the randomly selected apparently healthy birds. The blood was transferred into sterile tubes and kept at room temperature on a slanted surface, which allowed the blood to clot, and the resultant supernatant designed serum was harvested by centrifugation at 12,000× In order to detect the ARV antibodies, the collected and processed sera samples were analyzed using a commercially available ARV-specific indirect ELISA kit  containing ARV antigen-coated plates, in accordance with the instructions supplied by the manufacturer.In brief, 100 µL of serially pre-diluted test samples (1:500) was dispensed into the 96-well microtiter plates coated with ARV antigens, and previously individually filled with the positive (PC) and negative controls (NC). The microplates were subsequently incubated for 30 min in order to form an antibody–antigen complex. After removing the solution content  from the wells, the microplates were washed 5 times with 350 µL of distilled water, then 100 µL of horseradish peroxidase conjugate (GOAT anti-chicken) was dispensed into each well, which binds to any attached chicken antibody, and incubated for 30 min and then washed as previously described. Thereafter, 100 µL of TMB  substrate was incubated in each well for 15 min, which allowed the color to develop at room temperature. Finally, the reaction in the wells was terminated by dispensing 100 µL of a blocking solution. An ELISA microtiter plate photometer reader, equipped with a 650 nm filter, was used to measure, and record the absorbance values and the optical density of each well. In order to investigate the seroprevalence of birds against various diseases, several parameters were considered including age, type of birds, regions and their geographic location, vaccination status, period of collection, and flock density. The seropositivity of indirect ELISA antibody titers, as well as their means and standard deviations, were calculated using a one-way analysis of variance (ANOVA) model. Additionally, ANOVA and post hoc t-tests were used to determine whether there were any significant differences in antibody titers within and between the different parameters investigated. Differences were considered statistically significant when their probability (P) values were equal to or less than 0.05. By utilizing these statistical techniques, it was possible to identify the factors that influence seroprevalence, and to determine the effectiveness of vaccination programs against avian reovirus in different regions and age groups of birds.10 Titer = 1.09 (log10 S/P) + 3.36 helped to measure the endpoint titers. Samples with titers greater than 396 (S/P ratio > 0.2) are considered positive.For each serum tested, the following formula /(OD of PC—OD of NC) was used to calculate the simple to positive S/P ratio, and by converting the resultant S/P ratio, this equation LogThe study evaluated a total of 826 serum specimens collected from different regions of Morocco to determine the prevalence of avian reovirus-specific antibodies. The results showed that 782/826 of the specimens were positive for ARV-specific antibodies, indicating a high prevalence of the virus in the sampled regions.A further analysis of the prevalence rates in different regions revealed that all regions had confirmed ARV-specific antibodies, with the highest rates being found in the Marrakech-Safi 265 (98.51%) and Rabat-Salé-Kénitra 78 (100%) regions. The prevalence rates were lower in the Casablanca-Settat and Oriental regions, with rates of 88.5% and 90.82%, respectively. Interestingly, when considering Tanger-Tétouan-Al Hoceïma and Fez-Meknès in the north, Casablanca-Settat and Rabat-Salé-Kénitra in the west, Marrakech-Safi in the south, and Oriental in the east, the prevalence rates were significantly higher in the north and south regions of Morocco, with rates of 96.77% and 98.51%, respectively, compared to the east (90.82%) and west regions (91.45%).p-value = 0.63) between the two groups. Moreover, the results showed that the highest antibody titer means were recorded in the vaccinated flocks, particularly in Tanger-Tétouan-Al Hoceïma, with an average of 6078.62 ± 240.2. On the other hand, the unvaccinated flocks from Fez-Meknès had the lowest antibody titer mean with a rate of 3838.8 ± 322.9. Interestingly, when the geographic location of these regions was taken into account, the average antibody titer means of 5037.10 ± 301.25 did not exhibit any significant differences.The study also evaluated the seropositivity rates in vaccinated and unvaccinated flocks, and found that the unvaccinated flocks had a higher seropositivity rate of 98% compared to 92.8% in vaccinated flocks. Furthermore, the study compared the prevalence of ARV-specific antibodies between breeding and broiler chickens and found no significant difference  compared to 2021 (3956.05). p < 0.05), while the difference between medium-density and high-density flocks is not significant.The findings indicate that flock density has a significant impact on antibody titers, with the medium density flocks showing the highest mean titer of 6135.27 ± 200, followed by low-density flocks with a mean titer of 5000.08 ± 317.72, and high-density flocks with a mean titer of 2879.42 ± 154.1. The difference between the mean titers of medium-density and low-density flocks is statistically significant  on the antibody titers, with means of 4975.55 ± 151.4 and 5470.03 ± 345.8, respectively. Regarding the age of chickens, the results indicate that the mean antibody titer varies significantly depending on the age of the chicken. Chickens aged between 21 and 26 weeks had the highest mean titer of 5853.26 ± 266.9, followed by chickens aged 1 day (5325.41 ± 301.6), and those aged between 8and12 weeks (3766.13 ± 219.1).The type of production, either breeders or broilers, did not demonstrate a significant effect (p < 0.01) with the test results, with a Pearson correlation coefficient of 0.104 (p < 0.001). Interestingly, there was a weak positive correlation (p < 0.05) between the age of the birds and the test results, with a Pearson coefficient of 0.017. On the other hand, there was no significant correlation between the density of the flock, the type of production, or the period of collection and the test results . A total of 826 serum samples were collected from 36 flocks for serological testing, out of which 14 flocks were unvaccinated against ARV, while the remaining flocks had received ARV vaccination. An indirect ELISA commercial kit was used for the testing of serum samples. The results revealed that 782 serum samples were positive for ARV-specific antibodies, indicating a high prevalence (94.6% ± 0.7) of ARV infections in breeder and broiler flocks across all regions during 2021 and 2022. The high prevalence of ARV infections in all the examined flocks suggests that ARV infections are common in poultry farms in Morocco. In the 782 serum specimens found to be positive, antibody titers ranged from 403 to 22,372, with the average being 5320.8. Based on the vaccination status, the prevalence of ARV infection was higher in non-vaccinated chickens (98%) compared to vaccinated chickens (92.8%), indicating that the seroprevalence rate is related to the effectiveness of vaccination against ARV in different flocks, which can be explained by the involvement of multiple factors. These factors include the effect of the surrounding environment, the quality of vaccination, which depends on the techniques used to administer the vaccine, such as the equipment used and the skill of the person administering it, and the vaccination program itself, which includes the type of vaccine used, the age at which prophylaxis is administered, and the method of vaccination. According to several studies, the success of vaccination is influenced by various factors, including careful storage conditions , the appropriate dosage and administration route, vaccination timing , vaccination coverage and compliance , and considering the potential immunosuppressive effects (avoiding the concurrent use of the S1133 vaccine strain with live Marek and Gumboro vaccines). Failure to adhere to each of these parameters can lead to a lower seropositivity rate, even if the flock is considered vaccinated ,17,18; aThe geographical location effect was investigated, and the results indicated that the prevalence of reovirus was significantly higher in medium-density flocks as compared to low-density and high-density flocks. Specifically, 98.5% of the sampled birds from medium-density flocks tested positive for the virus, whereas only 92.5% and 88.5% of the sampled birds from low-density and high-density flocks, respectively, were positive for reovirus. The increased prevalence of the virus in medium-density flocks, particularly among the non-vaccinated flocks, could be attributed to the higher likelihood of virus transmission and infection in such flocks. This is due to the close proximity of birds in medium-density flocks, which can facilitate the spread of the virus through contact, airborne particles, or contaminated materials.The seroprevalence of ARV infection was not significantly different among the different regions studied. The highest seroprevalence was observed in Rabat-Salé-Kénitra, where all of the sampled flocks were positive for ARV antibodies. In contrast, the lowest seroprevalence was observed in Casablanca-Settat, with only 88.5% of the sampled flocks being positive for ARV antibodies. For the other regions, the seroprevalence ranged from 90% to 97%, with 98.5% for Marrakech-Safi, 97.2% for Fez-Meknès, 95.8% for Tanger-Tétouan-Al Hoceïma, and 90.8% for Oriental. This indicates that the virus seems to have a relatively equal presence across all regions, indicating that geographical location does not have a significant effect on the spread and transmission of the virus. Regarding periods of collection, the prevalence of ARV specific antibodies was significantly higher in 2021 than in 2022, being, respectively, 97.9% and 91.9%. The higher seroprevalence observed in 2021 compared to 2022 can be explained by two factors: the high viral pressure in Moroccan farms due to the importation of day-old breeder chicks of broiler chickens that were likely already infected with the avian reovirus, and the shortage of vaccines. The appearance of lameness cases on the Moroccan territory in 2021 was initially thought to be due to avian reovirus infection, but the PCR results turned out to be negative. Further investigations revealed that the problem was caused by nutritional and genetic issues. These findings highlight the need for better biosecurity measures in the importation of day-old chicks, and the timely provision of sufficient vaccine stocks to prevent the spread of avian reovirus in Moroccan poultry farms .The serological testing results revealed a high seroprevalence of ARV in unvaccinated broiler and breeder flocks in Morocco, with values of 100% and 97.4%, respectively, indicating the endemic and circulating nature of this virus within breeder farms, independent of various factors. The high incidence of ARV infections in breeders is probably due to two main sources. Firstly, the flocks may become infected by the virus through the environment, as ARVs are commonly found in the poultry population and can be isolated from the respiratory and gastrointestinal tracts of chickens showing acute or asymptomatic infections . SecondlTo better understand the circulation of ARV infection in Moroccan poultry farms, we examined the prevalence of ARV antibodies in unvaccinated broiler breeders of different age groups. The highest prevalence of ARV antibodies was found in 1-day-old chicks, with a prevalence of 100%. This suggests that younger chickens have lower immunity due to their underdeveloped immune system . ChickenThe vaccinated breeder flocks displayed a similar pattern to the unvaccinated flocks, with a high seroprevalence rate of 98.1% at 1 day of age. This can be attributed to the vaccination of the parent birds before the chicks are imported from foreign countries. The seroprevalence rate then decreased to 81.1% at 8–12 weeks of age, which can be explained by the vaccination schedule that might not have been enough to sustain high antibody levels at this age. The increase in seroprevalence to 98.9% for the age group of 21–26 weeks can be explained by the close contact between birds or the transfer of birds to production facilities, which is a common practice at this stage, and may increase their exposure to environmental stressors suppressing the immune system, enhancing the exposure to the virus. The prevalence of ARV infection in the poultry population has been investigated in several countries. Comparable findings were reported in Romania, by Botuş et al. , Iran, bThe variations in the observed seroprevalence rates across different countries suggest the potential influence of multiple factors, including differences in management practices, vaccination schedules, environmental conditions, and potential exposure to other viral and bacterial infections . AdditioThe epidemiology of ARV infections in North Africa is not well understood, particularly in Algeria, Mauritania, and Tunisia, which have shared a molecular study of ARV  but not Although our study on reovirus seroprevalence in poultry shares similarities with other research, there are notable differences in the study design and population that complicate comparisons. These differences include the criteria for defining flock status as positive or negative, the antibody titer thresholds used to indicate reovirus seropositivity, the age of broilers and breeders during sampling, and the vaccination status of breeders . These dThe findings of this study provided valuable insights into the control and management of ARV infections in poultry farms in Morocco. The results served as a foundation for future research aimed at comparing the efficacy of different vaccination strategies in controlling ARV infections in poultry farms across the country; focusing on evaluating the long-term effectiveness of different ARV vaccines, and identifying the most suitable vaccination schedules for different age groups and breeds of poultry; and investigating the potential benefits of combining vaccination with other control measures, such as biosecurity protocols, to enhance the efficacy of ARV control programs. This study will also contribute to filling the current knowledge gaps regarding ARV infections in chickens in Morocco, supporting the sustainable development of the poultry industry in the country.


Successful recanalization (TICI 2b-3) and hemorrhagic transformation were similar in both groups. The BT group more frequently showed excellent outcomes at 3 months . Multivariate analysis showed that BT was independently associated with excellent outcomes  and lower mortality . Conclusions: Compared with dMT, BT was associated with excellent functional outcomes and lower 3-month mortality in this real-world clinical practice study conducted in a region belonging to the “Stroke Belt” of Southern Europe. Given the disparity of results on the benefit of BT in the current evidence, it is of vital importance to analyze the convenience of its use in each health area. The aim of this 4-year observational study is to analyze the outcomes of stroke patients treated with direct mechanical thrombectomy (dMT) compared to bridging therapy (BT) (intravenous thrombolysis [IVT] + BT) based on 3-month outcomes, in real clinical practice in the "Stroke Belt" of Southern Europe. In total, 300 patients were included (41.3% dMT and 58.6% BT). The frequency of direct referral to the stroke center was similar in the dMT and BT group, whereas the time from onset to groin was longer in the BT group (median 210 [IQR 160–303] vs. 399 [IQR 225–675],  Intravenous thrombolysis (IVT) in eligible patients with acute ischemic stroke (AIS) and large vessel occlusion (LVO) is currently recommended before mechanical thrombectomy (MT) by American and European guidelines . HoweverIn the recent years, observational studies, randomized clinical trials (RCT), and various meta-analyses have been carried out in order to determine if direct MT (dMT) may be more beneficial than BT in AIS with LVO ,7,8,9,10In Europe, the burden of vascular risk factors, cerebrovascular disease mortality, and stroke management protocols differ among the different areas. In Spain, situated in the south of Europe, there is a specific southern region of the country  where higher rates of cardiovascular factors and stroke mortality have been reported, called the “Stroke Belt” ,16,17. SThe main objective of the present real-world study is to compare the functional outcomes, clinical characteristics, and the complication rate of AIS patients who underwent dMT vs. BT in Spain’s Stroke Belt, a region with a particularly high incidence of stroke and a mixed mothership and drip-and-ship-model for MT. We retrospectively analyzed through the records of patients ≥ 18 years admitted to the SC with AIS in the anterior or posterior circulation and LVO who were treated with dMT or BT, according to current International Guidelines, in Almeria, province of Andalusia (Southern Spain), between February 2017 and February 2021. Almería has a referral SC, the Torrecárdenas University Hospital, serving a total population of 739,293 inhabitants. All the patients admitted to the SC benefiting from a mechanical thrombectomy were enrolled consecutively during the study period. AIS from the SC health area were transferred directly, following a mothership model, while those residing in other areas (approximately 60% of the population) were initially treated in one of the regional hospitals of the province, following a drip-and-ship model. From March 2021, all patients in the province switched to a mixed model depending on the stroke severity, with a mothership model if the RACE scale was greater than or equal to 5. The nearest regional hospital is 36 km away and the farthest regional hospital is 113 km from the SC. The variables included were: demographic data, vascular risk factors and comorbidities, previous treatments, AIS blood biomarkers, stroke characteristics and etiology according to the TOAST classification, NIHSS on admission and after 24 h, reperfusion treatments (MT and IVT), time periods , recanalization by TICI grades, number of passes during MT, in-hospital complications, and 3-month outcomes according to the modified Rankin Scale score (mRS).The primary outcome was excellent functional outcome at 3 months, defined as a mRS score of 0–1. Secondary outcomes were death due to any cause at 3 months, defined as a mRS of 6, hemorrhagic transformation during the first 36 hours  according to the ECASS classification, and proportion of patients with successful recanalization (TICI 2b–3) .t-test or the Mann–Whitney test, as appropriate. Comparisons between groups were analyzed with the two or Fisher’s exact test for dichotomous variables. The relationship between BT and 3-month excellent outcome was assessed using a multivariate binary logistic regression model. Those variables with p < 0.2 in the bivariate analysis performed according to prognosis were included in the multivariate analysis. Those variables that differed significantly in the bivariate analysis between treatment groups were also included. A backward procedure was followed as the modelling strategy, using the log likelihood ratiotest to assess the goodness of fit and compare nested models. Those variables that when eliminated produced a change of ≥15% in the odds ratios (OR) were considered confounding variables. The OR and the 95% confidence intervals (CI) were used to evaluate the association strength. A p-value of < 0.05 was considered statistically significant.The statistical analysis was performed using IBM SPSS Statistics for Windows, Version 25.0, software  for Windows. Continuous variables were expressed as mean SD or median (interquartile range [IQR]) and compared with Student’s The study protocol is in accordance with the ethical guidelines of the 1975 Declaration of Helsinki, including all subsequent amendments. The project was approved by the Clinical Research Ethics Committee of the Torrecárdenas University Hospital. The data collected for the study were processed in accordance with the General Data Protection Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016.A total of 300 patients were included in the study, median age (IQR) 71 (61–80) years, 114 (48%) women. Overall, 139 (41.3%) were treated with dMT and 176 (58.7%) received BT. Follow-up at 3 months was not achieved in 21 patients due to transfer to the usual home in a different region, or other impediments. p < 0.001) as well as a higher percentage of wake-up stroke . Similarly, the dMT group had a lower percentage of previous excellent mRS, a longer duration of stroke symptoms at the time of MT , a higher rate of awakening stroke and anticoagulation , and higher door-to-groin time , with a lower median ASPECTS score . The main reason for dMT was a time window from stroke onset above 4.5 hours. Other reasons were neoplastic disease and recent anticoagulation intake. A higher proportion of patients with anticoagulant therapy was found in the dMT group , with no differences in TICI 2b/3 (84.9% BT vs. 82.8% dMT. p = 0.63). The dMT group had greater rates of recanalization failure (TICI-0) compared to BT , without a greater rate of symptomatic bleeding between the two groups . Mortality at 3 months was almost twice as high in the dMT group (14.2% BT vs. 26% dMT). At the 3-month follow-up, the percentage of patients who obtained an excellent functional outcome was twice as high in the patients who received BT  adjusted by confounders. Higher age  and NIHSS score , prior anticoagulation , significant cerebral edema , and lower respiratory tract infection during hospitalization  were inversely associated with a mRS 0–1 at three months. Three or fewer passes during the MT was independently associated with an excellent outcome . The multivariate logistic regression analysis  showed tp = 015). Higher age (OR 1.04. 95% CI 1.01–1.09. p = 0.016) and NIHSS , lower respiratory tract infection during hospitalization , and use of antiplatelet agents (OR 0.4 % CI 0.67–0.96. p = 0.04) were also associated with death. Another multivariate logistic regression analysis was performed considering death in the first three months, showing BT to be a protective factor  at three months after adjustment for confounding variables, as well as lower mortality. This higher rate of excellent prognosis was not associated with a higher rate of significant hemorrhagic transformation.Previous RCTs have yielded conflicting evidence about the benefit of IVT prior to MT. While two large RCTs showed non-inferiority of dMT versus BT with no significant differences in symptomatic intracerebral hemorrhage, a third Asian RCT as well as three other European RCTs did not ,21,22,23When this question is analyzed in real-world studies, the balance tips toward a greater benefit of BT treatment regarding the 90-day functional outcome ,29,30,31Likewise, several meta-analyses have been conducted in order to find an answer regarding the best therapeutic option in AIS with LVO. Those that have benefited the BT group included mainly observational, real-world, non-randomized studies, showing a better functional outcome, higher recanalization rates, and lower mortality with BT ,12,34,35Several factors explain this heterogeneity between studies, such as differences in the clinical and ethnic-related characteristics of the population, stroke severity, recanalization rates according to the size and location of the occluded artery (anterior or posterior), the transfer model (mothership or drip-and-ship), stroke etiology, and resources in the health area ,18,36,37Most of the previous studies that have addressed whether IVT prior to MT improves the stroke outcome have not analyzed the results based on the patient transfer model ,14,15,43It is worth mentioning that this study has been carried out in an area within the so-called “Stroke Belt”, performed for the first time in this population group with specific characteristics and cardiovascular risk factors. In studies carried out in the 2010s, it was observed that Andalusia had higher rates of risk factors such as hypertension, diabetes, cardiovascular disease, overweight, obesity, and smoking, as well as worse socioeconomic data (education and income) ,16. It tOn the other hand, a future has been recently opened with the administration of intra-arterial fibrinolysis after the performance of MT, following recent results published by Renú. A. et al. . The intThe main limitation of our study is its retrospective nature, which limited or prevented complete follow-up of some patients. In addition, the treatment decision was based on clinical characteristics with the aforementioned differences between groups. Compared to dMT, BT was associated with an excellent functional outcome and lower mortality at 3 months in a study of real-world clinical practice in a region belonging to the “Stroke Belt” of Southern Europe. Given the previously reported disparity of findings on the benefit of IVT prior to MT in ischemic stroke patients with LVO, it is of vital importance to analyze the convenience of its use in each health area, given that the demographic characteristics, the facilities of the regional hospitals, the health organization, and the transport times to the thrombectomy center differ from one community to another.


The long-term trends of cognitive function and its associations with physical performance remain unclear, particularly in Asian populations. The study objectives were to determine cognitive trajectories in middle-aged and elderly Chinese individuals, as well as to examine differences in physical performance across cognitive trajectory groups.Data were extracted from the China Health and Retirement Longitudinal Study. A total of 5,701 participants  with a mean age of 57.8  years at enrollment were included. A group-based trajectory model was used to identify cognitive trajectory groups for each sex. Grip strength, repeated chair stand, and standing balance tests were used to evaluate physical performance. An ordered logistic regression model was employed to analyze differences in physical performance across cognitive trajectory groups.Three cognitive trajectory groups were identified for each sex: low, middle, and high. For both sexes, higher cognitive trajectory groups exhibited smaller declines with age. In the fully adjusted model, relative to the low trajectory group, the odds ratios (ORs) of better physical performance in the middle cognitive group were 1.37  during follow-up and 1.40  at the endpoint. The ORs in the high trajectory group were 1.94  during follow-up and 2.04  at the endpoint.Cognitive function was better preserved in male participants and individuals with higher baseline cognitive function. A higher cognitive trajectory was associated with better physical performance over time. The global population and life expectancy are growing, leading to an escalating aging problem. In 2018, 249 million individuals in China were aged 60 and older, accounting for 17.9% of the total population , resultiA growing public health concern, aging is associated with declines in bodily functions and physical performance . PreviouCognitive impairment is an early stage of dementia, determined by a wide variety of neurological, psychological, and emotional factors ,12. The Recently, studies have demonstrated that changes in cognitive function are associated with physical performance in the areas of mobility , balanceUtilizing data from the 2011, 2013, and 2015 waves of the China Health and Retirement Longitudinal Study (CHARLS), this study was conducted with the goal of identifying diverse patterns of cognitive trajectories and examining their associations with physical performance outcomes in middle-aged and elderly populations.The data for this study was obtained from the CHARLS, a nationally representative prospective cohort survey conducted by the National School of Development at Peking University. The baseline survey took place in 2011 and was followed by 2 additional surveys in 2013 and 2015, utilizing a multistage probability sampling design and a probability-proportional-to-size sampling technique . The CHAIn the present study, participants who completed all 3 waves of surveying were included . Among these individuals, those with incomplete demographic information (n=36), missing physical performance indicators , and incomplete cognitive function measurements  were excluded. Consequently, 5,701 participants were included in the data analysis.Physical performance was considered to be indicated by grip strength, repeated chair stands, and 2 standing balance tests. Each of these indicators was assigned a score of either 0 or 1, depending on whether they were completed successfully. The total physical performance score was calculated by summing the scores of the 3 indicators, and this was done at baseline, during follow-up, and at the endpoint. The physical performance score ranged from 0 to 3.Grip strength, an indicator of upper limb muscle strength, was measured using a dynamometer  and recorded in kilograms ,19. DuriRepeated chair stands, an indicator of lower limb muscle strength, were assessed using a standard chair with a height of 47 centimeters from the floor . ParticiThe standing balance test, which evaluates static balance, endurance, and postural control ability, was assessed using semitandem stands and full-tandem stands . In the Cognitive function in the CHARLS was assessed across 2 dimensions, evaluating orientation, attention, visuospatial abilities, and word recall ,24. The Baseline socio-demographic characteristics and health-related factors were considered for the adjusted analysis. Socio-demographic characteristics included age, sex , residence , province (of 28 provinces), marital status (married or not), and education level . Residence was defined based on China’s National Bureau of Statistics, with urban areas including cities, city suburbs, towns, town suburbs, and other special zones, while all other areas were considered rural ,25. For We utilized group-based trajectory modeling to identify distinct trajectory groups of cognitive function as they relate to age . TrajectTo assess whether the socio-demographic characteristics and health influence factors differed among the 3 cognitive trajectory groups, the chi-square test was employed for categorical variables, while the independent-samples t-test was utilized for continuous variables. Physical performance was an ordinal categorical variable with a score range of 0 to 3, determined by 3 independent measures of 0 or 1 on grip strength, repeated chair stands, and standing balance tests. An ordered logistic regression model was used to analyze the differences in physical performance of participants from the 3 cognitive trajectory groups at baseline, during follow-up, and at the endpoint, using the low trajectory group as the reference. Odds ratios (ORs) and 95% confidence intervals (CIs) of physical performance for each cognitive trajectory group were calculated. Age, marital status, education level, BMI, smoking status, and drinking status were included in adjusted model 1. The presence of chronic diseases, categorized as cardiovascular and cerebrovascular metabolic diseases, psychiatric and neurological diseases, and other diseases, was incorporated into adjusted model 2. Considering that 556 (9.7%) respondents had missing grip strength data due to 1-handed disability or inconvenience, sensitivity analyses were conducted on respondents with complete grip strength data in adjusted model 3 to avoid the manifestation of poor physical performance. All analytical procedures were stratified by sex and residential status. Additionally, we analyzed the relationship between the cognitive trajectory group and each original variable of physical performance to avoid ceiling effects.All statistical analyses were conducted using Stata version 14.0 . An association was deemed significant if the associated 2-sided p-value was less than 0.05.The study was approved by the Ethical Review Committee of Peking University (No. IRB00001052-11014), and all participants provided written informed consent.A total of 5,701 middle-aged and elderly Chinese individuals participated in this study , with an average baseline age of 57.8 years . Male participants demonstrated higher grip strength than female participants  and performed more successful repeated chair stands . The baseline cognitive function scores were 16.1±4.6 for male participants and 14.3±5.4 for female participants. Demographic characteristics and lifestyle information of the participants at baseline, by sex, are presented in The distribution of cognitive function scores for each cognitive trajectory group during the follow-up period is displayed in In the fully adjusted model, compared to the low group, the ORs of better physical performance in the middle and high groups at baseline were as follows: 1.92  and 2.92  for urban male, 1.12  and 1.86  for urban female, 1.28  and 2.07  for rural male, and 1.25  and 1.55  for rural female. During follow-up, the ORs of better physical performance in the middle and high groups were 1.04  and 2.21  for urban male, 1.32  and 1.77  for urban female, 1.71  and 2.53  for rural male, and 1.30  and 1.67  for rural female. At the endpoint, the ORs of better physical performance in the middle and high groups were 2.60  and 2.53  for urban male, 1.57  and 2.31  for urban female, 1.36  and 2.09  for rural male, and 1.42  and 2.06  for rural female . All treSensitivity analyses were performed for respondents with complete grip strength data, separated by sex and residential status . The resIn this study, we conducted a 4-year follow-up with 5,701 middle-aged and elderly individuals and observed a significant overall decline in cognitive function as they aged. We identified 3 distinct cognitive trajectories, which we labeled as low (19.4%), middle (43.5%), and high (37.2%). All 3 cognitive trajectories displayed parallel downward trends with age, suggesting that cognitive impairment is a chronic and irreversible degenerative pathological process . We discOur findings demonstrated that participants with better cognitive function at baseline tended to maintain a more stable level of cognitive function as they aged. This trend was consistent with the results of previous regional studies conducted among American and Mexican populations ,31. AddiThe finding that a decline in overall cognitive function is associated with a decline in physical performance aligns with previous studies ,29,37. PThe differences in physical performance between cognitive trajectory groups also widened among female participants over time, while those in male participants remained stable. However, the underlying mechanism requires further exploration. After stratifying by urban-rural residence, we found that urban participants were more likely to be categorized into the high cognitive trajectory group, while rural participants were more likely to be assigned to the middle-low cognitive groups. This, coupled with the facts that cognitive function declined more rapidly in the lower trajectory groups to which rural participants were more likely to be assigned  and thatThe present study had several potential limitations. First, although we found that the group with a higher cognitive trajectory exhibited better physical performance, the causal relationship and mechanism underlying this transition remain unknown and warrant further research. Second, while factors such as residence, sex, age, marital status, BMI, smoking, drinking, and chronic diseases were included in our analysis to control for confounding effects, other influential factors such as socioeconomic status and sleep duration could not be incorporated due to the design of the original CHARLS study. Third, approximately 60% of respondents from the original CHARLS dataset had missing values for key information such as cognitive function and grip strength and were therefore excluded from the present analysis. This exclusion may also impact the effect size and generalizability of our findings. Nonetheless, the CHARLS is a database that covers 150 counties/districts and 450 villages/residential committees in 28 provinces of China and possesses strong national representativeness. The relationship between physical performance and cognitive trajectory groups identified in the present study still provides a reliable basis for further research.Using CHARLS data, we discovered that improved cognitive function was associated with male sex, urban residency, high BMI, and high cognitive trajectory. Participants from the high cognitive trajectory group demonstrated better physical performance, suggesting that early interventions to maintain stable cognitive function may be crucial for preserving optimal physical performance in middle-aged and elderly individuals.


There was no thrombosis reduction , p = 0.10) or intervention number reduction , p = 0.84) with BH. Outcomes like pain, hematoma, and aneurism could not be merged due to a lack of data, reported as medians, as well as due to different definitions. The quality in general was poor and the heterogeneity among the studies prevented us from merging the outcomes.Adequate cannulation technique (CT) methods and successful puncture are essential for hemodialysis (HD) and arteriovenous fistula (AVF) maintenance. This systematic review and meta-analysis was designed to identify which CT allows better AVF primary patency and lower rates of complications in HD patients. The search was carried out on the CINAHL, MEDLINE, Cochrane Library, and Joanna Briggs Institute Library databases to identify all randomized controlled trials (RCTs) and observational studies comparing clinical outcomes of buttonhole (BH) versus rope ladder cannulation (RL) from 2010 to 2022. The Risk-of-Bias (Rob 2) tool was used for RCTs and the ROBINS-I was used for non-randomized studies. RevMan 5.4 was used for the meta-analysis. A total of five RCTs, one quasi-randomized controlled trial, and six observational studies were included. When compared with RL cannulation, BH cannulation significantly increased bacteremia ,  With the increase in the number of elderly patients, the exhaustion of vascular territory, and the emergence of diabetes as the primary cause of renal etiology, the establishment and preservation of suitable vascular access (VA) is essential for the successful treatment of patients with end-stage kidney disease (ESKD) on hemodialysis (HD) programs. A functioning VA is the lifeline ,2 that aHowever, challenges have been identified in the use of this cannulation technique such as severe pain with an impact on treatment time ,12 and aIn recent years, some studies have been published ,19,20 thThis systematic review and meta-analysis was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines . The proWe conducted searches on the EBSCO platform, accessing the Cumulative Index of Nursing and Allied Health Literature (CINAHL) and MEDLINE databases using the following Medical Subject Headings (MeSH) (2021) and strategy:  OR  OR  OR (MH “Kidney Failure Chronic”)) AND (((MH “Arteriovenous Fistula”) OR (MH “Catheterization”)) AND ((“Buttonhole”) OR (buttonhole) (OR constant site)) AND ((rope-ladder) OR (ropeladder) OR (rotating site))). We also considered other databases such as the Cochrane Library, ScienceDirect web, Joanna Briggs Institute Library Evidence-Based Practice Network (JBI), SCOPUS, ResearchGate, American Society of Nephrology (ASN), American Nephrology Nurses Association (ANNA), Sociedade Espanhola de Nefrologia (SEN), and Sociedade Brasileira de Nefrologia (SBN). We considered these databases because they publish randomized controlled trials (RCTs) and observational studies in the nephrology field. All the articles included in the review were available in full text. A flow chart is shown in We included all RCTs and quasi-experimental and prospective observational studies published between January 2000 and January 2022 that satisfied the following criteria: studies that compare CTs and thus define the advantages and risks of each CT; primary and full studies, or abstracts that include one or some outcomes. We reviewed articles in English that enrolled adults aged 18 years and older who underwent hemodialysis using an autogenous AVF. Patients who underwent BH were considered the experimental group, and patients who underwent RL or other CT were considered the control group.We excluded studies associating patient data from home hemodialysis with data from hospital or hemodialysis clinics and studies with incidents of patients on hemodialysis and qualitative studies.The primary outcome was the fistula primary patency, and according to Lee T. et al. , this waThe secondary outcome was considered fistula survival, for which failure was defined as AVF no longer used for successful HD. The numbers of interventions, thrombosis, bacteremia, cannulation pain related to CT, hematoma or infiltration, bleeding time, aneurysm, and unsuccessful cannulation were considered.First, the studies were independently selected by a reviewer (RP). The titles and content of the abstracts were assessed. Then, the second reviewer (AC) carried out the subsequent verification and validation. This selection was made strictly according to the inclusion and exclusion criteria defined in the protocol published elsewhere . All dupTo assess the risk of bias in RCT studies, we used the Revised Cochrane Risk-of-Bias Tool for Randomized Trials (RoB 2) tool . Accordin), and outcome analysis method. Outcomes: participant’s characteristics , context of dialysis , follow-up of the study (months), and primary and secondary outcomes obtained, including the pain scoring tool used to define the severity of pain. We performed a meta-analysis only when studies were sufficiently homogeneous in terms of participants, interventions, outcomes, measurement, and method of aggregation . We presented the results in a narrative form when statistical comparisons were not possible. Tables and figures were included to facilitate the presentation of the data. Meta-analysis was performed with the generic inverse variance method using Cochrane Collaboration Review Manager software (RevMan 5.4) for Windows.The studies selected for the systematic review were presented in a summary table with the following main attributes: the author’s name, year of publication, country, study design, sample size , sOnly four studies evaluated this outcome ,19,27,28All studies had a critical risk of bias in the first two domains . No basep < 0.1 e I2 = 81%), it was not possible to prove which CT allowed greater unassisted primary patency  p = 0.90).Four RCTs reported this outcome, and the results are shown in p < 0.1 e I2 = 78%), it was not possible to prove which CT allowed a lower rate of interventions (angioplasty or surgery) in AVF  p = 0.84).There were three studies  that repp > 0.1 e I2 = 0%). However, it was not possible to confirm which CT allowed a lower frequency of AVF thrombosis  p = 0.10).In the four studies that reported AVF thrombosis ,19,26,27All six RCTs  assessedp < 0.001) associated with BH .RCTs ,16,27,28observed  in the ip = 0.003) in local signs of infection associated with BH (50 per 1000/HD sessions) compared with the SN group (22.4/1000 HD sessions).There also seemed to be a higher rate of localized infection in the BH group. MacRae J. et al.  showed ap = 0.05) regarding pain reduction in the control group. In this study, eight patients asked to change to the usual practice because of pain associated with the BH. In contrast, three observational studies [p = 0.0049, and p < 0.001) when using BH. Of the eight studies evaluating cannulation pain, in five studies, the participants used analgesic cream.There were three RCTs ,26,27 T that ass studies ,32,33 (Tp = 0.003) compared with SN. Van Loon et al. [p < 0.0001). Apparently, there was an advantage associated with BH in reducing hematomas, but it was not possible to merge data because the presentation of the results was different. We understand that further studies are needed to assess the frequency of hematomas associated with CT.There were two RCTs ,26 Tabl and two n et al.  also fouThe three RCTs ,26,27 T failed tThere seemed to be unanimity of both RCTs  and obsep < 0.001) compared to standard CT mainly after the fourth month. In another study [Nurses perceived significantly higher levels of difficulty with both arterial and venous cannulation in the BH  group  ,34,35, rOther studies  also facp = 0.20) were found in AVF survival, even with longer follow-up. These are very important outcome indicators, but there was limited research and the few selected studies revealed considerable heterogeneity (I2 = 81%). Therefore, it is recommended that future studies perform more analysis on fistula survival.The results of this meta-analysis indicated that BH did not show evidence of superiority in primary patency or in the reduction in the number of interventions when compared with RL. In the study by MacRae et al. , no signThe results of this meta-analysis are inconclusive if BH reduces the number of cases of thrombosis; however, there was a clear trend toward a lower incidence of this event. Two previous systematic reviews ,39 conclp = 0.07). However, we must consider that there were only four trials with a small sample size (243 versus 246) and a small event size (17 versus 5), and these results showed a lack of statistical power. A retrospective observational study [The results indicated that BH has a higher risk of AVF bacteremia than RL. These results are similar to those of another systematic review , which sent size  versus 5al study .However, other studies ,39 did nThis study showed that BH did not reduce the incidence of pain, despite the reduction in injury caused by blunt needles. However, this can be explained by the increase in miscannulation and the “trampoline” effect ,30 usingThere is a clear trend in favor of BH in reducing the rate of hematomas and aneurysm development in RCTs and observational studies. This is in accordance with other studies ,39 that As limitations of this study, we found some constraints on the quality of the included studies, especially the observational studies. The multiple definitions of RL in the control group limited the results, with implications for decisions in clinical practice. Also, the follow-up lengths of the studies were short, and outcomes such as infection and new aneurysms occurred late. Another limitation was the small number of studies and participants, so it would have been useful to conduct a sequential analysis of trials ,42.This meta-analysis demonstrated that BH is significantly associated with higher bacteremia; however, it found no differences in AVF primary patency, number of interventions, or thrombosis. Therefore, BH should be exclusively reserved for home dialysis patients or those with anatomical constraints, as described by some authors ,43.To select the best CT for each person, it is necessary to adopt a decision model ,45 that


Negative correlations were found between the R-index and body weight, BMI, WHR, subcutaneous and visceral fat surface and the VAT/SAT ratio, and total body fat; positive links were found with fat free mass and total body water. Opposite associations were noted between GSSG level and the aforementioned body composition parameters. Oxidative stress characterized by a depleted reduced-to-oxidized glutathione index is associated with anthropometric and body composition parameters in women with PCOS. In particular, abdominal obesity expressed by the VAT/SAT ratio and/or WHR seems to have a negative impact on glutathione status, which may lead to a disruption of many biological cell processes. The observed negative association of Keap1 with R-index suggests that the elevated oxidative changes dependent on the VAT/SAT ratio may lead to Nrf2 activation to promote antioxidant enzyme expression. Although the GSH/GSSG index as well as the VAT/SAT ratio appear to be good indicators of oxidative status, studies on a larger group of patients should continue to confirm these links among women with PCOS.Metabolic disorders present in women with polycystic ovary syndrome (PCOS) and the associated risk of obesity may result in increased oxidative stress and reproductive failure. Therefore, we evaluated the concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione peroxidase (GPx), and reductase (GR), as well as nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein1 (Keap1) in the serum of 56 women with PCOS divided according to the visceral to subcutaneous fat surface ratio (VAT/SAT) and waist-to-hip ratio (WHR) values. Antioxidant parameter levels were measured by competitive inhibition enzyme immunoassay technique. As the VAT/SAT ratio and WHR increased, we observed significantly higher concentrations of GSSG and Keap1 protein and a lower value of the GSSG/GSH ratio (R-index), which is considered an index of cellular redox ( Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies and its prevalence—depending on the adopted diagnostic criteria and the population studied—ranges from 4 to 16% of reproductive-age women ,3. CharaIt is estimated that up to 60–70% of patients with PCOS are overweight or obese, with particularly increased central distribution of adipose tissue. Central accumulation of adipose tissue is characteristic of both lean and obese women with this disease entity . VisceraInsulin resistance in PCOS resulting in hyperglycemia and higher levels of free fatty acids can lead to increased reactive oxygen species (ROS) production, especially when accompanied by overweight and obesity. Because PCOS is also associated with reduced antioxidant levels, it is considered a state of increased risk for oxidative stress. Research conducted in this area suggests that there may be a strong relationship between impaired adipose tissue metabolism, insulin resistance, hyperandrogenism, inflammation, and oxidative stress in the pathogenesis of PCOS ,16,17,182O2) and lipid peroxide, oxidizing to disulfide. Regeneration of the active thiol form occurs with the participation of NADPH-dependent glutathione reductase (GR), belonging to flavoproteins. In addition to the regeneration of GSH from GSSG, the second process affecting the increase in glutathione concentration is its neosynthesis. The de novo synthesis of GSH in the cell is limited by the availability of its constituent amino acids, and in particular by the availability of the sulfur amino acid precursor, cysteine /22.5.Serum concentrations of antioxidant defense parameters were measured using immunoenzymatic methods following the manufacturer’s instructions.GSH and GSSG levels were determined using kits based on a sandwich enzyme-linked immunosorbent assay (ELISA) with two specific and high affinity monoclonal antibodies , which we have previously described in detail . To asseNuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or Nrf2 was measured using human an NFE2L2 ELISA kit  based on the double-antibody sandwich ELISA technique. The pre-coated antibody was an anti-human NFE2L2 monoclonal antibody, while the detection antibody was a biotinylated polyclonal antibody. Samples and biotinylated antibodies were added into ELISA plate wells. Then, avidin–peroxidase conjugates (HRP-streptavidin) were added to the wells. TMB substrate was used for coloration after the enzyme conjugate had already been thoroughly washed out of the wells. TMB  reacts to form a blue product from the peroxidase activity, and finally turns to yellow after adding the stop solution. The color intensity and quantity of target analyte in the sample were positively correlated. The levels of GR, GPx, and Keap1 proteins were determined in an analogous manner using a human glutathione reductase ELISA kit , a human glutathione peroxidase ELISA kit , and a human KEAP1 (Kelch-like ECH-associated protein 1) ELISA kit , respectively. The concentrations of NFE2L2, Keap1, GR, and GPx in the samples were calculated by comparing the O.D. of the samples to the standard curve. The intra- and inter-assay CVs were less than 8.0% and 10% for NFE2L2, Keap1, and GPx, whereas they were 10% and 12% for GR, respectively. Assay sensitivity was less than 0.094 ng/mL for NFE2L2, 14.063 pg/mL for Keap1, 0.260 ng/mL for GPx, and 35.000 pg/mL for GR, respectively.t-test and in the second case using the nonparametric Mann–Whitney test. The level of correlation between GSH, GSSG, as well as the R-index and anthropometric parameters was also calculated using Spearman’s rho. The relationship between Keap1 and the three aforementioned antioxidant defense parameters are presented graphically as a scatter plot.Statistical analysis included 40 parameters in four groups: baseline clinical features (13), anthropometric data and body composition (20), and antioxidant defense parameters (7). The normality of each parameter was checked using the Kolmogorov–Smirnov test. The study group was divided into two according to the visceral to subcutaneous fat surface ratio, with a cut-off point of 0.90, and independently into two groups according to the waist-to-hip ratio values, with a cut-off point of 0.85. For each of the identified subgroups, the mean values of the respective parameters were presented with the standard deviation (SD) if the distribution did not deviate from normal, or the median along with the interquartile range (1-3IQR) if the hypothesis of normality of distribution was rejected. In the first case, the groups were compared using the parametric Student’s All reported p-values were two-tailed, and values = <0.05 were considered significant. IBM SPSS v.28 statistical software was used . p < 0.01). Fasting glucose levels were also higher in these groups, but the difference was not statistically significant. p < 0.001) and WHR < 0.85 (p < 0.01). Additionally, lower VAT/SAT values were associated with lower levels of 17-OHP (p < 0.01). We did not observe significant differences in LH, FSH, T, E2, A, TSH, and DHEAS concentrations between the studied groups. We found that patients with higher VAT/SAT and WHR ratios had significantly lower levels of SHBG than patients with VAT/SAT ≤ 0.90 (p < 0.001). Additionally, this group of women was characterized by a significantly higher WHR compared with women with VAT/SAT ≤ 0.90 (p < 0.001). Similar relationships were observed in the group of women with WHR ≥ 0.85. The subjects’ detailed anthropometric data and body composition measures are shown in As expected, anthropometric and body composition indices differed significantly in both groups divided by VAT/SAT and WHR values. The PCOS group with increased VAT/SAT ratios had statistically higher subcutaneous and visceral adipose tissue content, BMI, total body fat, and muscle mass compared with the PCOS group with normal VAT/SAT ratio .The correlations of GSH, GSSG, and the R-index with anthropometric parameters and body composition in women with PCOS are shown in detail in p = 0.001; r = 0.405, p = 0.002, respectively) and negatively correlated with R-index values . In addition, associations between Nrf2 concentrations and insulin  and HOMA-IR levels  were on the border of significance.Increased concentrations of insulin and HOMA-IR levels observed in women with PCOS were positively correlated with oxidized glutathione concentrations . Other relationships between the selected antioxidant defense parameters were not confirmed.Compared with healthy counterparts, body fat distribution is different in women with PCOS due to the predominant accumulation of visceral fat and abdominal obesity ,12,32. AStudies determining glutathione status in patients with PCOS mainly concern its reduced form or the activity of glutathione peroxidase ,17. In t2O2, reduced glutathione is oxidized to disulfide (oxidized glutathione), which—with the participation of glutathione reductase and NADPH coenzyme—is reduced back to thiol, which is an important redox cycle in the cell [In the present study, we showed differences in the levels of the tested oxidative stress markers both when dividing the groups due to WHR, determining abdominal obesity, and VAT/SAT, determining an additional increased risk of metabolic syndrome. We demonstrated that reduced glutathione was slightly higher in the group of PCOS women with low WHR and VAT/SAT ratios. This finding is consistent with the results obtained by Uckan et al. , who fouthe cell . The effthe cell ,35. Datathe cell . Oxidizethe cell . In the In our study, GR levels were the lowest in patients with the highest VAT/SAT ratio, while GR correlated positively with GSSG concentration and negatively with the R-index value. This confirms the important role of this enzyme in the glutathione redox cycle. As we noted in the introduction, in addition to the regeneration of GSH from GSSG via GR, the concentration of this compound in cells is dependent on de novo synthesis. The neosynthesis of GSH in the cells is limited by the availability of cysteine, whose oxidative modifications are largely responsible for glutathione’s role in redox-dependent cell signaling. Containing cysteine residues, Nrf2 participates in the transcriptional regulation of many antioxidant genes, including enzymes that regulate the rate of glutathione synthesis. Activation of Nrf2 by pro-oxidant factors or specific Nrf2 activators  is associated primarily with conformational changes in the Keap1 inhibitory protein ,38,39,40The links between oxidative stress, glutathione, and the Keap1-Nrf2 system have not yet been described. We observed slightly increased Nrf2 levels in patients with abnormal VAT/SAT and WHR ratios. In addition, in the group with VAT/SAT > 0.90, we found significantly higher Keap1 protein levels. Moreover, the negative association of Keap1 with the R-index may suggest that the elevated oxidative changes observed in this group may lead to Keap1 dissociations from Nrf2 and the activation of this factor to promote antioxidant enzyme expression. The unchanged GPx levels observed in all the groups with PCOS and only slightly reduced GR in the groups with increased risk of metabolic disorders may be the result of a compensatory antioxidant response associated with the activation of the Keap1-Nrf2 system. It is currently known that, in addition to Keap1, there are other factors that can regulate Nrf2 gene transcription. These include phosphorylation of Nrf2 by protein kinases or acetylation of Nrf2 ,42. For A strength of the presented research was taking comprehensive measurements of glutathione status markers in the blood of women with PCOS in relation to body composition, with particular emphasis on visceral adipose tissue storage. Moreover, determining the ratio of GSH to GSSG allowed us to estimate the severity of oxidative stress in these patients. Assessment of Nrf2 and Keap1 proteins in the serum of patients with PCOS also seems to be important, although, on the other hand, it may be a certain limitation. Serum concentrations of this factor may not reflect the true cellular antioxidant response as Nrf2 functions mainly in the cell nucleus, and released forms do not always represent its free or active status. Another limitation of the study is that we did not measure serum levels of sulfur amino acids, which are important for the synthesis of this compound. However, we are planning to assess the amino acid profile—such as cysteine, cysteamine, cysteinyl-glycine, and homocysteine—in a future study of patients with PCOS. Finally, the lack of lipid profile and inflammatory markers may be a further limitation of our study. However, it is well known that patients with PCOS often have abnormal lipid levels, and chronic low-grade systemic inflammation is an important factor in this disease ,33,43,44In conclusion, oxidative stress characterized by a depleted reduced-to-oxidized glutathione index is associated with anthropometric and body composition parameters in women with PCOS. In particular, abdominal obesity expressed by the VAT/SAT ratio and/or WHR seems to have a negative impact on glutathione status, which may lead to a disruption of many biological cell processes. The observed negative association of Keap1 with the R-index suggests that the elevated oxidative changes dependent on the VAT/SAT ratio may lead to Nrf2 activation to promote antioxidant enzyme expression. Although the GSH/GSSG index, as well as the VAT/SAT ratio, appear to be good indicators of oxidative status, studies on a larger group of patients should continue to confirm these links among women with PCOS.


Lactobacillus, Bifidobacteria, Akkermansia, and Roseburia in CKD rats. Supplements of RMD should be advantageous in the treatment of gut leakage and microbiota dysbiosis in CKD.Microbiota-dysbiosis-induced gut leakage is a pathophysiologic change in chronic kidney disease (CKD), leading to the production of several uremic toxins and their absorption into the bloodstream to worsen the renal complications. We evaluate the benefits of resistant maltodextrin (RMD) and chitosan oligosaccharide (COS) supplements in cell culture and CKD-induced rats. The RMD exerted a significant anti-inflammatory effect in vitro and intestinal occludin and zonula occluden-1 up-regulation in CKD rats compared with inulin and COS. While all prebiotics slightly improved gut dysbiosis, RMD remarkably promoted the relative abundance and the combined abundance of  Chronic kidney disease (CKD) is a global health crisis. Patients with CKD suffer from the retention of serum uremic toxins that subsequently instigate systemic complications. Uremic toxins are mainly produced in the intestine by the gut microbiota and absorbed into the bloodstream. In CKD patients, harmful gut microbiota overgrowth leads to the production of uremic toxins, inflammation, and an increase in gut permeability ,2. The rSeveral approaches to suppress uremic toxin synthesis and absorption have been recently introduced to alleviate CKD progression or induce renal recovery. Among these, dietary supplementation with prebiotics and probiotics was one of the most promising methods ,4,5,6. PRecent studies disclosed the usefulness of other prebiotics in modifying gut microbiota and modulating gut and other systemic diseases. Chitosan, as a non-plant derived prebiotic, was used to treat bowel inflammation and metabolic syndrome ,12, and The resistant maltodextrin (RMD) and inulin used in this study were purchased from Banpong Novitat Co., Ltd. . RMD is an oligosaccharide with random 1–2, 1–3, 1–4, and 1–6 α and β glycosidic linkages, making it partially resistant to human digestive enzymes. The glycemic index of RMD was evaluated to be approximately 59, and it has a 2000 Da molecular weight. Inulin is a chain of fructose molecules linked together, with a glucose molecule at one end. The molecular weight of inulin typically ranges from approximately 500 to 4000 daltons.Chitosan oligosaccharide (COS) is an oligomer composed of β-(1-4)-linked d-glucosamine units. It is derived from the deacetylation and hydrolysis of chitin. The molecular weight of COS is less than 10,000 daltons, and each molecule consists of approximately 55 monomers. A small molecule COS used in this study was synthesized and provided by Assist. Prof. Dr. Rath Pitchyangura from the Department of Biochemistry, Faculty of Science, Chulalongkorn University, with a molecular weight of around 5–9 kDa.v/v FBS , 1% v/v penicillin, and 1% v/v streptomycin and incubated at 5% CO2 and 37 °C. The cells were seeded in a 96-well plate with 15,000 cells per plate and incubated for 24 h. The cells were washed with PBS  and new DMEM was added to the test substances for 24 h. The test substances included inulin, RMD, and COS at 10, 50, 100, 500, and 1000 µg/mL. PBS, 0.05% acetic acid, and 0.3% H2O2 were used as positive control, vehicle, and negative control for viable cells, respectively. After 24 h, 3--2,5-diphenyltetrazolium bromide (MTT) was added to the cells. A multidetection microplate reader was used to detect the absorbance at 570 nm on the culture plate. The survivability of cells exposed to different concentrations of the test substances were compared to the positive control.Caco-2, human colon adenocarcinoma cells were used in this study. The cells were cultures in sterilized DMEM  supplemented with 10% 2 culture surface) and incubated for 21 days with high glucose DMEM supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. Then, the cells were washed by PBS and the medium was replaced by FBS-free media. The cells’ TEER values were measured using an epithelial voltohmmeter (EVOM2) to obtain TEER values at 0 h. The cells in the transwell were treated with PBS, as a control, and the tested substances, including 100 µg/mL of inulin, COS, and RMD. A concentration of 60 ng/mL of TGF-β, growth factor, was used to see the TEER value in integrity-enhanced epithelial tissue. The study was carried out in 2 settings: normal and inflammation. In the inflammation setting, the cells were challenged by adding 50 ng/mL TNF-α, pro-inflammatory cytokine, in both the apical and basolateral sides to induce cell inflammation that disturbs the epithelial integrity of the cells 1 h before adding the test substances. The cells from each treatment were incubated for 24 h and then their TEER values were remeasured to obtain TEER values at 24 h. Fold changes were calculated from the ratio between TEER values at 24 h and TEER values at 0 h. The fold change in TEER values from each treatment was compared to the fold change in TEER from the control to calculate the %TEER to control.Caco-2 cells of 500,000 cells were seeded into transwell , along with the designated treatments for the experiment. Additionally, the rats were orally administered 54 mg of inulin per kg, 16 mg of COS per kg, or 54 mg of RMD per kg of rat. An approximately 0.5 mL blood sample was obtained from each rat’s tail vein using heparinized 1.5 mL microcentrifuge tubes. The blood was then centrifuged at 2500 G for 10 min to separate the plasma, which was subsequently stored at −80 °C. The rats’ fresh feces were collected and preserved in DNA/RNA Shield®® reagent  at −80 °C.During this period, the rats were provided with normal chow (CP 802), comprised of carbohydrate 52%, fat 19.77%, and protein 28.23%, and drinking water containing 1% 2. On the day of sacrifice, blood samples were collected via cardiac puncture. The kidneys were longitudinally split, with half of the specimens placed in 4% paraformaldehyde and stored at 4 °C, while the other half was collected in RNAlaterTM and stored at −80 °C. The jejunum section of the intestine was cut into 1 cm pieces and stored at −80 °C. Fecal samples were also collected and stored in DNA/RNA Shield®® reagent (ZYMO Research) at −80 °C. The remaining sections of the intestine and right femur bones were collected and preserved in paraformaldehyde at 4 °C.Blood samples were collected from all rats before the administration of cisplatin, at the start of the treatments, and on the day of sacrifice. At the end of the twelve-week study period, the rats were euthanized using long exposure to CORNA was extracted from the jejunum sections of the intestinal tissue, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was performed. RT-qPCR utilized primers specific to GAPDH (a housekeeping gene), claudin-1, occludin, and zonula occludens-1 (ZO-1). The goal was to measure the relative expression of each tight junction protein in response to different treatments.To determine the fold changes of each tight junction protein relative to the control group, the expression levels were calculated and normalized to the housekeeping gene. This analysis allowed for an assessment of the impact of each treatment on the expression of these crucial tight junction proteins.https://idal.uv.es/aclara/, accessed on 13 July 2023) [The concentrations of creatinine, calcium, and phosphate in the samples were analyzed using the automated Alinity ci system at the Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University. The serum parathyroid hormone (PTH) concentration from each treatment was determined using an enzyme-linked immunosorbent assay (ELISA) of a parathyroid hormone ELISA kit in 96-well plates purchased from Wuhan Fine Biotech Co., Ltd. . The optical densities were measured at a wavelength of 450 nm using multidetection microplate readers. In addition, the estimated creatinine clearance (eClCr)  was calcly 2023) .The tissue samples were subjected to permanent slide-making processes at the Department of Pathology, Faculty of Medicine, Chulalongkorn University. Sections of jejunum tissues were stained using the hematoxylin and eosin (H&E) staining technique to visualize the ZO-1 protein. Immunofluorescent staining was performed using a rabbit polyclonal antibody specific to ZO-1. The evaluation of the stained tissues was conducted by a pathologist affiliated with the Department of Pathology, Faculty of Medicine, Chulalongkorn University.The DNA extraction process from fecal samples involved using the ZymoBIOMICS™ DNA Miniprep Kit according to the manufacturer’s instructions provided by Zymo Research Corp and supplied by S.M.Chemical Supplies Co., Ltd., Bangkok, Thailand. The resulting DNA was then assessed for concentration and purity using a DeNovix™ UV-vis spectrophotometer  and stored at −20 °C until further analysis.To analyze the intestinal microbiome, the 16S/ITS Microbiome Profiling Service offered by Modgut Genomic Service at King Mongkut’s University of Technology Thonburi (Thailand), was utilized. The V3–V4 region of the 16S rRNA gene, serving as the target sequence, was amplified. The richness of microbial taxonomic groups in the samples was determined by the number of groups, while the evenness of their distribution indicated the evenness of the groups. For data visualization, an analysis of the alpha diversity, which includes the Shannon index and Pielou’s evenness, was conducted to summarize the ecological communities of the gut microbiota in terms of richness and evenness. The top relative abundance taxa were determined by calculating the average abundance of each taxon in each group divided by the total abundance of that taxon. The relative abundance of the bacteria was further analyzed and correlated with physiological parameters.t-test for comparing two independent groups, while the ANOVA with post hoc Bonferroni test was used for comparisons involving more than two groups. Non-parametric variables were analyzed using the Mann–Whitney U test. In an animal experiment, the Student t-test was employed to compare two independent groups, and the Kruskal–Wallis test was used to evaluate differences in means for non-parametric data. To examine correlations, Pearson correlation was employed. Significance was determined at p < 0.05. Figures, diagrams, and graphs were created using GraphPad Prism 9 (Windows 64-bit) v9.5.1.733.The statistical analysis in the current study was conducted using SPSS version 22.0. Continuous data were assessed using the Student The research adhered to the principles outlined in the Helsinki Declaration and followed the guidelines of good clinical practice when involving human participants, who provided fecal samples. The experimental procedures involving animals were conducted in accordance with the protocols of the Institutional Animal Care and Use Committee (IACUC). Ethical approval for the study was obtained from the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (IRB number 914/64). The protocols for animal experiments were approved by the Chulalongkorn University Animal Care and Use Committee (CU-ACUC protocol number 004/2563).In this study, we conducted an MTT survivability test on Caco-2 cells to assess the potential cytotoxicity of inulin, chitosan oligosaccharide (COS), and resistant maltodextrin (RMD). Our results demonstrated a significant decrease in cell viability at a concentration of 1000 µg/mL for inulin and COS, and at 500 µg/mL for RMD A. These To further investigate the effects of prebiotic treatments on intestinal barrier integrity, we employed the TEER assay. The TEER values of the Caco-2 cell monolayers were compared between each prebiotic treatment and the control group. As a positive control for TEER enhancement, TGF-β was included. In the normal setting, none of the prebiotic treatments exhibited a significant increase in the %TEER compared to the control group, as shown in All rats with CKD exhibited significantly higher serum creatinine levels compared to the control group, indicating the induction of CKD through cisplatin administration. The estimated creatinine clearance of CKD rats was about 41.1% lower than the control . However, no significant differences were observed between prebiotic-treated and untreated rats in terms of serum creatinine levels A, suggesThe RNA expression levels of occludin (OCLN) and zonula occluden-1 (ZO-1) in the jejunum of rats were analyzed. We found no significant differences in the RNA expression of OCLN among any of the groups. However, treatment with RMD resulted in a promotion of jejunal ZO-1 expression in rats with CKD, as shown in Immunohistochemistry studies also revealed that COS and RMD slightly rescued ZO-1 protein expression in the jejunum of rats with CKD, further supporting the potential protective effects of RMD on gut barrier integrity C.The Shannon index and Pielou’s evenness were evaluated to assess the diversity and evenness of bacterial communities at week 12 of the experiment. However, no significant differences were observed in the Shannon index and Pielou’s evenness among the different groups A.In contrast, when examining the control group, beta diversity analysis to compare the gut microbiota alteration between pre- and post-study in each treatment group using principal coordinate analysis (PCoA) of Bray–Curtis dissimilarity demonstrated no significant difference in diversity between week 0 and week 12. This suggests that the overall composition of bacterial communities in the control group remained relatively stable over the course of the experiment. The CKD group exhibited a significantly lower diversity at the 12th week, while inulin supplementation led to a relatively lesser but still significantly lower diversity. This indicates a notable change in the bacterial community composition over time in these groups. On the other hand, the rats treated with RMD did not show a significant change in beta diversity, similar to the control group, suggesting that RMD treatment maintained a relatively stable and consistent bacterial community composition throughout the 12-week period. Interestingly, the COS group displayed a significant increase in diversity, suggesting a notable shift in the composition of bacterial communities caused by the COS treatment.Cyanobacteria were found to be higher in abundance compared to the CKD group, while Proteobacteria were lower. By week 12, significant differences in relative abundance emerged. In the control group, Verrucomicrobiota and Campylobacterota were higher in abundance compared to the CKD group. Similarly, in the RMD group, a higher relative abundance of Verrucomicrobiota compared to CKD was observed. These findings indicate that both the control and RMD groups exhibited higher levels of Verrucomicrobiota compared to the CKD group at the end of the 12-week period. These results indicated the potential influence of RMD on the relative abundance of the specific bacterial phylum Verrucomicrobiota.Regarding the relative abundance of bacterial phyla in the fecal microbiota, comparisons were made between each treatment group and the CKD group A. At weeLactobacillus, Bifidobacterium, Roseburia, and Akkermansia. The relative abundances of these genera were aggregated for each treatment group. Remarkably, a significant increase was observed in the RMD treatment group compared to the other groups, as shown in The analysis of relative abundances at the genus level revealed slight differences in several bacterial genera across each treatment group; however, no significant differences were observed. While the relative abundances of various genera showed some variability, these differences did not reach statistical significance. Of particular interest were the beneficial bacteria, including r-values) were calculated. These correlation coefficients provide insights into the strength and direction of associations between variables. A heatmap was generated based on the correlation coefficients to visualize the relationships between the variables. The heatmap allows for a comprehensive overview of the correlation patterns among the measured parameters  production [RMD is a polymer of D-glucose derived from the enzymatical process of starch, especially from plants. RMD has been used as the placebo control in many studies focusing on prebiotic efficacy. However, indigestible, fermentable, resistant maltodextrin was proven to have prebiotic functions, as consumption of RMD increased gut microbiota richness, particularly the oduction ,41,42. Roduction ,43. HoweVerrucomicrobiota and ‘bad bacteria’ Campyrobacterota numbers. In addition, we found that the combination of ‘good bacteria’ Lactobacillus, Bifidobacteria, Akkermansia, and Roseburia increased significantly in RMD-fed rats compared to their CKD counterparts. This group of bacteria is responsible for SCFA synthesis and mucous membrane protection. These results indicated that RMD supplementation was better than dose-dependent inulin and COS in the modulation of gut dysbiosis.Our study showed that RMD treatment efficiently increased monolayer Caco-2 cell integrity in normal and inflammatory-induced conditions and promoted intestinal ZO-1 expression in CKD rats. These effects are stronger than inulin at the same dosage. The RMD supplement substantially boosted the relative abundance of gut microbiota in CKD rats, and partly suppressed Lactobacillus, Bifidobacteria, Akkermansia, and Roseburia are commonly recognized as beneficial microbiota. These bacteria have been found to enhance metabolism, reduce inflammation, improve intestinal barrier function, and maintain microbiota homeostasis. Their higher abundance is associated with positive outcomes in the treatment of diabetes mellitus, obesity, and inflammatory bowel disease (IBD) [Lactobacillus, Bifidobacteria, Akkermansia, and Roseburia have been strongly linked to poorer glomerular function, malnutrition, and fatal complications [Gut bacteria such as se (IBD) ,45,46. Iications ,46,47. TOscillibacter and Lachnoclostridium showed a negative correlation, while Sellimonas, Eubacterium nodatum, and NK 4A214 exhibited a positive correlation with serum creatinine. Serum creatinine is commonly utilized as a marker of kidney function, particularly glomerular filtration rate. Normally, creatinine production is independent of gut microbiota activity, as it primarily originates from muscle metabolism and glomerular filtration. However, recent research has indicated that the gut microbiota can influence various aspects of human health, including metabolism and inflammation, which may indirectly impact kidney function [Based on the correlation study between the relative abundance of gut microbiota and serum creatinine or tight junction protein expression C, signiffunction . It is iAlistipes and Anaerovorax on the expression of intestinal tight junction protein expression. Previous studies reviewed the increase in Alistipes abundance in bowel inflammation related to the downregulation of intestinal claudin-1, ZO-1, and occludin expression [Anaerovorax was associated with low tight junction protein expression in heat stress [Alistipes and Anaerovorax could reflect the severity of gut leakage and uremic toxin absorption in CKD patients.Moreover, we demonstrated the deleterious effects of pression ,50, whilt stress . Regardit stress ,53. We sWe proposed the resistant type of maltodextrin as a desirable prebiotic supplement in CKD patients to improve gut integrity, lower uremic toxin absorption, and modulate beneficial gut microbiota growth. RMD is generally safe and has no contraindication for hypertension, diabetes mellitus, or renal insufficiency. We also expected that COS may be useful in the management of CKD, but the optimal preparation and dosage should be validated. Our future plan includes utilizing COS and RMD as components of synbiotics to alleviate complications of CKD in animal models.