Multilocus sequence typing (MLST) is a molecular genotyping method based on nucleotide sequencing. The procedure of this method characterizes isolates of bacterial species using the DNA sequencing of multiple housekeeping genes(usually seven). For each housekeeping gene, the different sequences present within a bacterial species are assigned as distinct alleles.For each isolate, the alleles at each of the loci define the allelic profile or sequence type (ST). MLST has the advantages of being robust (based on genetic data) and electronically portable to generate data that allow rapid and global comparisons between different laboratories. In this paper, the principle, method, data analysis, application, advantages and flaws of MLST are introduced.

Objective To clone,express and characterize a tegument protein gene of Schistosoma japonicum(Sj29),and investigate the immune protection of the recombinant protein against S.japonicum in mice.Methods The gene coding for Sj29 protein was amplified by PCR,and the sequence was analyzed by bioinformatics tools.Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c(+).The recombinant plasmid was transformed into E.coli BL21(DE3) and induced the recombinant with IPTG.The recombinant protein(rSj29) was purified by His-binding-resin affinity chromatography and characterized by Western blotting.Three groups each with 10 BALB/c mice were immunized subcutaneously three times(two weeks interval) respectively with 100 ?l recombinant rSj29(0.1 mg/ml),adjuvant or PBS.At the 15th day after the final inoculation,each mouse was challenged by 40 ?2 cercariae of S.japonicum.At the 53th day after infection,the mice were sacrificed to obtain the number of adult worms,number of eggs in liver and feces.Serum samples were collected at pre-immunization and certain time after immuniza-tion,and were analyzed for IgG by ELISA.The localization of rSj29 in worms of different developmental stages was demonstrated by immunofluorescent technique.mRNA expression level of Sj29 gene in worms of different developmental stages and three groups after infection was detected by quantitative real-time PCR.Results A 576 bp Sj29 gene fragment was obtained.The recombinant protein rSj29 with Mr 22 900 was expressed in the form of inclusion body.The recombinant rSj29 can be recognized by sera of mice immunized with rSj29 and sera of infected mice.The number of adult worms(15.4?5.9),number of hepatic eggs(40 143.3?2 995.9) and number of fecal eggs(3 803.9?110.9) in re-combinant protein group were significantly higher than those of PBS control group(20?3.4,49 318.1?6 648.3,5 238.1? 303.5,respectively)(P

OBJECTIVETo discuss the impact of number of retrieved lymph nodes and lymph node ratio(LNR) on the prognosis in patients with stage II and III colorectal cancer.

METHODSClinicopathological data of 507 patients with stage II and III colorectal cancer were analyzed retrospectively. Follow-up was available in all the patients.

RESULTSThe total number of retrieved lymph nodes was 5801, of which 1122 had metastasis. There was a positive correlation between metastatic lymph nodes and retrieved lymph nodes(r=0.171, P<0.01). In stage II colorectal cancer there was a significant difference in 5-year survival rate between patients with more than 12 lymph nodes retrieved and those with less than 12 lymph nodes retrieved(P<0.01). LNR also affected the 5-year survival rate of patients with stage II and III colorectal cancer(P<0.05). In patients with similar LNR, the 5-year survival rate differed significantly among different regions of lymph node metastasis(P<0.05). LNR influenced the prognosis independent of the number of lymph nodes retrieved.

CONCLUSIONSThe number of retrieved lymph nodes is a prognostic factor for stage II and III colorectal cancer. More than 12 lymph nodes should be retrieved for better staging and prognosis. LNR is also a prognostic factor in stage II and III colorectal cancer. Regions of lymph nodes metastasis should be considered when evaluating the prognosis of patients using LNR.

Colorectal Neoplasms ; diagnosis ; pathology ; surgery ; Female ; Follow-Up Studies ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; diagnosis ; pathology ; Male ; Neoplasm Staging ; Prognosis ; Retrospective Studies

To investigate the T cell distribution characters of TCR Vbeta repertoire in Ph(+) and Ph(-) CML. The 24 subfamilies of the TCR Vbeta genes were amplified in peripheral blood T cells from 13 patients with CML (Ph+ b3a2, 5 cases; Ph+ b2a2, 5 cases; Ph(-), 3 cases) by RT-PCR, to analyze the usage of Vbeta subfamilies in different CML patients. The results showed that the expression pattern of Vbeta repertoire was different in normal individuals and in patients with CML which only have part of Vbeta subfamily T cells. 4 - 16 (mean 10.2) Vbeta subfamily T cells were detected in the Ph+ b3a2 CML, 8 - 11 (mean 8.8) Vbeta subfamily T cells in the Ph(+) b2a2 CML and 5 - 6 (mean 5.7) in Ph(-) CML. Moreover, the expression of Vbeta subfamily T cells was different among these three types CML.Vbeta10 and Vbeta16 were detected in the all cases with Ph(+) b3a2 and Ph(+) b2a2 CML, whereas Vbeta9 and Vbeta22 could be found in the most cases with Ph(+) b3a2 CML or Vbeta24 and Vbeta8 in Ph(+) b2a2 CML. In patients with Ph(-) CML, Vbeta24 were detected in all samples, and Vbeta9, Vbeta10, Vbeta13 and Vbeta22 were found in the most cases. The results suggest that skew distribution of TCR Vbeta subfamily T cells was existed in peripheral blood of Ph(+) and Ph(-) CML patients. The selected usage of TCR Vbeta is different in various types of CML patients. It may relate to difference of CML cells associated antigen and individual special immunity reaction.
Adolescent ; Adult ; Female ; Genes, T-Cell Receptor beta ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; immunology ; Leukemia, Myeloid, Chronic, Atypical, BCR-ABL Negative ; genetics ; immunology ; Male ; Middle Aged ; RNA, Neoplasm ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes ; metabolism

To observe the expression and clonal expansion of TCR Vbeta subfamily T cells induced by AML-M(2a) cells in vivo and in vitro, complementary determining region 3 (CDR3) of TCR beta with variable region genes was amplified by using RT-PCR in both peripheral blood mononuclear cells (PBMNC) and T cells from mixed lymphocyte and tumor culture (MLTC) from four AML-M(2a) patients. The positive products were further analyzed to identify the clonality of T cells by genescan. The results showed that the similarity distribution of TCR Vbeta subfamily T cells was found Vbeta in PBMNC and MLTC. One or two clonality expansions of T cells could be found in predominant TCR Vbeta subfamily T cells induced by A ML-M2a cells from 3 cases in vivo and in vitro. It was concluded that clonal expansion of TCR Vbeta subfamily T cells stimulated selectively by AML-M(2a) cells may be a specific immune response of patient's T cells to AML-M(2a) cells associated antigen. The clonal proliferation of TCR Vbeta subfamily T cells were affected somewhat by environmental difference in vivo and in vitro.
Gene Rearrangement, T-Lymphocyte ; Genes, T-Cell Receptor beta ; Humans ; Leukemia, Myeloid, Acute ; immunology ; Lymphocyte Activation ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes ; immunology

Objective: To investigate whether insulin stimulates the translocation of glucose transporter-4 (GLUT4) and glucose uptak e in ischemic myocardium. Methods: Plasma concentration of gluc ose, lactate, free fatty acid and insulin were determined by autoanalyser, and G LUT4 was studied by Western blotting analysis. Results: Insulin increased GLUT4 significantly in sarcolemma of ischemic myocardium ［(25±4)% vs (40±6)%］, and GLUT4 content in intracellular membrane decreased proporti onally. The glucose uptake increased significantly in insulin-ischemic myocardi um. The uptake of insulin-ischemic myocardium was almost 2 times that of ischem ic myocardium. Conclusion: Insulin stimulation results in GLUT4 translocation and increases glucose uptake in ischemic myocardium. When myocardi al ischemia occurs, insulin is helpful in increasing myocardial glucose uptake a nd utilization.

Objective: To investigate whether there is additi ve effects of hyperinsulinemia and ischemia on expression of canine myocardial G LUT4 gene in vivo. Methods: The expression of myocardial GLU T4 was determined by semiquantitative immunoblotting.The expression of GLUT4 mRN A was determined by semiquantitative Northern blotting. Results: Dramatic changes were seen in GLUT4 mRNA and GLUT4 expression in the ischemic hearts.After infusing insulin for 8 h,regional GLUT4 mRNA and GLUT4 levels in is chemic hearts were 2.5, 2.3-fold that of expression in normal hearts(P<0.01 ). Myocardial glucose uptake in ischemic hearts was increased by 4-fold when co mpared with normal hearts(P<0.01). Conclusion: There are not only additive effects of hyperinsulinemia and low-flow ischemia on canine myoc ardial GLUT4 mRNA and GLUT4 expression in vivo, but also increase of myocar dial glucose uptake. Enhanced GLUT4 expression may be an important protective m echanism by which myocardial cells enhance glucose uptake and metabolism during low-flow ischemia.

OBJECTIVETo construct glypican-3 (GPC-3) short hairpin RNA (shRNA) and investigate the effects of GPC-3 transcription silencing on hepatoma cell invasion and angiogenesis mechanisms.

METHODSGPC-3-specific shRNA and non-target control shRNA were constructed and transfected into the human hepatoma cell lines HepG2, MHCC-97H, and Huh7. shRNA-mediated silencing of GPC-3 expression was confirmed at the mRNA and protein levels by fluorescence quantitative reverse transcription (FQRT)-PCR and western blotting, respectively. The effect of silenced GPC-3 expression on cell proliferation was detected by EdU and sulforhodamine B assays, on migration by wound healing (scratch) assay, on invasion by transwell chamber assay, and on apoptosis by luminescence assay of caspase-3/7 activity. The effect of silenced GPC-3 expression on angiogenesis-related signaling factors was detected by FQRT-PCR (for the glioma-associated oncogene homolog-1 hedgehog signaling factor, GLI1, and the beta-catenin Wnt signaling factor, b-catenin), immunofluorescent staining (for the insulin-like growth factor-II, IGF-II), and ELISA (for the vascular endothelial growth factor, VEGF). Pairwise comparisons were made by the independent sample t-test, and multiple comparisons were made by one-way ANOVA.

RESULTSIn all cell lines, transfection with the GPC-3-specific shRNA significantly reduced GPC-3 mRNA levels (% reduction as compared to the non-target control shRNA: HepG2, 89.2+/-6.0%, t = -25.753, P less than 0.001; MHCC-97H, 75.3+/-4.9%, t = -26.487, P less than 0.001; Huh7, 73.6+/-4.6%, t = -27.607, P less than 0.001); the GPC-3 protein levels were similarly reduced. The GPC-3 shRNA-silenced cells showed significantly reduced proliferative, migratory and invasive capacities, as well as significantly increased apoptosis. The shRNA-mediated GPC-3 silencing was accompanied by significant down-regulation of b-catenin mRNA (HepG2, 46.9+/-0.6%; MHCC-97H, 67.5+/-2.7%; Huh7, 56.3+/-8.4%) and significant up-regulation of GLI1 mRNA (HepG2, 49.2+/-28.6%; MHCC-97H, 54.6+/-24.4%; Huh7, 31.6+/-15.7%). At 72 h after transfection, the HepG2 cells showed significant down-regulation of VEGF protein (54.3+/-1.5%, t = 46.746, P less than 0.001).

CONCLUSIONGPC-3 contributes to migration, invasion, angiogenesis, and apoptosis of hepatoma cells, possibly through its interactions with the Wnt/b-catenin and Hedgehog signaling pathways. GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma.

Apoptosis ; Carcinoma, Hepatocellular ; blood supply ; metabolism ; pathology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Gene Silencing ; Glypicans ; genetics ; Humans ; Liver Neoplasms ; blood supply ; metabolism ; pathology ; Neoplasm Invasiveness ; Neovascularization, Pathologic ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo understand the biochemical characteristics, virulence genes and pathogenicity of Shigella flexneri Xv isolated in Beijing.

METHODS61 strains of S. flexneri Xv isolated from diarrhea patients in Beijing were systematically determined through biochemical reactions and serological tests. Application of PCR technique in detection of virulence genes on ipaH, sen, virF, ial and pulsed-field gel electrophoresis (PFGE) was used to identify the related characteristics and on rat lung slices to determine its pathogenicity.

RESULTSAll of the S. flexneri Xv could ferment glucose, mannitol, melibiose and arabinose. Using serum agglutination, we found that the antigen structure was (IV: 7, 8). IpaH, sen, virF and ial that carried rates of virulence genes appeared to be 100%, 81.97%, 75.41% and 80.30%, respectively. Among 61 strains of S. flexneri Xv, the PFGE typing of Shigella bacteria could be divided into 25 belt types while the results from rat lung slices showed inflammatory change of Xv.

CONCLUSIONS. flexneri Xv was found that it carried high rate of Shigella virulence genes, exhibiting genetic polymorphism and highly invasive.

Animals ; Humans ; Microbial Sensitivity Tests ; Rats ; Shigella flexneri ; classification ; isolation & purification ; pathogenicity ; Virulence ; genetics

OBJECTIVETo investigate the prevalence and subtypes of influenza viruses in Liaoning regions from November 1999 to March 2005.

METHODSInfluenza virus was isolated by embryonated eggs together with cell culture and subtypes, identified by HI test.

RESULTSDuring the study in 1999 - 2005, a total number of 2713 swab specimens were collected in different cities in Liaoning regions in which 188 strains were identified for influenza viruses with an average rate as 7.0%. A total number of 1466 swab specimens were collected by both Centers for Disease Control and Prevention in Dalian city and Liaoning province, and 167 strains were identified positive with an average rate of 11.4%. Influenza A3, A1 and B/Yamagata all appeared before March 2002 which were predominant strains. However, since then Influenza A1 has never appeared again in Liaoning regions and B showed some changes, from Yamagata to Victoria, the characteristics on the prevalence of influenza appeared only in the period of November to February.

CONCLUSIONIt was meaningful to analyze the surveillance data of influenza in different years in Liaoning regions in order to better understand the characteristics of influenza and the shifting of subtype.

China ; epidemiology ; Humans ; Influenza A virus ; classification ; isolation & purification ; Influenza B virus ; classification ; isolation & purification ; Influenza, Human ; epidemiology ; Population Surveillance ; Seasons