Objective To observe the relation between the changes of plasma TpP and D-dimer in patients with acute cerebral infarction( ACI).Methods D-dimer and thrombus precursor protein (TpP) levels in plasma of 55 cases with ACI during early phase and 25 normal healthy subjects were detected,and 24/55 cases were measured dynamically. D-dimer levels were detected by automated latex D-dimer immunoassay. Plasma TpP level was determined using a new assay,the TpP_ TM(USA),which is based on an ELISA method.Results There was significant difference(P

Objective:To investigate the role of NOD-like receptor protein 3 (NLRP3) in the regulation of phagocytosis in Vibrio vulnificus ( V. vulnificus)-infected macrophages. Methods:Expression profiles of phagocytosis-related genes in PBS- and V. vulnificus-infected J774A.1 cells were analyzed by RNA-Seq. NLRP3-knockout (NLRP3 KO) J774A.1 cells were constructed using CRISPR-Cas9 gene-editing system. The phagocytosis of V. vulnificus and pHrodo RED-labelled Escherichia coli ( E. coli) bioparticles in parental and NLRP3 KO J774A.1 cells was detected by flow cytometry. Real-time PCR was performed to measure the expression of Fgr2 b gene at mRNA level in PBS- and V. vulnificus-treated parental and NLRP3 KO J774A.1 cells. Results:The expression of 18 phagocytosis-related genes was upregulated in V. vulnificus-infected J774A.1 cells than in PBS-treated J774A.1 cells ( P<0.05). There was a 5 bp deletion in the exon 2 of NLRP3 gene in NLRP3 KO J774A.1 cells, resulting in frameshift mutation and complete loss of NLRP3 expression. NLRP3 KO J774A.1 cells exhibited enhanced phagocytosis of V. vulnificus and pHrodo RED-labelled E. coli bioparticles than parental J774A.1 cells ( P<0.05). Besides, the expression of Fgr2 b gene at mRNA level was significantly increased in V. vulnificus-infected NLRP3 KO J774A.1 cells than in parental J774A.1 cells ( P<0.05). Conclusions:The phagocytosis of V. vulnificus in macrophages could be negatively regulated by NLRP3, which was possibly mediated through the regulation of Fgr2 b gene expression.

ObjectiveTo generate the prokaryotic expression systems of vwA1 and vwA2 genes of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai,and to determine the correlation among the target recombinant expressed products rVwA1 and rVwA2 and platelet-associated hemorrhage.MethodsThe vwA1 and vwA2 genes of L.interrogans strain Lai were amplified using high fertility PCRs and then the amplification products were sequenced.The prokaryotic expression systems of vwA1 and vwA2 genes were generated by using routine genetic techniques.The expression and dissolubility of rVwA1 and rVwA2 proteins were examined by SDS-PAGE plus Bio-Rad Gel Image Analyzer.Ni-NTA affinity chromatography was used to extract the expressed rVwA1 and rVwA2.Real-time fluorescence quantitative RT-PCRs were performed to determine the changes of vwA1-mRNA and vwA2-mRNA levels in the leptospires of L.interrogans strain Lai before and after infection of human umbilical vein endothelial cell line (HUVEC).The ability of leptospiral rVwA1 binding to human platelets was detected by flow cytometry.A hydrolytic test plus SDS-PAGE were applied to examine the cleavage of leptospiral rVwA2 by a human von Willebrand factor lysase (ADAMTS13).ResultsThe nucleotide and putative amino acid sequences of the cloned leptospiral vwA1 and vwA2 genes were 100％ identities compared to the reported corresponding genes.The generated prokaryotic expression systems of vwA1 and vwA2 genes could express soluble rVwA1 and rVwA2,respectively.When L.interrogans strain Lai infected HUVEC for 8 h,both the vwA1-mRNA and vwA2-mRNA levels were significantly up-regulated (P＜0.05).The result of flow cytometry showed that the leptospiral rVwA1 was able to combine with human platelets with a 60.8％ binding rate. Human recombinant vWF-A2 ( rhvWF - A 2 ),but not the leptospiral rVwA 2,could be hydrolyzed by human ADAMTS 13.Conclusion The vwA1 and vwA2 genes may play roles during infection of L.interrogans species,and the function of vwA1 gene product is referring to the hemorrhage in leptospirosis.

Objective To study the role of Vibrio vulnificus cytolysin(rVvhA) induced THP-1 apoptosis and calcium influx.Methods CCK-8 cell proliferation kit,Fluo3/AM staining and AnnexinV/PI staining were performed to identify the apoptosis and calcium influx induced by rVvhA in THP-1 cells.Results rVvhA could induce THP-1 apoptosis and up-regulate the cellular calcium concentration.BAPTAAM could enhance the calcium influx induced by rVvhA in THP-1.Conclusion rVvhA had cytotoxic to THP-1 cells by inducing apoptosis and triggering extracellular calcium influx.

Objective To investigate the molecular and cell signal transduction mechanisms by Lactobacillus cell wall extract(LCWE)inducing human-β-defensin-2(hBD-2)expression in human vaginal epithelial cells.Methods The induction of hBD-2 in human vaginal epithelial cells(WZV-1)by LCWE was observed using real-time PCR and Western blot.After stimulating WZV-1.the activation of NF-κB and p38MAPK signaling pathways were determined by Western blot.The induction of hBD-2 in WZV-1 cells by LCWE was observed with signaling pathways inhibitors of NF-κB and p38MAPK using real-time PCR and Western blot.Results The results showed that LCWE significantly upregulated hBD-2 expression in the time and dose-dependent manner.The maximal stimulatory effect of LCWE on the expression of hBD-2mRNA in WZV-1 cells were observed at the concentration of 50μg/ml after treatment for 8 h.After stimulation by 50μg/ml LCWE,Western blot analysis demonstrated that the phosphorylation of p38MAPK increased at 0.5 h significantly,peaked at 1 h,moreover the concentration of NF-κB in nucleus increased at 0.5 h significantly(P<0.05),peaked at 2 h.Blocking with inhibitor of NF-κB and(or)p38MAPK pathways results in decreased levels of HBD-2 expression.Conclusion These findings suggest that p38MAPK and NF-κB pathways play the important roles in induction of hBD-2 expression by LCWE in human vagihal epithelial cells.

Experiment examination plays an important role in evaluation of clinical microbiology experiment teaching.Standardized experiment examination system of clinical microbiology is a fair,objective and scientific assessment of students' learning effect and an important mean to evaluate teaching quality.The standardized experimental evaluation system includes organization,standardized examination contents,standardized procedures and operations,standardized invigilation and record,standardized scoring criteria,correction and prevention measures.

Objective To construct a shuttle plasmid for inducible gene expression in Borrelia burgdorferi (B.burgdorferi) with an advantage of flexible genetic manipulation.Methods The IPTG-inducible lac repressor/operator system from Escherichia coli (E.coli) was adopted and modified in the current study.The plasmid shuttle vector was developed by inserting multiple cloning sites,FLAG and HA tags into the shuttle vector by molecular cloning approaches.The target gene was inserted at the site under the control of the promoter (Tn5 derivate) in plasmid pQE30.This promoter contained two lac operators and a codonoptimized lacI gene driven by flaB promoter.Results A plasmid shuttle vector,pJJ275,was successfully constructed with the ability to express target genes in B.burgdorferi in the presence of IPTG.By using this system,a HA-tagged rpoS gene was introduced into the typical infectious strain B.burgdorferi B31.The target gene expression induced by IPTG was confirmed at transcriptional and translational levels.The RpoS dependent virulence factor of Borrelia,OspC,was also detected,indicating that the expressed protein was functional.Conclusion The constructed plasmid shuttle vector can express exogenous genes in B.burgdorferi with an inducible feature and an advantage of flexible genetic manipulation.It can be applied for genetic manipulation of B.burgdorferi involved in gene regulation and complementation.

Objective To construct a mutant strain of Streptococcus pneumoniae ( S.pneumoniae) with ciaH gene-knockout (ΔciaH) and to analyze the correlation between the ciaH gene and the bacterial re-sistance against β-lactam antibiotics.Methods The ciaH gene segament of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR product was sequenced after T-A cloning.A suicide plasmid pEVP3ciaH was constructed for the deletion of ciaH gene and then transformed into the ATCC 6306 strain by using the CaCl2 method .The mutant strain of S.pneumonia strain ATCC6306 with ciaH gene-knockout (ΔciaH) was genera-ted through homologous recombination , insertion inactivation and amphemycin screening , which was further identified by PCR , sequencing analysis and laser confocal microscopy .Double agar dilution method was used to detect the minimal inhibitory concentrations ( MICs ) of penicillin G ( PCN ) and cefotaxime ( CTX ) against theΔciaH mutant strain and the wild type strain .The differences between the MICs were further ana-lyzed.The changes of ciaH gene expression at mRNA level after treatment with 1/4 MIC of PCN or CTX were detected by real-time fluorescent quantitative RT-PCR ( qRT-PCR ) .Results The ciaH gene in the genomic DNA of the generated ΔciaH mutant strain was inactivated by insertion as indicated by PCR and se-quencing analysis .Results of the immunofluorescence assay showed that the ΔciaH mutant strain did not ex-press the CiaH protein .The MICs of PCN and CTX against the ΔciaH mutant strain were 32 μg/ml and 64μg/ml, respectively, which were significantly higher than that of the wild type strain (0.06 μg/ml and 1μg/ml) (P<0.01).The expression of ciaH gene at mRNA level was significantly elevated after treatment S.pneumoniae ATCC6306 strain with 1/4 MIC PCN or CTX (P<0.01).Conclusion The CiaH protein in the CiaH/CiaR two-component signaling system is involved in the resistance of S.pneumoniae against β-lac-tam antibiotics.

Objective To investigate the role of recombinant Vibrio vulnificus cytolysin (rVvhA) in inducing THP-1 cells damage and study the pathway of associated calcium influx .Methods Inverted mi-croscope, CCK-8 cell proliferation kit, Fluo3/AM staining and caspase activity detection were performed to analyze the damage of THP-1 cells induced by rVvhA and the pathway of calcium influx .Results rVvhA had cytotoxic effects on THP-1 cells in a dose-dependent manner .The concentrations of extracellular K +and LDH were respectively up-regulated after 1 h and 6 h of 12 μg/ml rVvhA intervention .Verapamil , Mibe-fradil and SKF-96365 could not prevent the influx of free Ca 2+induced by rVvhA .The activities of caspase-3 and caspase-9 were singanificantly enhanced by rVvhA in a time-dependant manner .Conclusion rVvhA can induce THP-1 cells damage through triggering extracellular calcium influx via porous channel on cell membrane.Moreover, rVvhA might induce THP-1 cell apoptosis through activating caspase-9/3-dependent pathway .

Objective To determine the functional motif of the recombinant Ricin B(rRicin B) in Vibrio vulnificus cytolysin (VVC) and understand its molecule pathogenic mechanism.Methods The motif of VVC was predicted through bioinformatics analysis and cloned into a procaryotic expression vector pET28a-rRicin B.The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG to express rRicin B.The expressed protein was further analyzed by SDS-PAGE and purified by Ni2+-NTA agarose.Renaturation of the rRicin B were also carried out for further analysis.ELISA assay and confocal microscope was applied to identify the activity of the rRicin B on human Hela cells.Results Ricin B motif located in the 336-465 amino acids of Vibrio vulnificus cytolysin with a relative molecular weight of 20×103.The result of ELISA showed that the antigenicity of rRicin B was 28.71 U/L after renaturation.FITC labeled rRicin B could bind to the cell membrane and enter the cytoplasm of human Hela cells.Conclusion The Ricin B motif in Vibrio vulnificus cytolysin bearing the similar ability with the natural Ricin B can bind to the cell membrane and enter the cytoplasm.This feature may play an important role in the activity of pore-forming and the cytotoxicity of Vibrio vulnificus cytolysin.