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

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

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

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 explore the effective therapy for maintenance hemodialysis patients in dificiency syndrome in end-stage renal disease.

METHODSNinety-seven cases were divided into an observation group (51 cases) and a control group (46 cases) randomly, and routine western medicine was used in both of them. On this base, moxibustion was used in Zusanli (ST 36) and Sanyinjiao (SP 6) in paper-tube-moxibustion equipment in the observation group. Evaluate the therapeutic effect on symptoms by comparing the symptom scores in two groups before and after treatment.

RESULTSAll the symptom scores in the observation group were improved after treatment, and the differences were significant (all P < 0.05). Among all symptoms, the most improved ones included lassitude and fatigue, short breath and aversion to talk, poor appetite, soreness and softness of waist and knees, aversion to cold, cold extremities and so on. In the aspect of therapeutic effect on symptoms, the total effective rate in observation group (64.7%, 33/51) was higher than that in control group (23.9%, 11/46), and the difference was significant (P < 0.05).

CONCLUSIONMoxibustion can improve the clinical symptoms of maintenance hemodialysis patients in end-stage renal disease, and can generate some therapeutic effect to the dificiency syndrome of this disease.

Acupuncture Points ; Adult ; Aged ; Female ; Humans ; Kidney Failure, Chronic ; therapy ; Male ; Middle Aged ; Moxibustion ; Renal Dialysis ; Young Adult

OBJECTIVETo detect and identify the potential specific serum biomarkers for diagnosis of papillary thyroid cancer.

METHODSSamples of 35 patients with papillary thyroid carcinoma, 40 patients with benign thyroid nodule and 34 healthy individuals were analyzed using the SELDI-TOF ProteinChip System and bioinfomation technology to find the differential peaks which were separated by HPLC and then further analyzed by LC-MS/MS. The protein sequences were analyzed by SEQUEST software and searched in Bioworks database.

RESULTSThe top six mass-to-charge ratio (M/Z) peaks with the smallest P value were 6651, 6452, 7653, 7932, 15 106 and 15 848 Da, respectively. The 6651 and 6452 Da proteins were weakly expressed in papillary thyroid carcinoma but highly expressed in benign thyroid nodules and healthy individuals. The differences had statistical significance (P < 0.01). The 7653, 7932, 15 106, 15 848 Da proteins were highly expressed in papillary thyroid carcinoma but weakly expressed in benign thyroid nodules and healthy individuals. The differences were statistically significant (P < 0.01). Combination of these six proteins, using the method of leave-one-out to make crossing detection, the specificity of discriminating papillary thyroid carcinoma and non-cancer was 88.0%, and its sensitivity was 92.5%. The 6651 and 6452 Da proteins were identified as apolipoprotein C-I and apolipoprotein C-III, respectively. The 7653 and 15 106 Da proteins were identified as the same protein-alpha-globin, and the 7932 and 15,848 Da proteins were identified as the same protein-beta-globin.

CONCLUSIONThe detection of differentially expressed apolipoprotein C-I, apolipoprotein C-III, alpha-globin, and beta-globin may have utility for diagnosis of papillary thyroid carcinoma and are worthy of further investigation.

Adult ; Apolipoprotein C-I ; blood ; Apolipoprotein C-III ; blood ; Biomarkers, Tumor ; blood ; Carcinoma, Papillary ; blood ; diagnosis ; Female ; Humans ; Male ; Middle Aged ; Protein Array Analysis ; Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Thyroid Neoplasms ; blood ; diagnosis ; alpha-Globins ; metabolism ; beta-Globins ; metabolism

OBJECTIVETo discuss the impacts of moxibustion for regulating spleen and stomach function on the survival quality of the patients of end stage renal disease (ESRD) with maintenance hemodialysis (MHD).

METHODSOne hundred and nine cases of uremia with MHD from 3 hemodialysis centers were randomized into an observation group (58 cases) and a control group (51 cases). The regular hemodialysis and conventional medication were used in the two groups. In the observation group, on the basis of the common treatment, moxibustion was applied to Zusanli (ST 36) and Sanyinjiao (SP 6), 2-3 times a day, the treatment of 4 weeks made one session. Totally, 3 sessions were required and the follow-up lasted for 3 months. KDQOL-SF (kidney disease quality of life short form,KDQOL-SFTM 1. 3) was adopted for the questionnaire investigation on survival quality before treatment, after treatment and at the end of follow-up separately in the two groups.

RESULTSAfter treatment, the survival quality scores in terms of physical functioning (83.62+/-13.27 vs 79.32+/- 22. 17), general health (58. 88+/- 20.24 vs 48.82+/-20.89) and vitality (77.07+/-15.56 vs 70. 59+/-22.61) in the observation group were higher than those in the control group (all P<0. 05). In comparison before and after treatment in the same group, the survival quality scores in terms of physical functioning, general health, vitality and symptoms/problems were all improved in the observation group (all P<0. 05). At the end of follow-up, the survival quality scores in terms of physical functioning, general health, mental health, social functioning, vitality, effects of kidney disease and cognitive function were higher in the observation group as compared with those in the control group (all P<0. 05). In comparison of the results at the end of follow-up with those before treatment, the survival quality scores in terms of vitality, symptoms/problems and cognitive function in the observation group were improved (all P< 0. 05). The differences were not significant in all of the 19 fields of survival quality evaluation before and after treatment, and after follow-up in the control group (all P>0. 05).

CONCLUSIONMoxibustion for regulating spleen and stomach function improves the survival quality of the patients with hemodialysis in terms of physical functioning, general health and vitality, which benefits the psychological condition of the patients, resulting in the improvements of the survival quality in the fields of mental health, social functioning, effects of kidney disease and cognitive function.

Adult ; Aged ; Female ; Humans ; Kidney Failure, Chronic ; physiopathology ; therapy ; Male ; Middle Aged ; Moxibustion ; Quality of Life ; Renal Dialysis ; Spleen ; physiopathology ; Stomach ; physiopathology