BACKGROUND:The pathogenesis of inflammatory bowel disease involves inflammation,immune activation,visceral hypersensitivity,and dysbiosis of the gut microbiota.Inflammation promotes the release of inflammatory mediators by immune cells,damaging the enteric nervous system.Enteric glial cells are an important component of the intestinal nervous system and are excellent cells for studying intestinal neuroinflammation.Primary enteric glial cells play a crucial role in exploring cell therapies for intestinal nervous system diseases.Currently,the methods for obtaining these cells are mostly cumbersome.Therefore,finding a convenient and fast method for extracting this cell is crucial.OBJECTIVE:To establish a method for optimizing the isolation,culture,and identification of mouse enteric glial cells.METHODS:0-7-day-old C57BL/6 neonatal mice were euthanized by excessive inhalation of isoflurane.After soaking in 75％alcohol for disinfection,the duodenum(1 cm below the pylorus to 1 cm above the Qu's ligament)was removed by laparotomy at the midline of the abdomen.A 1 mL syringe was filled with DPBS and the intestinal contents were repeatedly rinsed until the intestine became translucent,and the mesentery and blood vessels were peeled off.The duodenum was cut to a size of 1 mm and digested in 0.25％EDTA trypsin for 20 minutes.Then an equal amount of DMEM/F12 complete culture medium was added to terminate digestion.The liquid was filtered through a 100 μm cell filter,centrifuged,and the cells were resuspended in 1 mL of DMEM/F12 complete culture medium.When the cell adhesion growth density reached 80％,cells were digested for subculture.When cells were cultured to the third generation,glial fibrillary acid protein labeled with enteric glial cells was used for identification by immunofluorescence method.RESULTS AND CONCLUSION:The isolated and cultured cells were full of colloids,with protrusions extending outward and passable.Glial fibrillary acid protein staining was positive.This method can successfully isolate and culture enteric glial cells and is easy to operate,providing a stable model for the study of the pathophysiology of the enteric nervous system.

BACKGROUND: Post-stroke disability diminishes the physical activity (PA) level of survivors, potentially affecting their long-term prognosis. This study endeavors to explore the correlation between daily PA level and the all-cause mortality in patients with a history of stoke in the United States. METHODS: Data of stroke survivors were sourced from the National Health and Nutritional Examination Survey (NHANES) 2007-2018. The population was stratified into three groups based on their PA level. Kaplan-Meier method with log-rank tests for significance was used for survival analysis. Weighted Cox proportional hazards regression models were employed to estimate the hazard ratios (HRs) for all-cause mortality. Subgroup analysis was conducted to strengthen the results. RESULTS: A total of 1395 participants were recruited, comprising 679 males and 716 females, with a median age of 68 years. Based on their PA levels, 779 individuals were classified as inactive, 156 as insufficiently active, and 460 as sufficiently active. Following a median observation period of 59 months, there were 476 recorded deaths, with 349, 47, and 80 cases in the three respective groups. Compared to the inactive group, the HRs and 95% confidence intervals (CIs) for all-cause mortality in participants who were insufficiently active and sufficiently active were 0.58 (0.40, 0.84) and 0.47 (0.33, 0.67), respectively. The Kaplan-Meier curve revealed a significant difference in overall survival between the three groups, as confirmed by the log-rank test (P < 0.0001). Subgroup analysis further validated our results and demonstrated that the protective impact of PA on stroke prognosis varies according to distinct characteristics. CONCLUSIONS The results indicate that increased levels of PA are associated with a protective effect on long-term mortality among stroke survivors. Further prospective longitudinal studies are necessary to elucidate the optional PA level and special exercise guideline targeting this population.
Humans ; Male ; Female ; Aged ; Exercise ; Middle Aged ; Nutrition Surveys ; Stroke/mortality* ; United States/epidemiology* ; Survivors/statistics & numerical data* ; Aged, 80 and over ; Mortality

BACKGROUND:The pathogenesis of inflammatory bowel disease involves inflammation,immune activation,visceral hypersensitivity,and dysbiosis of the gut microbiota.Inflammation promotes the release of inflammatory mediators by immune cells,damaging the enteric nervous system.Enteric glial cells are an important component of the intestinal nervous system and are excellent cells for studying intestinal neuroinflammation.Primary enteric glial cells play a crucial role in exploring cell therapies for intestinal nervous system diseases.Currently,the methods for obtaining these cells are mostly cumbersome.Therefore,finding a convenient and fast method for extracting this cell is crucial.OBJECTIVE:To establish a method for optimizing the isolation,culture,and identification of mouse enteric glial cells.METHODS:0-7-day-old C57BL/6 neonatal mice were euthanized by excessive inhalation of isoflurane.After soaking in 75％alcohol for disinfection,the duodenum(1 cm below the pylorus to 1 cm above the Qu's ligament)was removed by laparotomy at the midline of the abdomen.A 1 mL syringe was filled with DPBS and the intestinal contents were repeatedly rinsed until the intestine became translucent,and the mesentery and blood vessels were peeled off.The duodenum was cut to a size of 1 mm and digested in 0.25％EDTA trypsin for 20 minutes.Then an equal amount of DMEM/F12 complete culture medium was added to terminate digestion.The liquid was filtered through a 100 μm cell filter,centrifuged,and the cells were resuspended in 1 mL of DMEM/F12 complete culture medium.When the cell adhesion growth density reached 80％,cells were digested for subculture.When cells were cultured to the third generation,glial fibrillary acid protein labeled with enteric glial cells was used for identification by immunofluorescence method.RESULTS AND CONCLUSION:The isolated and cultured cells were full of colloids,with protrusions extending outward and passable.Glial fibrillary acid protein staining was positive.This method can successfully isolate and culture enteric glial cells and is easy to operate,providing a stable model for the study of the pathophysiology of the enteric nervous system.

Objectives:Fenofibric acid is extracted from the widely used hypolipemic fenofibrate,nowadays being approved for marketing around numerous nations and regions,nonetheless not in China.Present trial evaluated the efficacy and safety in the Chinese hypertriglyceridemia population. Methods:This is a multi-center,randomized,double-blind,placebo-controlled phase Ⅲ clinical trial.Patients from 3 different cohorts,including severe hypertriglyceridemia(HTG),moderate HTG and mixed-dyslipidemia(MD),were randomized at 1:1 ratio to receive fenofibric acid 135 mg or placebo daily for 12 weeks.The primary endpoint was the percentage change of triglyceridemia(TG)from baseline at week 12.Secondary endpoints were the percentage changes of other blood lipid indexes.At the same time,the incidence of medical adverse events was observed. Results:Among the three cohorts of patients with severe HTG(n=52),moderate HTG(n=23)and MD(n=52),the TG levels in the fenofibric acid-treated group decreased by(49.12±29.19)%,(49.95±25.19)%and(49.79±19.28)%,respectively from baseline to 12 weeks,while the corresponding placebo groups decreased by(18.88±40.69)%,(8.11±29.86)%and increased by(10.42±73.04)%,respectively from baseline to 12 weeks.The differences between treatment and placebo groups were statistically significant(P<0.017 for severe HTG cohort,P<0.05 for moderate and MD cohort).The high-density lipoprotein cholesterol(HDL-C)in the fenofibric acid-treated group increased by(25.51±21.45)%,(24.55±24.73)%,and(23.60±27.38)%,and the placebo group increased by(1.91±20.42)%,(2.40±9.32)%and(7.13±19.12)%,respectively,the differences between the two groups were statistically significant(all P<0.05).In the fenofibric acid group,adverse events with incidence>5%included upper respiratory tract infection(10.9%),abdominal pain(6.3%),and increased serum creatinine levels(6.3%),rates of adverse events were similar between the two groups(P>0.05). Conclusions:Fenofibric acid can significantly reduce triglycerides and elevate HDL-C levels safely in Chinese patients with severe to moderate HTG without statin or MD patients on top of statin therapy.

Objective:To explore the role and the intervention effect of emodin in intestinal neuropathy in rats with severe acute pancreatitis (SAP) through the nucleotide binding oligomerization domain like receptor protein 3/cysteine containing aspartic acid protease-1 (NLRP3/Caspase-1) pathway.Methods:Forty male healthy SD rats aged 6-8 weeks with a weight of approximately 200g were randomly divided into control group, SAP model group, emodin treatment (EMO) group, and NLRP3 knockdown group. SAP were induced by retrograde injection of sodium deoxycholate into the pancreatic duct of rats and serum amylase of which were detected. The effective NLRP3 knockdown sequence was screened for NLRP3 knockdown animal experiments. Fluorescence quantitative polymerase chain reaction was used to detect the expression of NLRP3, Caspase-1, gasdermin-D (GSDMD), interleukin (IL)-1β, IL-18 and tumor necrosis factor-α(TNF-α) in the small intestine of each group. Immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein (GFAP) in the small intestine of each group.Results:The amylase levels of the control group, SAP group, EMO group, and NLRP3 knockdown group were (277.73±24.92) U/L, (1018.57±282.89) U/L, (625.43±134.40) U/L, and (391.01±27.63) U/L, respectively. The SAP and EMO groups were significantly higher than the control group ( P<0.001), while the EMO and NLRP3 knockdown groups were significantly lower than the SAP group (all P<0.001). Compared with control group, the expression levels of NLRP3, Caspase-1, IL-1β, IL-18, TNF-α and GSDMD in SAP group were increased, with statistical significance (all P<0.001). Compared with SAP group, the NLRP3 knockdown group showed the expressionlevels of the above 6 genes were all decreased, and EMO group showed decreased gene expressing levels of NLRP3, IL-1β, IL-18 and TNF-α, with statistical significance (all P<0.05). The relative expression of GFAP in small intestine of control group, SAP group, EMO group and NLRP3 knockdown group were (1.00±0), (1.66±0.11), (1.13±0.02) and (1.13±0.02), respectively. Among them, the expression of GFAP in SAP group was increased compared with the control group; The expression of GFAP in EMO group and NLRP3 knockdown group was lower than that in model group, and the differences were statistically significant (all P<0.05). Conclusions:Emodin and knocking down NLRP3 can both promote the repair of SAP small intestine injury through the NLRP3/Caspase-1 signaling pathway, and thus play a protective role in the intestine.

Objective:To investigate the clinical features and associated influencing factors of cognitive impairment in middle-aged and elderly Chinese adult patients undergoing maintenance hemodialysis (HD).Methods:A cross-sectional study was conducted among HD patients from 11 centers in Beijing city from April 2017 to June 2017. A neuropsychological battery covering domains of attention/processing speed, executive function, memory, language, and visuospatial function was applied in cognitive function assessment. Patients were classified as normal cognitive function group and cognitive impairment group according to the fifth version of the diagnostic and statistical manual of mental disorders criteria (DSM-V). Multivariate binary logistic regression was used to analyze the independent influencing factors of cognitive impairment. Results:A total of 613 HD patients were included in the study, and the prevalence of cognitive impairment was 80.91% (496/613). Attention impairment (81.05%) and memory impairment (63.51%) were the most common impaired domains, and 79.23% was concomitant impairment across two or more cognitive domains among those with cognitive impairment. Compared with the patients in the normal cognitive function group, the patients in the cognitive impairment group had senior age, longer dialysis vintage, higher proportion of diabetes, hypertension, and stroke, higher level of serum intact parathyroid hormone (iPTH), lower education level, and lower urea clearance index (Kt/V) (all P<0.05). Factors were independently associated with cognitive impairment including increasing age ( OR=1.110, 95% CI 1.072-1.150, P<0.001), education time>12 years (with education time<6 years as reference, OR=0.323, 95% CI 0.115-0.909, P=0.032), history of diabetes ( OR=2.151, 95% CI 1.272-3.636, P=0.004), history of stroke ( OR=2.546, 95% CI 1.244-5.210, P=0.011), increased dialysis vintage ( OR=1.016, 95% CI 1.010-1.022, P<0.001), reduced Kt/V( OR=0.008, 95% CI 0.002-0.035, P<0.001), and increased iPTH level ( OR=1.002, 95% CI 1.002-1.003, P=0.012). Conclusions:The prevalence of cognitive impairment in middle-aged and elderly adult Chinese patients undergoing HD is high. Memory and attention are the most commonly impaired domains. Increasing age, low education level, history of diabetes and stroke, increased dialysis vintage, reduced Kt/V and increased serum iPTH are the independent influencing factors associated with cognitive impairment.

Objective:To investigate the association between cognitive impairment and all-cause mortality in middle and elderly adult patients undergoing maintenance hemodialysis (HD).Methods:A prospective cohort study was conducted. Patients from 11 HD centers in Beijing between April and June 2017 were enrolled. Baseline data were collected, and a series of neuropsychological batteries covered 5 domains of cognitive function were applied for the assessment of cognitive function. The patients were then classified as normal and cognitive impairment groups according to the fifth version of the Diagnostic and Statistical Manual of Mental Disorders criteria (DSM-V) and followed-up until June 2018. The clinical characteristics of the two groups of patients were compared. Kaplan-Meier survival analysis was used to compare the difference in the cumulative survival rate between the two groups. Multivariate Cox regression model was used to analyze the independent influencing factors of all-cause mortality, to determine the relationship between cognitive impairment and different cognitive domain impairments and all-cause death.Results:A total of 613 patients were enrolled, of which 496(80.91%) patients had cognitive impairment. Compared with the normal cognitive function group, the patients in the cognitive impairment group tended to be older, longer dialysis vintage, a higher proportion of diabetes, hypertension, and stroke, increased serum iPTH level, and lower education level and urea clearance index (Kt/V) (all P<0.05). After (49.53±8.42) weeks of follow-up, Kaplan-Meier survival analysis showed that the cumulative survival rate of cognitive impairment group was significantly lower than that of cognitive normal group (Log-rank χ2=8.610, P=0.003). Multivariate Cox regression analysis showed that history of diabetes ( HR=2.742, 95% CI 1.598-4.723, P<0.001), coronary heart disease ( HR=1.906, 95% CI 1.169-3.108, P=0.010), dialysis vintage (every increase of 1 month, HR=1.007, 95% CI 1.003-1.011, P=0.001), serum level of albumin (every increase of 1 g/L, HR=0.859, 95% CI 0.809-0.912, P<0.001), cognitive impairment ( HR=2.719, 95% CI 1.088-6.194, P=0.032) were independently associated with all-cause mortality. Multivariate Cox regression analysis on different cognitive domains also indicated that memory impairment ( HR=2.571, 95% CI 1.442-4.584, P<0.001), executive function impairment ( HR=3.311, 95% CI 1.843-5.949, P=0.001) and three, four, five domains combined impairment ( HR=5.746, 95% CI 1.880-17.565, P=0.002; HR=12.420, 95% CI 3.690-41.802, P<0.001; HR=13.478, 95% CI 3.381-53.728, P<0.001) were independently related to all-cause mortality. Conclusions:Cognitive impairment is an independent risk factor of all-cause mortality in middle and elderly adult patients undergoing maintenance hemodialysis, and the risk is significantly increased in patients with the impairment of the domains of memory, executive function, or in the combination of three to five cognitive domains.

Endo-β-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-β-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×10⁶ U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; Gene Expression ; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Streptomyces ; enzymology ; genetics

Objective To investigate the prevalence of mcr-1 gene,a plasmid-mediated polymyxin resistance gene,in Escherichia coli(E.coli) strains isolated in Dongyang of Zhejiang Province and to under-stand the epidemiological characteristics of E.coli strains carrying mcr-1 gene in order to provide local clini-cians with a theoretical basis for prevention and control of the spread of mcr-1-bearing E.coli strains. Meth-ods A total of 315 E.coli strains were collected in the People′s Hospital of Dongyang, Zhejiang Province from January to December 2016. All strains were isolated from specimens of blood,urine,respiratory tract, etc. PCR was performed to detect the genes confering resistance to polymyxin (mcr-1 gene), β-lactamase and carbapenem. Minimal inhibitory concentrations (MIC) of antibiotics against mcr-1-positive strains were determined by micro-broth dilution method. Conjugation test was performed to confirm whether the mcr-1 gene was located on the transferable plasmid. Multilocus sequence typing (MLST) was used for molecular typing of mcr-1-positive strains. Results Five mcr-1-positive strains were identified from 315 E.coli strains with a positive rate of 1.6%. Two out of the five mcr-1-positive E.coli strains contained β-lactamase resist-ance genes,blaTEM-1and blaCTX-M-14. Both of them were resistant to the first, second and third generation of cephalosporins and one was also resistant to cefepime. All of the five mcr-1-positive E.coli strains were sen-sitive to ciprofloxacin and levofloxacin,but resistant to ticarcillin/clavulanic acid. No carbapenem resistance genes were detected. One transconjugant was successfully obtained by transconjugation assay. MLST analysis showed that a total of four sequence types were identified, including ST131 (two strains), ST43 (one strain),ST69 (one strain) and ST349(one strain). Conclusion Only 1.6% of all E.coli strains isolated in Dongyang area of Zhejiang Province carry mcr-1 gene,indicating that there is no epidemic of mcr-1 gene-positive E.coli infection. The coexistence of mcr-1 gene and β-lactamase resistance genes in E.coli strains isolated in Dongyang suggests that local clinicians should avoid antibiotic abuse to prevent the spread of drug-resistant E.coli.