[ ABSTRACT] AIM:To study the process of promoting mouse embryonic stem cells ( ESC) to specify to definitive endoderm by up-regulating of Nodal signal pathway in order to find the best cultivated systems of differentiation of mouse ESC to definitive endoderm cells.METHODS:The cells were divided into different groups based on the culture medium:ESC group ( serum-free medium +LIF) , natural differentiation group ( serum-free medium) and activin A group ( serum-free medium +50μg/L activin A).The cells and the sterilized coverslips with cells were collected at 1, 3, 5 and 7 d of the cultivation.The proportion of CXCR4 +cells was detected by flow cytometry.The expression of CXCR4 was determined by immunocytochemical method, and the protein expression of OCT4 and CXCR4 was detected by Western blot.RE-SULTS:The proportion of CXCR4 +cells showed no dramatic change in ESC group along with the extending of cultivation day, while there were gradually increased in natural differentiation group and activin A group and the highest level was ob-served at 5 d.Among the 3 groups, the proportion of CXCR4 +cells at 5 d was the highest in activin A group.The brown or tan staining in the cells observed under microscope was considered as positive CXCR4 by immunocytochemistry.The pro-tein levels of OCT4 and CXCR4 in ESC group along with the extending of cultivation days was observed.The expression levels of OCT4 were gradually decreased in the cells in natural differentiation group and activin A group, while those of CX-CR4 were gradually increased with the highest level at 5 d.It was highest in the cells in activin A group.CONCLUSION:The proportion of definitive endoderm was the highest at 5 d of the induction during in vitro mouse ESC differentiation.Up-regulation of Nodal signal pathway by adding activin A at the early stage of mouse ESC differentiation promotes mouse ESC to specify to definitive endoderm with CXCR4 molecular marker.

AIM:To investigate the effect of microRNA-429 (miR-429) on the expression of occludin (Ocln) in intestinal epithelial cells ( IECs) and intestinal epithelial barrier function in diabetic mice.METHODS:Diabetes mel-litus mouse model was induced by intraperitoneal injection of streptozocin.The expression of miR-429 in IECs of diabetic mice was inhibited by antagomiRNA-429 injected via tail vein.The expression of miR-429 and mRNA expression of Ocln were detected by real-time PCR.The protein expression of Ocln was determined by Western blotting and immunohistochem-istry.The urinary lactulose/mannitol ratio was measured by gas chromatography.The plasma LPS concentrations in the mice were measured by chromogenic end-point TAL kit.RESULTS:The results of real-time PCR confirmed that the ex-pression of miR-429 in IECs of diabetic mice was remarkably inhibited by tail-vein administration of antagomiRNA-429, and resumed to similar level of normal mice on the 6th day after the administration.After suppressing the level of miR-429, the expression of Ocln in IECs of diabetic mice increased significantly (P<0.05), while the urinary lactulose/mannitol ra-tio and the plasma LPS concentration decreased obviously ( P<0.05 ) .CONCLUSION:AntagomiRNA-429 effectively suppresses miR-429 expression in IECs of diabetic mice, and then enhances the expression of Ocln and partially resumes the intestinal epithelial barrier function.

OBJECTIVETo explore the changes in cellular immune function and the safety of physical therapy in patients with systemic lupus erythematosus (SLE) complicated by herpes zoster (HZ) virus infection. Methods A retrospective analysis was conducted among 10 SLE pateints with HZ virus infection, with 30 SLE patients without HZ infection as the control group. The results of routine laboratory tests and T lymphocyte subset counts (before and during infection and after cure of infection) were compared between the two groups.

RESULTSThe proportion of patients with significantly increased absolute neutrophil count and white blood cell count before HZ infection, and those with decreased lymphocyte percentage and elevated C-reactive protein during infection differed significantly between the two groups (P<0.05). Before HZ infection, the proportions of patients with increased CD3%, decreased CD16CD56%, abnormal 4/8 ratio (P<0.01) and those with increased CD8% and CD8count and decreased CD16CD56count differed significantly between the two groups (P<0.05). During HZ infection, the proportion of patients with increased CD3% and CD8% and decreased CD4% and 4/8 ratio count (P<0.05) and those with decreased CD16CD56% and increased CD8count differed significantly between the two groups (P<0.01). After cure of HZ infection, the proportion of patients with decreased CD16CD56% was significantly different between the two groups (P<0.01). The treatment time of HZ infection averaged 5∓1.3 days with a cure rate of 100% postherpetic neuralgia occurred in none of the patients after the treatment.

CONCLUSIONPatients with SLE complicated by HZ infection are in an immunosuppressive state, suggesting the need of monitoring T lymphocyte subsets in SLE treatment. The combined use of antiviral drugs and physical therapy can be safe and effective for treatment of SLE complicated by HZ infection.

To analyse the familial aggregation and genetic predisposition of Shen-yin deficiency syndrome (SYDS) in families with diabetes mellitus type 2 (DM2). Methods One hundred and forty-one DM2 patients were collected from 32 family lines in Nanjin area, in which the probands were differentiated as DM2 with SYDS. On them, genetic analysis on the characteristics of SYDS was conducted using pedigree analysis, morbidity and heritability of the first-degree relatives of the probands were calculated, and the action of familial SYDS factor on the genesis of the syndrome was assessed by multiple factors regression analysis. Results The morbidity rate of SYDS in the first-degree relatives of the probands was 33.71%, and the heritability, calculated by Falconer formula, was 80.6%. The fitting result of regression analysis showed that familial factor played an important role in SYDS genesis (OR = 5.61, P = 0.001), but DM2 itself is not an independent risk factor for it. Conclusion DM2 with SYDS shows the tendency of familial aggregation and genetic predisposition, genetic factor is associated with the genesis of the syndrome. Pedigree research is a good method for exploring the relationship between syndrome and genetic factor.
Adult ; Diabetes Mellitus, Type 2 ; genetics ; Diagnosis, Differential ; Female ; Genetic Predisposition to Disease ; Humans ; Male ; Medicine, Chinese Traditional ; Pedigree ; Yin Deficiency ; genetics

Two clinical ablation protocols,2C3L and stepwise,have been routinely used in our group to treat atrial fibrillation (AF),but with a less than 60％ long-term arrhythmia-free outcome achieved in patients.The goal of this study was to examine the underlying mechanism of low success in clinical outcome.MRI images from one patient were used to reconstruct a human atrial anatomical model,and fibrotic tissue was manually added to represent the arrhythmia substrate.AF was induced with standard protocols used in clinical practice.2C3L and stepwise were then used to test the efficacy of arrhythmia termination in our model.The results showed that re-entries induced in our model could not be terminated by using either 2C3L or the stepwise protocol.Although some of the induced re-entries were terminated,others emerged in new areas.Ablation using only the 2C3L or stepwise method was not sufficient to terminate all re-entries in our model,which may partially explain the poor long-term arrhythmiafree outcomes in clinical practice.Our findings also suggest that computational heart modelling is an important tool to assist in the establishment of optimal ablation strategies.

OBJECTIVETo detect the level of fasting plasma homocysteine (Hcy) in patients with oligospermia and/or asthenospermia and to investigate its clinical significance.

METHODSSemen quality analyses and fasting plasma Hcy determination were performed for 86 infertility patients (21 with oligospermia, 32 with asthenospermia and 33 with oligo-asthenospermia) and 19 normal fertile volunteers. The results were compared.

RESULTSThe level of plasma Hcy was significantly higher in the infertility patients than in the normal controls (P < 0.05) and negatively correlated with sperm concentration (r = -0.433, P < 0.01), the percentage of grade a sperm (r = -0.303, P < 0.05) and the percentage of grade a+b sperm (r = -0.339, P < 0.01).

CONCLUSIONThe increased level of human plasma Hcy directly or indirectly affects spermatogenesis and correlates negatively with oligospermia and/or asthenospermia.

Adult ; Asthenozoospermia ; blood ; Case-Control Studies ; Homocysteine ; blood ; Humans ; Male ; Oligospermia ; blood ; Sperm Count ; Sperm Motility

Objective Development and application of a real time fluorescent quantitative PCR (FQ-PCR) assay for detecting WU polyomavirus in children with low respiratory tract infections.Methods The VP2 gene of WU polyomavirus was selected as the detection target,from which the real time primers and probes were designed.The standard curve was established by using recombinant plasmid as template.And the FQ-PCR assay for specific detection of WU polyomavirus was established.The speciflcity,sensitivity and reproducibility of the method were evaluated. Furthermore,the clinical specimens from children with respiratory tract infections collected in Wenling First People's Hospital were quantitatively detected using this method.Results In this study,the FQ-PCR method was established to detect a specific fragment in VP2gene of WU polyomavirus.The standard curve coefficient R2 was 0.998.And this method can detect as low as 50 copies recombinant plasmid.The clinical specimens of sputum and throat swab from children with respiratory tract infections were quantitatively detected using this method.7 sputum specimens were detected as WU polyomavirus positive in 700 sputum specimens,the positive ratio was 1.00％.No positive specimens were detected in 146 specimens of throat swabs and 846 blood samples from same patient population.Conclusion .The results indicated that the FQ-PCR assay method established in this study was specific,rapid and sensitive for detecting WU polyomavirus in children with lower respiratory tract infections.The sputum specimen is more suitable to be used for gene detection of WU polyomavirus than throat swab or blood.

Background:For Chinese patients with hepatocellular carcinoma (HCC),surgical resection is the most important treatment to achieve long-term survival for patients with an early-stage tumor,and yet the prognosis after surgery is diverse.We aimed to construct a scoring system (Shanghai Score) for individualized prognosis estimation and adjuvant treatment evaluation.Methods:A multivariate Cox proportional hazards model was constructed based on 4166 HCC patients undergoing resection during 2001-2008 at Zhongshan Hospital.Age,hepatitis B surface antigen,hepatitis B e antigen,partial thromboplastin time,total bilirubin,alkaline phosphatase,γ-glutamyltransferase,α-fetoprotein,tumor size,cirrhosis,vascular invasion,differentiation,encapsulation,and tumor number were finally retained by a backward step-down selection process with the Akaike information criterion.The Harrell's concordance index (C-index) was used to measure model performance.Shanghai Score is calculated by summing the products of the 14 variable values times each variable's corresponding regression coefficient.Totally 1978 patients from Zhongshan Hospital undergoing resection during 2009-2012,808 patients from Eastern Hepatobiliary Surgery Hospital during 2008-2010,and 244 patients from Tianjin Medical University Cancer Hospital during 2010-2011 were enrolled as external validation cohorts.Shanghai Score was also implied in evaluating adjuvant treatment choices based on propensity score matching analysis.Results:Shanghai Score showed good calibration and discrimination in postsurgical HCC patients.The bootstrap-corrected C-index (confidence interval [CI]) was 0.74 for overall survival (OS) and 0.68 for recurrence-free survival (RFS) in derivation cohort (4166 patients),and in the three independent validation cohorts,the CIs for OS ranged 0.70-0.72 and that for RFS ranged 0.63-0.68.Furthermore,Shanghai Score provided evaluation for adjuvant treatment choices (transcatheter arterial chemoembolization or interferon-α).The identified subset of patients at low risk could be ideal candidates for curative surgery,and subsets of patients at moderate or high risk could be recommended with possible adjuvant therapies after surgery.Finally,a web server with individualized outcome prediction and treatment recommendation was constructed.Conclusions:Based on the largest cohort up to date,we established Shanghai Score-an individualized outcome prediction system specifically designed for Chinese HCC patients after surgery.The Shanghai Score web server provides an easily accessible tool to stratify the prognosis of patients undergoing liver resection for HCC.

Objective To provide a longitudinal evaluation tool based on the frequency of aggressive be-havior for the aggression assessment of schizophrenia patients.Methods The Life History of Aggression was translated and revised to form the Life History of Aggression-Chinese Version(LHA-CV)based on 369 patients diagnosed with schizophrenia in the Chengdu community and compulsory medical insti-tution.The reliability of LHA-CV was analyzed by means of split-half reliability,test-retest reliability and inter-evaluator consistency.The validity was analyzed by item analysis,construct validity and crite-rion validity.Results Item analysis found that LHA-CV had good homogeneity and discriminant validity.Exploratory factor analysis found that the Kaiser-Meyer-Olkin(KMO)test value was 0.80,and the Bartlett's sphericity test χ2=1203.46(P<0.05),and it revealed four factors including non-physical ag-gression,physical aggression,self-directed aggression and antisocial behavior/consequences.The factor loadings for all 11 items were greater than 0.40.Confirmatory factor analysis was performed on the factor model,Chi-square degree of freedom(χ2/df)was 3.61,root mean square error of approxima-tion(RMSEA)was 0.07,goodness-of-fit index(GFI)was 0.92,comparative fit index(CFI)was 0.90,incremental fit index(IFI)was 0.90,and the discriminant validity of each factor was good.The criterion validity test showed the total score of LHA-CV was positively correlated with the aggressive behavior level of MacArthur Community Violence Instrument,the total score of Buss-Perry Aggression Scale,and the score of Antisocial Personality Disorder Subscale of Personality Diagnostic Question-naire-4th Edition Plus(PDQ-4+_ASPD,P<0.05).The Cronbach's α coefficient of non-physical aggres-sion,physical aggression,self-directed aggression,antisocial behavior/consequences and LHA-CV total score were 0.82,0.73,0.74,0.56 and 0.79,respectively.The test-retest reliability,Spearman-Brown split-half reliability and intra-class correlation coefficient of LHA-CV total score were 0.82(P<0.05),0.66 and 0.99,respectively.Conclusion LHA-CV has good reliability and validity,and can be used as an evaluation tool for longitudinally assessing aggressive behavior in schizophrenia patients.

This study was aimed at understanding the detection rate,drug resistance characteristics,virulence characteris-tics,multi-locus sequence typing,and other molecular epidemic and pathogenic characteristics of Campylobacter jejuni and Campylobacter coli in children in Guangdong Province from 2020 to 2022.Anal swabs or stool samples of suspected infection cases in children from 2020 to 2022 were collected from two hospitals in Guangzhou,Guangdong Province.Campylobacter was isolated and cultured through the filtration method,and identified with a microbial mass spectrometry system;antibiotic resist-ance was analyzed with the agar dilution method;bacterial genome nucleic acids were extracted,and whole-genome sequencing was conducted;and drug resistance genes,virulence genes,multi-locus sequence typing,and phylogenetic analysis based on whole-genome single nucleotide polymorphisms were analyzed from whole-genome sequencing results.First,53 strains of Campy-lobacter were detected through continuous routine monitoring in this study,with a positive detection rate of 2.94％.Among them,Campylobacter jejuni accounted for 81.13％(43/53)and Campylobacter coli accounted for 18.87％(10/53).In addition,16 strains of Campylobacter were screened through multi-pathogen surveillance,including 11 strains of Campylobacter jejuni and 5 strains of Campylobacter coli.Drug resistance ex-periments and whole genome sequencing were conducted on 46 Campylobacter isolates,including 33 isolates of Campylobacter jejuni and 13 isolates of Campylobacter coli.The resistance rate of Campylobacter to erythromycin,a widely used clinical treatment,was21.73％(10/46);that to tetracycline was 80.43％(37/46);those to the quinolone antibiotics nalidixic acid and ciprofloxacin were 76.08％(35/46)and 71.73％(33/46)respectively;and that to chloramphenicol was lowest,at 2.17％(1/46).The drug resistance rate was generally higher for Campylobacter coli than Campylobacter jejuni,and the differences in the indicators of erythromycin,gentamicin,streptomycin,telithromycin,and clindamycin were statistically significant.A total of 30 isolates of multidrug-resistant Campylobacter were detected,including nine multidrug-resistant phenotypes.Whole-ge-nome sequence analysis indicated that 46 Campylobacter isolates carried antibiotic resistance genes for antibiotics such as quino-lones,tetracyclines,β-lactams,and aminoglycosides,and carried 128 virulence factor genes in five categories.All 46 isolates of Campylobacter were identified as 35 ST type through MLST typing,and phylogenetic analysis indicated no obvious dominant ST type.Campylobacter coli had more SNPs than Campylobacter jejuni.In conclusion,the positive detection rate of Campy-lobacter in Guangzhou City,Guangdong Province stabilized from 2020 to 2022,and the detection rate of Campylobacter jejuni was higher than that of Campylobacter coli.Campylobacter isolates were resistant to tetracyclines and quinolone,and showed a wide spectrum of multi-drug resistance,which was relatively severe among Campylobacter coli.Resistance genes and drug-resistant phenotypes were correlated and had predictive significance.The virulence genes of Campylobacter jejuni were more a-bundant than those of Campylobacter coli,possibly because of the higher detection rate and pathogenicity of Campylobacter jejuni.The phylogenetic tree showed clear branches with high genetic diversity and no clearly dominant clonal group.