Objective To investigate the role of spo0A gene in growth and sporulation of Clostridium difficile clinical isolates. Methods ClosTron gene knock-out system was used to knock out the spo0A gene of C. difficile strain C25. Bacterial growth curve was plotted by measuring D600 with spectrophotometer in different phases of bacterial growth. Malachite green staining technique was used to count the number of vegetative cells and spores under optical microscope. The sporulation rate was calculated. Results The spo0A mutant and its C25 parental strain showed similar patterns of growth. However, after knock-out of spo0A gene, an asporogenous phenotype was built, while the parental strain could produce spores as usual.Conclusions The spo0A gene plays a key role in sporulation but not growth of C. difficile strain.

Objective To evaluate the effects of ulinastatin postconditioning and combination of ulinastatin preconditioning and postconditioning on myocardial apoptosis in patients undergoing cardiac valve replacement with cardiopulmonary bypass (CPB).Methods Eighty New York Heart Association (NYHA) class Ⅱ or Ⅲ patients of both sexes,aged 21-59 years,scheduled for cardiac valve replacement with CPB,were randomly divided into four groups (n =20 each):normal saline control group (group C),ulinastatin preconditioning group (group U1),ulinastatin postconditioning group (group U2) and ulinastatin preconditioning plus postconditioning group (group U3).In group U1,uinastatin 20000 U/kg was infused via the central vein at 500-1000 U·kg-1 · min-1 after endotracheal intubation until 10 minutes before blocking the ascending aorta.In group U2,ulinastatin 10000 U/kg was infused via the aortic root at 4000-5000 U· kg-1 · min-1 at 5-7 minutes before opening the aorta.In group U3,ulinastatin preconditioning and postconditioning were performed as described in groups U1 and U2.In group C,the same volume of normal saline was infused instead of ulinastatin.Blood samples were taken from the radial artery at 10 minutes before blocking the ascending aorta,40 minutes after blocking the ascending aorta,45 minutes after opening the aorta and at the end of operation for determination of plasma concentrations of tumor necrosis factor-alpha (TNF-α) and soluble tumor necrosis factor receptor 1 (sTNF-R1).Myocardial tissues were obtained from the right atrial appendage at 45 minutes after opening the aorta for determination of the expression of TNF-α,bcl-2,bax,caspase-3,and apoptosis.The bcl-2/bax ratio and apoptotic index were calculated.Results Plasma concentrations of TNF-α and sTNF-R1 and the expression of TNF-α,bax,caspase-3 and apoptotic index were lower and the expression of bcl-2 and bcl-2/bax ratio were higher in groups U1,U2 and U3 than in group C and they were lower in group U3 than in groups U1 and U2 (P ＜ 0.05).Conclusion Ulinastatin postconditioning can inhibit myocardial apoptosis in patients undergoing cardiac valve replacement with CPB,and the efficacy of combination of ulinastatin preconditioning and postconditioning is stronger than that of ulinastatin postconditioning.The mechanism is involved in balancing the expression of bax and bcl-2 and down-regulating the expression of TNF-α and its receptor.

ObjectiveTo investigate the effects of ulinastatin postconditioning and combining ulinastatin postconditioning with pretreatment on myocardial inflammatory response in patients undergoing cardiac valve replacement under CPB.MethodsEighty NYHA class Ⅱ or Ⅲ patients of both sexes aged 21-59 yr undergoing cardiac valve replacement under CPB were randomly divided into 4 groups ( n =20 each): group control (group C) ; group ulinastatin pretreatment ( group U1 ) ; group ulinastatin postconditioning (group U2 ) and group ulinastatin pretreatment and postconditioning combined (group U3 ).Ulinastatin 20 000 U/kg was infused via central vein at 500-1000 U·kg-1 ·min-1 after tracheal intubation until 10 min before cross-clamping of ascending aorta in groups U1 and U3.Ulinastatin 10 000 U/kg was infused into root of aorta at 4000-5000 U· kg- 1 · min- 1 at 5-7 min before declamping of aorta in groups U2 and U3.Blood samples were obtained from radial artery before cross clamping of ascending aorta,at 40 min after aortic cross-clamping,at 45 min after declamping of aorta (T3) and at the end of operation for polymorphonuclear leukocyte (PMN) count,routine analysis of blood and determination of plasma concentrations of IL-10,TNF-α,IL-1 and IL-6 (by ELISA).Myocardial specimens were obtained at 45 min after declamping of aorta for determination of IL-1β and IL-6 expression by immune-histochemistry.Results Ulinastatin pretreatment and/or postconditioning significantly increased plasma IL-10 concentration and decreased plasma IL-1,IL-6,TNF-α concentrations and PMN count and myocardial IL-1β and IL-6 expression in groups U1,U2 and U3 as compared with group C.Plasma IL-10 concentration was significantly higher and plasma IL-1,IL-6 and TNF-α concentrations,PMN count and myocardial IL-1β and IL-6 expression were lower in group U3 than in groups U1 and U2.ConclusionUlinastatin postconditioning can inhibit myocardial imflammatory response in patients undergoing valve replacement under CPB.The protective effect can be augmented by combining ulinastatin postconditioning with pretreatment.

Objective To study the correlation between EPHX2 gene rs751141 polymorphisms and coronary heart disease (CHD) in Chinese population.Methods Totally 108 patients having more than one major coronary vessel with at least 50％ stenosis defined by Coronary angiography were selected as CHD group and control group consisted of 112 healthy subjects.Rs751141 polymorphisms were detected by PCR and gene sequence.Results Three kinds of genotypes at the rs751141 were detected,and no deviation was observed from HardyWeinberg equilibrium.There was statistical difference between the two groups regarding the distribution of the genotype frequencies and the frequency of allele C (P ＜ 0.05).Conclusion It suggests that rs751141 polymorphism is associated with CHD in Chinese population and the C allele might be a risk factor of CHD.

Objective Sporadic cases occurring in mall geographic unit could lead to extreme value of incidence due to the small population bases,which would influence the analysis of actual incidence.Methods This study introduced a method of hierarchy clustering and partitioning regionalization,which integrates areas with small population into larger areas with enough population by using Geographic Information System (GIS) based on the principles of spatial continuity and geographical similarity (homogeneity test).This method was applied in spatial epidemiology by using a data set of thyroid cancer incidence in Yiwu,Zhejiang province,between 2010 and 2013.Results Thyroid cancer incidence data were more reliable and stable in the new regionalized areas.Hotspot analysis (Getis-Ord) on the incidence in new areas indicated that there was obvious case clustering in the central area of Yiwu.Conclusion This method can effectively solve the problem of small population base in small geographic units in spatial epidemiological analysis of thyroid cancer incidence and can be used for other diseases and in other areas.

Objective To evaluate the effects of ulinastatin on cardiac function in heart valve replacement patients with cardio-pulmonary bypass (CPB).Methods 120 patients received valve replacements were divided into 4 groups at random.Group U 1,preconditioning group:ulinastatin parenteral solution (20 000 U/kg) was injected into the central veins for 10 min before the ascending aorta was clamped.Group U2,postconditioning group:ulinastatin ( 10 000 U/kg) was injected into the aortic root for 5 min before the aortic clamp was opened.Group U3,combined the treatments of group U1 and group U2.Group C was served as control without using ulinastatin.The ST-T of ECG at different 8 time points was recorded from preanesthesia to the end of operation.The dosage of vasoactive agents in the 4 groups was recorded after the aortic clamp was opened.Blood samples were taken from the radial artery at 4 time points during 1O min before the ascending aorta was clamed to the end of operation for determining the serum concentration of H-FABP,IMA,CK-MB,MDA and SOD.The changes in myocardium were examined by microscope.Results The automatic reheating rate of heart in group U1,group U2,and group U3 were 70％,73％ and 90％ respectively,which were all higher than group C (33％) after the aortic clamp was opened in 3 -5 min.The scores of reperfusion arrhythmia,change of ST segments in ECG ( elevation or depression),the dosage of vasoactive drugs ( dopamine and adrenaline) and their using time,the concentration of MDA,H-FABP,IMA and CK-MB in group U1 and group U2 were ＜ than those of group C ( P ＜0.05 ),but was ＞ than those of group U3 ( P ＜0.05 ).The activity of SOD in group U1 and group U2 were ＞ than those of group C ( P ＜ 0.05 ),but was ＜ than those of group U3 ( P ＜ 0.05 ).There were no significant differences between group U1 and group U2( P ＞0.05 ).The myocardium in group C had focal coagulative necrosis.The damage of myocardium in group U3 was minor,the cytoplasm and nucleus was homogeneous,and the boundaries were distinct.Conclusion Ulinastatin parenteral solution preconditioning and postconditioning could improve heart function after valves replacement on CPB.The protective effects were not significantly different regarding ulinastati was administered into the central veins before the ascending aorta was clamped vs.it was injected into the aortic root before the aortic clamp opening.Combined these 2 administration methods and dosages could produce collaborative protection.

Objective: To study the association between eating at night and skipping breakfast with college students anxiety symptoms, and to provide reference basis for preventing and alleviating college students anxiety symptoms. Methods: A cross sectional survey was conducted among 9 960 freshman from three universities in Kunming and Dali, Yunnan Province. The dietary frequency questionnaire was used to evaluate the dietary behavior of college students. The Depression Anxiety Stress Scale-21 (DASS-21) was used to evaluate the anxiety symptoms of college students. The association of late night snack and breakfast skipping with the association of anxiety symptoms in college students used generalized linear model and Logistic regression model. Results: The proportion of college students who reported eating at night and breakfast skipping in the last month was 72.5%(7 217/9 960) and 61.6%(6 131/9 960) respectively. The detection rate of anxiety symptoms in college students was 28.9%(2 875/9 960). There was a statistical significance between eating at night with anxiety symptoms( OR =1.40-2.54), and breakfast skipping with anxiety symptoms( OR =1.23-1.60)( P <0.05). The interaction between eating late at night and breakfast skipping was positively correlated with college students anxiety symptoms (multiplicative interaction, β=0.06, 95%CI=0.02- 0.10 , P<0.01; additive interaction, OR=2.00, 95%CI=1.59-2.51, P <0.01). Conclusion The study suggests that the college students who eat at night and frequently skipped breakfast are more likely to have anxiety symptoms. It suggested to promote the formation of healthy eating habits of college students, so as to reduce the occurrence of anxiety sympotoms.

Objective To investigate the effect of circ_0114427 on apoptosis and inflammation of alveolar epithelial cells induced by lipopolysaccharide(LPS)and its mechanism.Methods Hu-man alveolar epithelial cells were cultured in vitro and transfected with si-circ_0114427,microRNA(miR)-330-5p mimics or co-transfected with si-circ_0114427 and anti-miR-330-5p,and the cell-swere then treated with 10 mg/L LPS for 24 hours.The expression levels of circ_0114427 and miR-330-5p in the cells were detected using real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).The protein levels of Cleaved-caspase3 and Cleaved-caspase9 were evaluated using western blot.Enzyme-linked immunosorbent assay(ELISA)was used to assess the expression of in-terleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α).Cell apoptosis was analyzed using flow cytometry.The interaction between circ_0114427 and miR-330-5p was validated using a dual lucifer-ase reporter assay.Results After LPS treatment of alveolar epithelial cells,the expression of circ_0114427 was upregulated,while miR-330-5p expression was downregulated in in the cells(P＜0.05).Knockdown of circ_0114427 or upregulation of miR-330-5p could inhibit LPS-induced apop-tosis,Cleaved-caspase3,Cleaved-caspase9,IL-6,and TNF-α levels in alveolar epithelial cells(P＜0.05).Circ_0114427 directly interacted with and negatively regulated miR-330-5p.Downregulation of miR-330-5p reversed the inhibitory effect of circ_0114427 knockdown on LPS-induced cell apoptosis and inflammation.Conclusion Knockdown of circ_0114427 can inhibit LPS-induced apoptosis and inflammation in alveolar epithelial cells,possibly through upregulation of miR-330-5p.

Objective To investigate the effect of circ_0114427 on apoptosis and inflammation of alveolar epithelial cells induced by lipopolysaccharide(LPS)and its mechanism.Methods Hu-man alveolar epithelial cells were cultured in vitro and transfected with si-circ_0114427,microRNA(miR)-330-5p mimics or co-transfected with si-circ_0114427 and anti-miR-330-5p,and the cell-swere then treated with 10 mg/L LPS for 24 hours.The expression levels of circ_0114427 and miR-330-5p in the cells were detected using real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).The protein levels of Cleaved-caspase3 and Cleaved-caspase9 were evaluated using western blot.Enzyme-linked immunosorbent assay(ELISA)was used to assess the expression of in-terleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α).Cell apoptosis was analyzed using flow cytometry.The interaction between circ_0114427 and miR-330-5p was validated using a dual lucifer-ase reporter assay.Results After LPS treatment of alveolar epithelial cells,the expression of circ_0114427 was upregulated,while miR-330-5p expression was downregulated in in the cells(P＜0.05).Knockdown of circ_0114427 or upregulation of miR-330-5p could inhibit LPS-induced apop-tosis,Cleaved-caspase3,Cleaved-caspase9,IL-6,and TNF-α levels in alveolar epithelial cells(P＜0.05).Circ_0114427 directly interacted with and negatively regulated miR-330-5p.Downregulation of miR-330-5p reversed the inhibitory effect of circ_0114427 knockdown on LPS-induced cell apoptosis and inflammation.Conclusion Knockdown of circ_0114427 can inhibit LPS-induced apoptosis and inflammation in alveolar epithelial cells,possibly through upregulation of miR-330-5p.

Objective:To explore the effects of conditioned medium of human bone marrow mesenchymal stem cells (BMSCs) on the proliferation, adhesion and differentiation of immortalized human Müller cell line (MIO-M1).Methods:The differentiation was induced in the third-passage BMSCs with osteogenic, chondrogenic and adipogenic medium and identified by alizarin red, alcian blue and oil red O staining, respectively.The expression levels of mesenchymal stem cell markers CD73, CD90 and CD105 and hematopoietic cell markers CD34, CD45 and human leukocyte antigen-DR (HLA-DR) were assayed by flow cytometry.The expressions levels of Müller cell markers SOX9, glutamine synthetase (GS), vimentin and cellular retinaldehyde-binding protein (CRALBP), retinal stem cell markers SOX2, nestin and CHX10, and cell proliferation marker cyclin D3 (CCND3) in MIO-M1 cells were detected by immunofluorescence staining.The MIO-M1 cells were divided into standard medium group, 293T conditioned medium group, and BMSC conditioned medium group and were incubated in the medium according to grouping.The cellular area, circularity, elongation factor and perimeter were analyzed quantitatively.The cell cycle was detected by flow cytometry, and the cell proliferation was determined by neurospora experiment and 5-ethynyl-2'-deoxyuridine (EdU) staining.The expression of vascular cell adhesion molecule 1 (VCAM-1) at protein and mRNA levels in the culture supernatant was detected by enzyme linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR), respectively.The expression of retinal neuron markers protein kinase C (PKCα), Rhodopsin, microtubule-associated protein 2 (MAP2) and β-tubulin (Tuj1) was detected by immunofluorescence staining and qRT-PCR.Results:CD73, CD90, CD105 showed an enhanced expression, and CD34, CD45 and HLA-DR showed weakened expression in the BMSCs.The BMSCs differentiated into osteoblasts, chondrocytes and adipocytes.Expression of SOX9, GS, vimentin and CRALBP, SOX2, CHX10, nestin and CCND3 was found in the MIO-M1 cells.Compared with standard medium group and 293T conditioned medium group, MIO-M1 cells cultured in BMSC conditioned medium group changed into an elongated spindle-shaped or multipolar morphology with reduced cell area, increased elongation index and decreased circularity, showing statistically significant differences among them ( F=6.973, 12.370, 6.311; all at P<0.01). There were increased neurospheres formed by MIO-M1 cells in BMSC conditioned medium group compared with standard medium group and 293T conditioned medium group at different time points ( Fgroup=134.300, P<0.001; Ftime=82.910, P<0.001). Compared with the standard medium group and 293T conditioned medium group, the EdU-positive rate and proliferation index of MIO-M1 cells in BMSC conditioned medium group were significantly increased, with statistically significant differences ( F=6.973, 74.110; all at P<0.05); the VCAM-1 protein expression in cell supernatant and the relative expression level of VCAM-1 mRNA in BMSC conditioed medium group were significantly increased ( F=13.720, 7.896; all at P<0.05); the mRNA expression levels of PKCα, Rhodopsin, Tuj1 and MAP2 were higher in MIO-M1 cells of BMSC conditioned medium group under the condition of differentiation ( F=14.490, 5.424, 14.330, 7.405; all at P<0.05). Conclusions:BMSCs conditioned medium can change the morphology of MIO-M1 cells and promote their proliferation, adhesion and differentiation into retinal neurons.