Objective To investigate the distribution of ompA gnne in 15 Chinese reference standard strains belonging to 15 serogroups of Leptospira interrogate, and to express recombinant OmpA ( rOmpA ) and to identify immunogenicity and immunoprotection of rOmpA. Methods Genomic DNAs from different leptospiral strains were extracted by phenol-chloroform method. Entire ompA gene fragments from the strains were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of ompA gene from L. interrogans strain 56601 was constructed, and the expression and yield of rOmpA were determined by SOS-PAGE plus Bio-Rad Agarose Image Analyser. Rabbits were immunized with rOmpA for obtaining antiserum, and immunodiffusion test was used to measure the antiserum's titer. Western blot assay was performed to determine the immunoreaetivity of rOmpA with the antiserum against rOmpA and antiserum against whole cell of L. interrogans strain 56601, while mi-croscopic agglutination test (MAT) was applied to detect the cross agglutination to the 15 L. interrogans strains. A leptospire adhering cell model and a leptospire infecting guinea pigs model were used to determine the adhesion-bloc-king effect of rOmpA antiserum and immunoprotection of rOmpA. Results All the 15 L. interrogans strains, but not L. biflexa strain Patoe Ⅰ , had sequence conserved ompA genes. The yield of rOmpA was approximate 20% of the total bacterial proteins, rOmpA could induce rabbits to produce antibody and immunodiffusion titer of the anti-serum was 1:4. Both antisera against rOmpA and against whole cell of L. interrogans strain 56601 were able to pro-duce positive Western blot signs to rOmpA, and the former offered 1 : 20-1 : 320 MAT titers to the 15 L. interrogans strains. 1: 10-1:160 dilutions of rOmpA antiserum could efficiently block L. interrogans strain 56601 adhering to J774A. 1 cells, and 100 μg and 200 μg rOmpA displayed 50.0% and 75.0% immunoprotective rates in the infee-ted guinea pigs. Conclusion ompA gene only exists in genomes of different pathogenic L. interrogans serogroups. rOmpA has relatively stronger antigenicity, cross immunoreactivity and certain immunoprotection, implying that this recombinant protein may be used as a candidate antigen for developing universal genetic engineering vaccine of L. interrogans.

Objective To determine the ability of biofilm formation of Staphylococcus ephtermidis isolates and analyze the correlation between the icaA gene and its expression and biofilm formation. Methods Collecting 205 Staphylococcus epidermidis isolates identified with normal laboratory tests (coagulase-negative, biochemical identification, polymyxin-resistant and novobiocin-sensitive ), the suspected isolates were con-formed with API-Staph. Biofilm production was assessed by incubating the strains on Congo Red Agar (CRA) plates and quantitative biofilm production determined by a 96-well tissue culture plate and biofilm morphous were detected by scanning electron microscope ( SEM ) ; Amplifying partial fragments of icaA genes with PCR; Analyzing the expression levels of icaA gene with RT-PCR through Bio-Rad system and Quantity One software. Results 24 isolates showed positive in CRA tests, 22 isolates were positive in semiquantita- tive adhesion assays and 28 isolates existed icaA gene among 205 isolates of Staphylococcus epidermidis. The icaA-positive strains demonstrated biofilm formation (microcolonies on silica films ) while icaA-negative strains only adhered as individual cells under scanning electron microscope. All 22 strains which showed positive in semiquantitative adhesion assays harbored the icaA gene. The expression levels of icaA gene with RT-PCR in 6 Staphylococcus epidermidis isolates showed a higher tendency in 4 strains which demonstrated positive in semiquantitative adhesion assays than 2 negative strains in semiquantitative adhesion assays. Conclusion The isolations of Staphylococcus epidermidis have the abilities of forming biofilm, and the icaA gene and its normal expression is the important molecular biology foundation of biofilm formation. Other fac-tors maybe involve in the expression of icaA gene in Staphylococcus epidermidis isolates.

Objective To investigate the mechanisms of Chlamydia pneumoniae (C. pn)-induced foam cell formation, the expression of ATP binding cassette transporter AI ( ABCA1 ) and perexisome prolif-erator-activated receptor γ (PPARγ) were examined. Methods THP-1 monneytes were induced into mac-rophages after the addition of 160 nmol/L phorbol myristate acetate (PMA) for 72 h. THP-1-dorived macro-phages when co-cuhured 50 mg/L low density lipoprotein (LDL) were designated randomly in four groups: control (uninfected) group, C. pn infection group, rosiglitazone + C. pn infection group and rosiglitazone group. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellnlur choles-terol ester were detected by enzyme-flnoreseence. The expression of ABCA1, PPARγ, mRNA and protein were determined by RT-PCR and Western blot, respectively. Results C. pn down-regulated the expression of ABCA1, PPARγ at mRNA and protein levels in a concentration-dependent manner in THP-1-derived mac-rophages when co-incubated with LDL. Resiglitazone not only concentration-dependently alleviated the down-regulation of ABCA1 expression by C. pn infection (P<0.05), but also markedly suppressed the accumula- tion of lipid droplets and cholesteryl ester by C. pn at higher concentrations ( 10 and 20 μaol/L). Condu-sion C. pn induces foam cell formation by down-regulating the expression of ABCA1 via PPART pathway, which may provide a new evidence for the development and progression of atherosclerosis initiated by C. pn infection.

Marburg virus (MARV) is a lethal virus that causes fatal hemorrhagic fever.It belongs to the Filoviridae family which also includes Ebola virus.MARV is similar to Ebola virus in structure and infection mechanism.Moreover,the diseases caused by them have similar clinical symptoms.However,researches on MARV are less than those on Ebola virus.In this review,we focus on the viral structure,especially the structure of MARV glycoprotein (GP) which determines its infectivity,functions of MARV GP as well as protective antibodies and vaccines against this protein.

Objective To understand the correlations between HIV-1 subtypes and changes in CD4+T cell count over time in patients with different subtypes of HIV-1 infection.Methods A total of 94 patients who were diagnosed with HIV-1 infection in Nanjing and received at least twice CD4+T cell counting test before antiretroviral therapy (ART) were recruited in this study.Descriptive analysis was used to present the rates of CD4+T cell decline for different HIV-1 subtypes.Logistic regression analysis and nonparametric test were conducted to investigate the factors responsible for CD4+T cell decline and to analyze the correlations between the rates of CD4+T cell decline and HIV-1 subtypes.Results The median monthly rate of CD4+T cell decline was-2.20 [interquartile range (IQR):-11.36-2.13] cell/μl.Of all patients,25.5% (24/94) had a significant decline (≥30%) in CD4+T cell count.Compared with the patients infected with CRF01_AE,those infected with CRF07_BC (OR=0.28,95%CI: 1.7-6.5) or other subtypes (OR=0.16,95%CI: 1.0-2.9) had a lower risk of significant decline in CD4+T cell count.In addition,results of the nonparametric test showed that the patients infected with CRF01_AE (M=-21.54,IQR:-30.97——11.92 cell/μl) had a faster CD4+T cell loss than those infected with CRF07_BC (M=-11.26,IQR:-14.06——5.63 cell/μl) (P=0.033).Conclusion HIV-1 subtype is associated with the rate of CD4+T cell decline.It is important to monitor the changes in CD4+T cell count in patients infected with CRF01_AE and to carry out timely ART.

Objective To investigate the distribution of cytotoxin-associated gene A (cagA) of Helicobacter pylori (Hp) and the polymorphism of EPIYA motifs in patients with upper gastrointestinal diseases in Changchun area of China and to evaluate the association between EPYIA motifs patterns and gastrointestinal diseases.Methods Hp strains were isolated from clinical samples.Their cagA gene was analyzed by PCR and sequencing analysis.Nucleotide sequence of cagA gene was translated into amino acid sequence by using DNAMAN software,and then the amino acid sequence was imported into software MEGA6.0 for multiple comparisons and construction of a phylogenetic tree.Results A total of 60 Hp strains were isolated and identified from gastric mucosa specimens collected from 298 patients.Hp infection was not correlated with patient's age or sex (P>0.05).The isolation rate of Hp in peptic ulcer disease (PUD) group was higher than that in non-peptic ulcer dyspepsia (NUD) group (P<0.05).Of the 60 Hp strains,90% (54/60) carried cagA gene.Twenty-three out of 26 successfully sequenced strains (88.4%) were East Asian-type including 22 containing EPIYA-ABD motif and one containing EPIYA-ABBD motif.The other three strains (11.6%) were Western type including two carrying EPIYA-ABC motif and one carrying EPIYA-BC motif.Results of the phylogenetic tree showed that the sequences of cagA gene were clustered into two groups,East Asian-type and Western-type groups.East Asian-type strains caused no disease cluster of statistical significance.All Western-type Hp strains were isolated from patients with peptic ulcer disease (PUD).Four mutant Hp strains were detected in the PUD group and the amino acid mutations preferentially occurred in the EPIYA-B segment.Conclusion The positive rate of Hp cagA gene is 90% in this region.Its distribution is not related to the type of gastrointestinal diseases.EPIYA-ABD (84.6%,22/26) is the predominant EPIYA motif.The amino acid mutation of EPIYA-B segment is closely related to peptic ulcer disease.Neither significant change in the sequence of 3' region of Hp cagA gene nor regional difference is observed in those Hp strains circulating in Changchun area of China.

Objective To investigate the correlation between Streptococcus pneumoniae (S.pneumoniae) StkP kinase and drug resistance and to analyze the binding ability of StkP extracellular region (EC-StkP) to β-lactam antibiotics.Methods A stkP gene knockout (ΔstkP) mutant was constructed from S.pneumoniae strain ATCC6306 by insertional inactivation method.E-test was performed to detect the minimum inhibitory concentrations (MIC) of penicillin (PCN) and cefotaxime (CTX) against ΔstkP mutant and its wild-type strain.Bioinformatic softwares were used to predict the EC-StkP of S.pneumonia strain ATCC6306,to generate the three-dimensional structure model of EC-StkP and to analyze the correlation between the structure and functions of EC-StkP.PCR was performed to amplify the extracellular segment of stkP (EC-stkP) gene and the product of it was sequenced after T-A cloning.A prokaryotic expression system of EC-stkP gene was constructed.SDS-PAGE in combination with a gel image analysis system was used to detect the expression of the recombinant EC-StkP (EC-rStkP).The expressed EC-rStkP was extracted by Ni-NTA affinity chromatography.The binding abilities of EC-rStkP to PCN and CTX were detected by isothermal titration calorimetry (VT-ITC) and surface plasmon resonance (Biacore).Results S.pneumonia strain ATCC6306 was sensitive to PCN (MIC=0.06 μg/ml) and CTX (MIC=0.12 μg/ml),but its ΔstkP mutant was resistant to the two antibiotics (PCN MIC=16 μg/ml,CTX MIC=32 μg/ml).The 295 aa segment was predicted as the extracellular region at C-end of StkP of S.pneumoniae strain ATCC6306,containing four penicillin-binding proteins and Ser/Thr kinase-associated (PASTA) domains.The cloned EC-stkP segment and the EC-stkP segment in GenBank shared 99.6% similarity in nucleotide sequence and 100% in amino acid sequence.The constructed prokaryotic expression system for EC-stkP gene expressed EC-rStkP in soluble form.Both PCN and CTX could bind to EC-rStkP and CTX was better than PCN in term of binding ability.Conclusion The stkP gene of S.pneumonia is closely related to drug resistance and the encoded protein,Ser/Thr kinase StkP,can recognize and bind to β-lactam antibiotics.

Objective To investigate the effects of stromal cell-derived factor 1 (SDF-1) and an CXC chemokine receptor 4 (CXCR4) antagonist (AMD3100) on the invasion and migration capabilities of the huaman choriocarcinoma cell line JAR for further elucidating the role of SDF-1/CXCR4 axis in the pathogenesis of preeclampsia.Methods JAR cells were divided into four groups: SDF-1 group (treated with 50 ng/ml of SDF-1),SDF-1+AM3100 mixed group (first treated with 100 ng/ml of AMD3100 for 2 hours and then treated with 50 ng/ml of SDF-1),AMD3100 group (treated with 100 ng/ml of AMD3100) and blank control group (without any treatment).RT-PCR was performed to detect the expression of CXCR4 at mRNA level in JAR cells.Western blot assay was used to measure the expression of CXCR4 and p-AKT at protein level.MTT assay was used to analyze the effects of different concentrations of SDF-1 (10,30,50 and 100 ng/ml) on the proliferation of JAR cells at different time points (0,24,48,72 h).Transwell invasion assay and wound-healing assay were used to test the changes in invasion and migration capabilities of JAR cells after different treatments.Results (1) Results of the RT-PCR showed that the expression of CXCR4 at mRNA level in JAR cells was increased in the SDF-1 group (1.839±0.083) as compared with that in the blank control group (1.372±0.086),AMD3100 group (0.694±0.045) or SDF-1+AM3100 mixed group (0.703±0.093).Moreover,the differences between the SDF-1 group and the other three groups were statistically significant (F=30.67,P<0.05).Compared with the blank control group,the expression of CXCR4 at mRNA level in JAR cells was decreased in the AMD3100 group (P<0.01).(2) Results of the Western blot assay showed that the expression of CXCR4 and p-AKT at protein level in JAR cells were enhanced in the SDF-1 group as compared with that in the blank control group,AMD3100 group or SDF-1+AM3100 mixed group.Compared with the blank control group,the expression of CXCR4 and p-AKT at protein level in JAR cells were inhibited in the AMD3100 group.(3) Results of the MTT assay showed that SDF-1,especially at the concentration of 50 ng/ml,could enhance the proliferation of JAR cells (P<0.05) and its best effect on proliferation was seen at 48 h.(4) Results of the Transwell invasion assay showed that the number of transmembrane cells in the SDF-1 group (70.49±2.42) was more than that in the blank control group (54.36±2.26),AMD3100 group (21.68±8.31),or SDF-1+AMD3100 mixed group (28.18±4.61).The differences between the SDF-1 group and the other three groups were statistically significant (F=116.26,P<0.01).Compared with the blank control group,the number of transmembrane cells was reduced in the AMD3100 group (P<0.05).(5) Results of the wound-healing assay showed that the relative migration distance was increased in the SDF-1 group (1.162±0.034) as compared with that in the blank control group (0.823±0.101),AMD3100 group (0.160±0.047),or SDF-1+AMD3100 mixed group (0.183±0.064).The differences between the SDF-1 group and the other three groups were statistically significant (F=30.500,P<0.05).Compared with the blank control group,the relative migration distance was decreased in the AMD3100 group (P<0.01).Conclusion The invasion and migration of huaman choriocarcinoma JAR cells can be enhanced by SDF-1,but inhibited by AMD3100.This study indicates that the blocked biological axis of SDF-1/CXCR4 may play an important role in the pathogenesis of preeclampsia through inducing abnormal activation of PI3K/AKT pathway,which results in inhibited invasion and migration of trophoblast cells and placenta abnormality.

Members of the tripartite-motif (TRIM) protein family share a highly conserved domain architecture known as RBCC motif,which is composed of a RING finger domain,one or two B-box domains,a coiled-coil domain as well as diverse types of C-terminal regions.TRIM proteins can not only maintain the normal physiological functions of the body,but also regulate the development of various diseases.In the current review,we focus on recent advances in the structures of TRIM proteins and their functions in the development of viral infection,cancer and neurodegenerative disease.

Innate immune cells have a critical role in defense against infections and diseases.Central to this is the broad specificity with which they can detect pathogen-associated patterns and danger-associated patterns via the pattern recognition receptors (PRRs) they express.TLR is one of the most largely studied families of PRRs.Activation of TLR promotes both innate inflammatory responses and the induction of adaptive immunity.Given the characteristics of TLR,various Toll like receptor agonists are used in vaccine design to accelerate the immune response and produce long-lasting protection.