This study established an indirect ELISA method for antibody detection based on human rotavirus group A(RVA)VP6 protein,to provide technical support for RVA antibody detection.An ELISA method for detecting RVA antibodies was established through a checkerboard assay for optimization of the reaction conditions with VP6 protein of RVA as the diagnostic antigen.Serum samples from healthy children and patients infected with RVA were collected and detected with the ELISA method.The optimal work-ing conditions for ELISA involved coating the ELISA plate with VP6 protein at a concentration of 2 μg/mL and diluting the serum at a ratio of 1∶250.The critical value of negative and positive samples was 0.137.The ELISA method had good specificity,sensitivity,and repeatability,and showed a 95%consistency rate with the western blotting antibody detection method.We tested 370 serum samples collected from children,which showed an antibody positivity rate of 81.1%.We additionally tested the acute and recovery phase sera from 15 patients infected with RVA,and observed a significantly higher RVA antibody titer in the recovery phase serum than the acute infection phase serum.The ELISA antibody detection method showed good specificity,sensitivity,and reproducibility,and therefore can be used for RVA antibody detection and auxiliary diagnosis of RVA infection.

This study expressed the VP6 protein of human group A rotavirus in a prokaryotic expression system and prepared mouse polyclonal antibodies to this protein.The human rotavirus VP6 gene was amplified through RT-PCR from fecal samples infected with group A rotavirus and cloned into the pET-32a vector to construct the recombinant pET-32a-VP6 prokaryotic expression plas-mid.The recombinant plasmid was transformed into Escherichia coli BL21(DE3).VP6 protein was expressed through induction with isopropyl-β-D-thiopyranoside(IPTG),purified with nickel nitrilotriacetic acid(Ni-NTA)affinity chromatography,and identified through SDS-PAGE and western blotting.Polyclonal antibodies to the VP6 protein were obtained by immunization of BALB/c mice with VP6 protein.Antibody titers were determined through indirect ELISA.The recombinant expression plasmid pET-32a-VP6 was con-structed,and VP6 protein was expressed primarily in inclusion bodies.Polyclonal antibodies to VP6 protein with a titer of 1∶32 000 were obtained by immunization of mice with VP6 protein purified by affinity chromatography.The antibodies specifically reacted with VP6 protein.The VP6 protein of human group A rotavirus was expressed and purified,and mouse polyclonal antibodies to this protein were prepared,thus providing a foundation for the preparation of a diagnostic antigen for human group A rotavirus and the establishment of serological detection methods.

This study established an indirect ELISA method for antibody detection based on human rotavirus group A(RVA)VP6 protein,to provide technical support for RVA antibody detection.An ELISA method for detecting RVA antibodies was established through a checkerboard assay for optimization of the reaction conditions with VP6 protein of RVA as the diagnostic antigen.Serum samples from healthy children and patients infected with RVA were collected and detected with the ELISA method.The optimal work-ing conditions for ELISA involved coating the ELISA plate with VP6 protein at a concentration of 2 μg/mL and diluting the serum at a ratio of 1∶250.The critical value of negative and positive samples was 0.137.The ELISA method had good specificity,sensitivity,and repeatability,and showed a 95%consistency rate with the western blotting antibody detection method.We tested 370 serum samples collected from children,which showed an antibody positivity rate of 81.1%.We additionally tested the acute and recovery phase sera from 15 patients infected with RVA,and observed a significantly higher RVA antibody titer in the recovery phase serum than the acute infection phase serum.The ELISA antibody detection method showed good specificity,sensitivity,and reproducibility,and therefore can be used for RVA antibody detection and auxiliary diagnosis of RVA infection.

This study expressed the VP6 protein of human group A rotavirus in a prokaryotic expression system and prepared mouse polyclonal antibodies to this protein.The human rotavirus VP6 gene was amplified through RT-PCR from fecal samples infected with group A rotavirus and cloned into the pET-32a vector to construct the recombinant pET-32a-VP6 prokaryotic expression plas-mid.The recombinant plasmid was transformed into Escherichia coli BL21(DE3).VP6 protein was expressed through induction with isopropyl-β-D-thiopyranoside(IPTG),purified with nickel nitrilotriacetic acid(Ni-NTA)affinity chromatography,and identified through SDS-PAGE and western blotting.Polyclonal antibodies to the VP6 protein were obtained by immunization of BALB/c mice with VP6 protein.Antibody titers were determined through indirect ELISA.The recombinant expression plasmid pET-32a-VP6 was con-structed,and VP6 protein was expressed primarily in inclusion bodies.Polyclonal antibodies to VP6 protein with a titer of 1∶32 000 were obtained by immunization of mice with VP6 protein purified by affinity chromatography.The antibodies specifically reacted with VP6 protein.The VP6 protein of human group A rotavirus was expressed and purified,and mouse polyclonal antibodies to this protein were prepared,thus providing a foundation for the preparation of a diagnostic antigen for human group A rotavirus and the establishment of serological detection methods.

Objective To investigate the effect of left bundle branch area pacing(LBBaP)on new-onset atrial fibrillation(NOAF)and atrial high rate episodes(AHREs)in patients with atrioventricular block(AVB).Methods Eighty-four patients with Ⅲ°AVB for pacemaker implantaion were divided into the LBBaP group(n=42)and the RVSP group(n=42)based on the site of the ventricular leads.The two groupswere compared in terms of the pre-and post-operative QRSd,ventricular pacing parameters,complications,incidence of stroke,NOAF and AHREs.Results(1)The incidence of postoperative NOAF and AHREs in the LBBaP group was significantly lower compared with RVSP group(P<0.05).(2)The p-QRSd in the LBBaP group was significantly shorter compared with RVSP group(P<0.05).(3)The two groups showed no significant differences in ventricular pacing parameters,incidence of complications and stroke events(P>0.05).Conclusion LBBaP is superior to right ventricular pacing in reducing the incidence of postoperative AHREs and NOAF in patients after implantation and improving the prognosis of patients.

Objective:A rare case of δ-globin gene (HBD) mutation in Guangdong Province was analyzed to provide reference for avoiding misdiagnosis of δ-thalassemia in clinic.Methods:The patient was admitted to Guangdong Maternal and Child Health Hospital, and the peripheral blood sample was collected for hematological phenotypes [mean erythrocyte volume (MCV), mean erythrocyte hemoglobin content (MCH), hemoglobin (Hb)] and Hb typing analysis. The routine deletion and mutation of α-thalassemia and β-thalassemia genes were analyzed by PCR-flow fluorescence hybridization. At the same time, DNA sequencing was used to analyze the type of HBD mutation.Results:The results of hematological phenotypes analysis showed that MCV was 87.9 fl, MCH was 29.3 pg, and Hb content was 140 g/L. The results of Hb typing showed that the contents of Hb F, Hb A 2, Hb A 2 variant, and Hb A were 0.4%, 1.3%, 0.6%, and 97.7%, respectively. No abnormality was found in α-thalassemia and β-thalassemia genes by routine deletion and mutation detection. According to DNA sequencing analysis, the patient had HBD: c.349 C>G variant. Conclusion:The low Hb A 2 content (reference value is 2.5% - 3.5%) in this case is due to the mutation of HBD, HBD: c.349 C>G variant is rare in Chinese population.

Objective:To investigate the treatment of an embryo with the partial dissolution of zona pellucida on day 2 and summarize the identification and treatment of embryos without zona pellucida during culture.Methods:A case of one live baby from an embryo with the partial dissolution of zona pellucida transferred on day 2 was retrospectively analyzed.Results:Through reducing the transplant cleaning process, the patient got pregnant and delivered a live girl baby at 39 +3 weeks of gestation. Conclusion:The early dissolution of zona pellucida may affect the compaction process of embryos, but a good outcome can still be obtained by carefully selecting embryos and reducing the washing process of embryo transfer.

Objective:To investigate the treatment of an embryo with the partial dissolution of zona pellucida on day 2 and summarize the identification and treatment of embryos without zona pellucida during culture.Methods:A case of one live baby from an embryo with the partial dissolution of zona pellucida transferred on day 2 was retrospectively analyzed.Results:Through reducing the transplant cleaning process, the patient got pregnant and delivered a live girl baby at 39 +3 weeks of gestation. Conclusion:The early dissolution of zona pellucida may affect the compaction process of embryos, but a good outcome can still be obtained by carefully selecting embryos and reducing the washing process of embryo transfer.

Objective:To investigate the regulatory effects and mechanism of mannose-binding lectin(MBL) on autophagy during the differentiation of 3T3-L1 adipocytes, and provide the feasibility for targeting autophagy to prevent obesity and related pathological conditions in natural immunity.Methods:3T3-L1 preadipocytes were cultured in vitro and induced to differentiation. Cell differentiation and lipid accumulation were analyzed by oil red O staining and CCK-8 was used to detect the effect of different concentrations of MBL (0, 1, 5, 10 μg/ml) on cell proliferation ability at different differentiation stages. Western blot was used to analyze the expression of MBL(10 μg/ml) on the key autophagy factors LC3B, Beclin1 and p62 protein at different stages of differentiation, and the changes of lipid droplet accumulation under the intervention of MBL were observed by oil red O staining. The protein and mRNA expression of autophagy key factors under the intervention of different concentrations of MBL were detected by Western blot and qRT-PCR. And autophagy flow analysis based on autophagic degradation was used to further illustrate the autophagic activity. The expression and phosphorylation of adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) signaling molecules were analyzed by Western blot. Results:The results of oil red O staining showed that 3T3-L1 preadipocytes could achieve complete differentiation after 10 days of induction. CCK-8 showed that the concentration of MBL (1-10 μg/ml) in the experimental group had no effect on cell proliferation at different differentiation stages. During the differentiation of 3T3-L1 preadipocytes, Western blot and qRT-PCR showed that the expression of autophagy-related proteins and mRNA levels was enhanced in the MBL treated group, and presented a concentration-dependent relationship. Oil red O staining showed that the lipid droplets in adipocytes at different stages of differentiation are reduced to varying degrees under the intervention of MBL. Fluorescence microscopy results further confirmed that MBL enhanced the autophagy activity of adipocytes by increasing the synthesis of autophagosomes. Moreover, under the intervention of MBL, the phosphorylation level of AMPK was significantly up-regulated, while the phosphorylation level of mTOR was significantly down-regulated, also showing a concentration-dependent relationship.Conclusions:MBL accelerates the autophagy process during the differentiation of 3T3-L1 adipocytes through AMPK/mTOR signaling pathway, reduces lipid accumulation, providing a possible functional pathway for the treatment of obesity and related metabolic diseases.

Objective:To evaluate the recurrence and progression of patients with pT1 high grade urothelial carcinoma of bladder (UCB) and glandular differentiation.Methods:We retrospectively analyzed the clinical and pathological information of 208 patients diagnosed as pT1 high grade urothelial carcinoma in the Fifth Central Hospital of Tianjin from January 2006 to February 2019.Among them, 78 cases were diagnosed as glandular differentiation (UCGD), the other 130 patients without histologic variants were served as control. The UCGD group included 62 male and 16 female, whose median age was 67 years old (range 38-81 years old). The control group contained 105 male and 25 female, whose median age was 66 years old (range 40-82 years old). Kaplan-Meier and Cox proportional hazard regression analyses were used to evaluate the predictors of oncologic outcomes.Results:The disease recurrence rate and progression rate in UCGD group were 65.4% (51/78) and 28.2% (22/78), higher than 38.5%(50/130) and 14.6%(19/130) of control group ( P<0.05). The median recurrence time in UCGD group was 41 months while 55 months in the control group. The median progression time in UCGD group was 39 months while 54 months in the control group. According to the univariate analysis, largest tumor size ( P=0.030), UCGD ( P=0.003) and lymphovascular invasion (LVI) ( P=0.032) were associated with disease recurrence. UCGD ( P=0.036) and LVI ( P=0.011) were associated with progression. Additionally, Cox multivariate analysis revealed that UCGD ( P=0.001), LVI ( P=0.038) were the independent factors of disease recurrence. UCGD ( P=0.007) and LVI ( P=0.037) were also found to be the independent factors of disease progression. Conclusions:Patients with T1 stage UCB and UCGD are at higher risk of disease recurrence and progression. Therefore, these patients should be followed up closely after being diagnosed and undergo individual treatment according to the situation.