The amino acid sequences of the OmpA protein isolated from Escherichia coli QML2206-1(E.coli QML2206-1)in our laboratory were analyzed for homology with different strains of OmpA proteins using bioinformatics software,and the OmpA protein was analyzed for its physicochemical properties,transmembrane structure and signal peptide prediction,B-cell anti-genic epitope prediction,secondary and tertiary structure prediction.The OmpA gene fragment was ligated with pET-32a vector to construct a prokaryotic expression vector,which was purified by a nickel column affinity purification system after prokaryotic expression and optimization of ex-pression conditions in BL21(DE3).The purified recombinant protein was fully mixed with Freund's adjuvant to immunize mice,and the levels of mouse-specific IgG antibody and the expression levels of cytokines CD4,CD8 and IL-4 in mouse serum were detected by ELISA,and the immuno-protective effect was evaluated by mouse attack protection test.OmpA protein is a hydrophilic protein with no transmembrane structural domains and a secondary structure consisting mainly of irregular coils(47.98％)and α-helices(29.77％),with 12 antigenic epitopes that can bind to anti-bodies produced by B cells.The recombinant protein OmpA with a relative molecular mass of a-bout 55 kDa was successfully obtained by prokaryotic expression,and the highest expression was induced by IPTG concentration of 0.000 4 mmol/L for 6 h at 37 ℃.The serum-specific IgG anti-body potency of recombinant protein immunized mice was up to 1∶32 000;the expression levels of CD4,CD8 and IL-4 in the serum of immunized mice were elevated compared with those of the con-trol group.The survival rate of mice was 80％and 40％after attack with minimum lethal dose(MLD)and 2 times minimum lethal dose(2MLD),respectively.OmpA recombinant protein has good antigenicity and certain immunoprotective effects,and this study provides a technical basis for the next step in the development of a genetically engineered subunit vaccine against yak-appli-cable E.coli based on OmpA protein.

Objective To investigate the protective effects of Kaempferol(KAE)against hepatic lipid de-position induced by a high-fat diet,as well as the underlying mechanisms.Methods C57BL/6J male mice were fed a high-fat diet for 22 weeks to establish a chronic nonalcoholic steatohepatitis(NASH)model.KAE was admin-istered during the last 8 weeks as an interventional agent to evaluate its effects.Liver lipid deposition was assessed,and the expression levels of endoplasmic reticulum(ER)stress-related proteins,activation of the Farnesoid X Re-ceptor(FXR)signaling pathway,and the expression of lipid synthesis-related genes were analyzed.In vitro,pal-mitic acid(PA)was used to stimulate AML-12 cells to induce lipid accumulation.Additionally,siRNA targeting FXR was transfected into AML-12 cells to investigate the role of the ER stress-FXR signaling pathway in mediating the effects of KAE.Results The intervention of kaempferol inhibited the rapid weight gain induced by a high-fat diet,reduced serum total cholesterol,triglyceride levels,and ALT activity,effectively alleviated large-scale lipid aggregation in the liver,thereby exerting a protective effect against hepatic lipid deposition in NASH.Mechanisti-cally,KAE decreased hepatic ER stress,promoted the expression of FXR and its activation marker SHP,thereby suppressing the expression of FASN and reducing hepatic lipid synthesis.In vitro,KAE treatment significantly re-versed the inhibitory effect of excessive ER stress on FXR activity,as evidenced by the upregulation of FXR activ-ity leading to decreased FASN expression and reduced steatosis in AML-12 cells.Moreover,FXR knockdown mark-edly abolished the protective effects of KAE on lipid deposition in AML-12 cells exposed to PA,by eliminating the promoting effect of KAE on SHP expression and the SHP-mediated suppression of SREBP1c.Conclusions KAE treatment alleviated ER stress,thereby enhancing FXR/SHP signaling and subsequently suppressing lipid synthesis to reduce hepatic lipid accumulation.These findings suggest that KAE holds therapeutic potential for the manage-ment of hepatic steatosis in NASH.

The amino acid sequences of the OmpA protein isolated from Escherichia coli QML2206-1(E.coli QML2206-1)in our laboratory were analyzed for homology with different strains of OmpA proteins using bioinformatics software,and the OmpA protein was analyzed for its physicochemical properties,transmembrane structure and signal peptide prediction,B-cell anti-genic epitope prediction,secondary and tertiary structure prediction.The OmpA gene fragment was ligated with pET-32a vector to construct a prokaryotic expression vector,which was purified by a nickel column affinity purification system after prokaryotic expression and optimization of ex-pression conditions in BL21(DE3).The purified recombinant protein was fully mixed with Freund's adjuvant to immunize mice,and the levels of mouse-specific IgG antibody and the expression levels of cytokines CD4,CD8 and IL-4 in mouse serum were detected by ELISA,and the immuno-protective effect was evaluated by mouse attack protection test.OmpA protein is a hydrophilic protein with no transmembrane structural domains and a secondary structure consisting mainly of irregular coils(47.98％)and α-helices(29.77％),with 12 antigenic epitopes that can bind to anti-bodies produced by B cells.The recombinant protein OmpA with a relative molecular mass of a-bout 55 kDa was successfully obtained by prokaryotic expression,and the highest expression was induced by IPTG concentration of 0.000 4 mmol/L for 6 h at 37 ℃.The serum-specific IgG anti-body potency of recombinant protein immunized mice was up to 1∶32 000;the expression levels of CD4,CD8 and IL-4 in the serum of immunized mice were elevated compared with those of the con-trol group.The survival rate of mice was 80％and 40％after attack with minimum lethal dose(MLD)and 2 times minimum lethal dose(2MLD),respectively.OmpA recombinant protein has good antigenicity and certain immunoprotective effects,and this study provides a technical basis for the next step in the development of a genetically engineered subunit vaccine against yak-appli-cable E.coli based on OmpA protein.

Objective To investigate the protective effects of Kaempferol(KAE)against hepatic lipid de-position induced by a high-fat diet,as well as the underlying mechanisms.Methods C57BL/6J male mice were fed a high-fat diet for 22 weeks to establish a chronic nonalcoholic steatohepatitis(NASH)model.KAE was admin-istered during the last 8 weeks as an interventional agent to evaluate its effects.Liver lipid deposition was assessed,and the expression levels of endoplasmic reticulum(ER)stress-related proteins,activation of the Farnesoid X Re-ceptor(FXR)signaling pathway,and the expression of lipid synthesis-related genes were analyzed.In vitro,pal-mitic acid(PA)was used to stimulate AML-12 cells to induce lipid accumulation.Additionally,siRNA targeting FXR was transfected into AML-12 cells to investigate the role of the ER stress-FXR signaling pathway in mediating the effects of KAE.Results The intervention of kaempferol inhibited the rapid weight gain induced by a high-fat diet,reduced serum total cholesterol,triglyceride levels,and ALT activity,effectively alleviated large-scale lipid aggregation in the liver,thereby exerting a protective effect against hepatic lipid deposition in NASH.Mechanisti-cally,KAE decreased hepatic ER stress,promoted the expression of FXR and its activation marker SHP,thereby suppressing the expression of FASN and reducing hepatic lipid synthesis.In vitro,KAE treatment significantly re-versed the inhibitory effect of excessive ER stress on FXR activity,as evidenced by the upregulation of FXR activ-ity leading to decreased FASN expression and reduced steatosis in AML-12 cells.Moreover,FXR knockdown mark-edly abolished the protective effects of KAE on lipid deposition in AML-12 cells exposed to PA,by eliminating the promoting effect of KAE on SHP expression and the SHP-mediated suppression of SREBP1c.Conclusions KAE treatment alleviated ER stress,thereby enhancing FXR/SHP signaling and subsequently suppressing lipid synthesis to reduce hepatic lipid accumulation.These findings suggest that KAE holds therapeutic potential for the manage-ment of hepatic steatosis in NASH.

Background Transforming growth factor-β1(TGF-β1) plays an important role in corneal woundhealing.The effects of TGF-β1 on the synthesis of extra cellular matrix (ECM) vary upon different concentrations.Previous studies focused on the effects of high concentration of TGF-β1 on keratocytes under the two-dimensional culture condition,and the effect of low concentration of TGF-β1 on the synthesis of ECM in keratocytes remains unclear.Objective This study was to investigate the growth of Pellet,a three-dimensional model of corneal stroma cells in vitro,and its ECM synthesis under a low concentration of TGF-β1.Methods Bovine corneal stromal cells were isolated from fresh bovine eyeballs by two-step digestion by collagenase and cultured using DMEM/F12 medium with 10％ fetal bovine serum (FBS).Pellets derived fresh bovine keratocytes with culture medium containing 0.25 ng/ml TGF-β1 +5％ FBS and 0.50 ng/ml TGF-β1 +5％ FBS were established,respectively.The morphology of Pellets was observed under the natural light at 48 hours,1 week,2 weeks and 3 weeks after culture.In 3 weeks after culture,the cell structures was observed by hematoxylin-eosin staining,and Calcein-AM/propidium (Calcein-AM/PI) staining was used to assay the cell viability.Real-time flurorescence quantitative PCR and immunofluorescence technology were applied to analyze the expressions of α-smooth muscle actin (α-SMA),fibronectin (FN),type Ⅰ collagen (Col Ⅰ) and type Ⅲ collagen (Col Ⅲ) mRNA and proteins.RT-PCR was employed to detect the expressions of lumican (LUM) mRNA and keratocan (KERA) mRNA in the cells.Results Cells in Pellet clustered throughout the culture duration.Hematoxylin-eosin staining showed the mass red-dyed collagen fibers in both 0.25 ng/ml TGF-β1 +5％ FBS group and 0.50 ng/ml TGF-β1 +5％ FBS group,and most cells possessed complete structures.The death rate of the cells was (33.60±1.65)％ in the 0.25 ng/ml TGF-β1 +5％ FBS group and (30.90±0.78) ％ in the 0.50 ng/ml TGF-β1 +5％ FBS group,showing an insignificant difference between them (t =0.144,P=0.887).The expressions of α-SMA,FN and Col Ⅲ proteins in 0.25 ng/ml TGF-β1 +5％ FBS group were lower than those in the 0.50 ng/ml TGF-β1 + 5 ％ FBS group (tα-SMA =4.622,P =0.010;tFN =2.973,P =0.040;tCol Ⅲ =7.845,P＜0.001),but the expression of Col Ⅰ in 0.25 ng/ml TGF-β1 +5％ FBS group was higher than that in 0.50 ng/ml TGF-β1+5％ FBS group (tColⅠ =4.022,P=0.016).The ratio of Col Ⅲ/Col Ⅰ in 0.25 ng/ml TGF-β1+5％ FBS group was lower than that in the 0.50 ng/ml TGF-β1 +5％ FBS group in both mRNA and protein level (tmRNA =-3.039,P =0.038;tprotein =3.215,P =0.032).The expression of LUM mRNA and KERA mRNA were detected in Pellet at different time points.The expression of LUM mRNA in 0.25 ng/ml TGF-β1 +5％ FBS group increased over time.While in 0.50 ng/ml TGF-β1 +5％ FBS group,the expression of LUM mRNA peaked at 1 week but declined at 2 weeks.The expression of KERA mRNA in two groups were all peaked at 1 week but declined at 2 weeks.Conclusions Low-dose TGF-β1 in Pellet can maintain the normal growth of keratocytes and synthesize ECM.The expression of ECM tends to the normal condition after reducing the concentration of TGF-β1,implying a scarless expression.

Background Extracellular matrix (ECM) fibrosis leads to corneal scaring during the process of cornea wound healing.Transforming growth factor-β1 (TGF-β1) is known to mediate overproduce of ECM components.Our previous study developed a three-dimensional model for corneal stromal cells culture in vitro.Objective The hypothesis of this study was to apply TGF-β1 in the three-dimensional culture system to establish a corneal stroma ECM fibrosis model.Methods Fresh bovine corneas were extracted for the culture of bovine keratocytes in constructed three-dimension culture system.The Pellets were cultured in the DMEM/F12+ 10％ fetal bovine serum (FBS) medium with 0.5 ng/ml or 1.0 ng/ml TGF-β1 or without TGF-β1,respectively.Calcein AM/(propidium iodide) PI staining was employed to assay the cell viability 2 weeks after culture.The expressions of α-smooth muscle actin (α-SMA),type Ⅰ collagen (Col Ⅰ) and Col Ⅲ mRNA and protein in the cells were detected by real-time PCR and Western blot respectively 48 hours,1 week and 2 weeks after cultured.The results were statistically analyzed.Results Cultured for 48 hours in the Pellet system,corneal stromal cells clustered and was identified alive by Calcein-AM/PI staining in 2 weeks.The relative expression levels of α-SMA,Col Ⅰ and Col m mRNA were elevated in both the 0.5 ng/ml and 1.0 ng/ml TGF-β1 supplement groups in comparison with the only DMEM/F12+10％ FBS group,with marked difference among the three groups (Fgroup =696.745,P＜0.001;Fgroup =35.166,P＜0.001;Fgroup =33.677,P＜0.001),and the expression levels increased with the lapse of culture time (Ftime =5.863,P＜0.05;Ftime =298.614,P＜0.001;Ftime =607.472,P＜0.001).The synthetic rate of Col Ⅲ mRNA was obviously faster than that of Col Ⅰ mRNA.Western blot showed that only a trace of α-SMA,Col Ⅰ and Col Ⅲ were detected 48 hours and 1 week after culture.The expression levels of α-SMA,Col Ⅰ and Col Ⅲ in Pellet system in 0.5 ng/ml TGF-β1 medium were 0.395±0.208,1.060±0.175 and 0.629±0.382,and in 1.0 ng/ml TGF-β1 medium were 0.758±0.228,1.201 ±0.187 and 0.753±0.468,respectively 2 weeks after culture,significant differences were shown among the three groups (α-SMA:F=10.691,P＜0.05;Col Ⅰ:F=14.094,P＜0.05;Col Ⅲ:F=10.995,P＜0.05).Conclusions Addition of TGF-β1 and serum enhance the assembly and fibrosis of ECM,showing the higher expressions of specific fibrotic markers in bovine keratocytes Pellet.This culture systerm can be used as a candidate three-dimensional model for corneal stroma ECM fibrosis.

Objective To characterize the baseline status of Chinese diabetic patients based on data derived from Chinese cohort from SOLVETM study.Methods Patients with type 2 diabetes initiating basal insulin detemir at the decision of the physician were eligible for the study.Data on demographics,medical history,glycemic profile and treatment regimen at baseline were collected by physicians.Results A total of 3272 patients [female 42％,male 58％,mean age (56.2 ± 10.8) years] were included in the study.Their BMI was (25.3 ± 3.3) kg/m2.The duration of diabetes was 4.0 (0.1-27.0) years,and the duration of treatment with oral antidiabetic drugs (OADs) was 3.0(0.0-20.2) years.The proportions of subjects with diabetic macro-and micro-vascular complications were 15.8％ (515 cases) and 27.1％ (866 cases),respectively.The hemoglobin Al c (HbAl c) at baseline was (8.33 ± 1.70) ％,and the fasting blood glucose (FPG) was (9.5 ± 2.6) mmol/L.Conclusions A large proportion of patients with type 2 diabetes remain in poor glycemic control,and the prevalence of diabetic complications is high,which requires optimal therapeutic strategy for the patients with suboptimal glycemic control.

OBJECTIVETo determine the feasibility of water fluoridation to prevent caries in Kunming by investigating the epidemiological status of dental caries and dental fluorosis of children, and to provide the longitudinal reference data for the long-term epidemiology survey of dental caries and dental fluorosis in Kunming city.

METHODSThrough stratified cluster sampling method, 212 5-year-old children and 1149 12-year-old children were recruited in the survey. Dental caries condition of each child was clinically examined, dental fluorosis was examined in 12-year-old group.

RESULTSThe prevalence of dental caries of primary teeth in 5-year-old group was 73.6%, mean value was 4.47 +/- 4.39. The values of permanent teeth in 12-year-old group were 53.5% and 1.42 +/- 1.83. The prevalence of dental fluorosis in 12-year-old group was 4.1% and the average community fluorosis index was 0.03.

CONCLUSIONBased on the high prevalence of dental caries and the low prevalence of dental fluorosis, it is suggested that using water fluoridation to prevent caries is feasible and necessary in Kunming city.

Child ; DMF Index ; Dental Caries ; Dentition, Permanent ; Fluoridation ; Fluorosis, Dental ; Humans ; Prevalence ; Tooth, Deciduous