Objective To knock down hemolysin hlyA gene and insert green fluorescence protein gene of vibrio cholerae vaccine candidate NFYY101. Methods Amplified fragments of hlyA gene upstream (hlyAup) and downstream (hlyAdown),lacz-GFPuv,and hlyAup-laczGFPuv-hlyAdown, and plasmids treated with specific enzymes were utilized to construct recombinant plasmids pUC18-hlyAup-laczGFPuv-hlyAdown and pCVD442-hlyAup-laczGFPuv-hlyAdown. Following the construction of the recombinant suicide plasmids ,a vaccine candidate was constructed by homologous recombination ,while SM10λpir carrying pCVD442- hlyAup-laczGFPuv-hlyAdown was utilized as the donor strain to transfect NFYY101. Results Construction of recombinant suicide plasmids pCVD442- hlyAup-laczGFPuv-hlyAdown was satisfactory that hemolysin hlyA gene was knocked out and green fluorescence protein gene was successfully inserted of vibrio cholerae vaccine candidate NFYY101. Conclusion Construction of the recombinant suicide plasmid pCVD442-hlyAup-laczGFPuv-hlyAdown successfully can be used for knocked out the hlyA gene and added green fluorescence protein gene as genetic marker into the chromosomal DNA of vibrio cholerae vaccine candidate.

Objective To learn more about virulence genes and clonal complex group structure of Vibrio parahaemolyticus (VPH)strains separated from aquatic products in East China coastal areas between 2007 and 2012.Methods Seventy-nine strains separated from aquatic products in eastern coastal areas(Guandong,Fujian,Shanghai,Shandong,Jiangsu and Beijing)of China between 2007 and 2012 were identified as VPH by real-time-PCR with gene tlh.Gene tdh,trh and orf8 were also detected.Subgroup analysis and complex analysis were conducted of the VPH strains to build the minimum spanning tree respectively based on ST and pST types.Results In 79 VPH strains,gene tlh was positive while 8.86%(7 /79)of the isolates of gene tdh were positive.The carrying rate of gene orf8 was 8.86%.These 79 strains were of 69 ST types,involving 3 clonal complex and 62 singleton.By amino acid(AA)-multilocus sequence typing(MLST),79 strains covered the 23 pST types,2 clonal complexes and 1 singleton.The 363 single nucleotide polymorphisms(SNPs)were divided into 68 patterns.Conclusion VPH strains from aquatic products in eastern coastal areas of China are characterized by high polymorphisms and a low carrying rate of virulence genes.ST3 type is dominating when MLST typing is used while pST1 type is the majority by AA-MLST typing.

Objective To find out more about the population structure and clonal complex of clinical Vibrio parahaemolyticus strains in Asia.Methods Clinical Vibrio parahaemolyticus strains data were screened in Asia with complete ST and pST types from PubMLST public database,their subgroup and complex were analyzed,and the minimum spanning tree based on ST and pST types respectively was completed.Results From the database,341 items of ST and pST types of Asian clinical Vibrio parahaemolyticus strains were screened,including 157 ST,most of which were of ST3 type.Totally 214 items of data came from China (including Hong Kong and Taiwan),and covered 133 ST,most of which were of ST3 type.eBURST software was used and 17 groups and 94 singletons were found.Software STRUCTURE analysis showed that the appropriate subset number of clinical V.parahaemolyticus strains in Asia was 7,and that the average distance between samples in each subgroup was 0.9113.Conclusion Clinical V.parahaemolyticus strains in Asia show high diversity and can be subdivided into seven subgroups.ST3 type is dominating when multilocus sequence typing(MLST)is used and pST2 type is the majority by AA-MLST typing.

Objective To evaluate the efficiency and safety of esophageal stents combined with radiotherapy compared with esophageal stents alone in the treatment of advanced esophageal cancer.Methods CBM,VIP,CNKI,Cochrane Library,Pubmed and Embase etc were searched by computer begining from the establishment of these datebases to December 2012.The related references as well as communicated with other researchers were also traced to obtain certain informations.Randomized and quasi-randomized controlled trials compared esophageal stents plus radiotherapy with esophageal stents alone in the treatment of advanced esophageal cancer were included.The statistical software RevMan 5.0 was used.Results Seven published articals were included (443 patients),and all trails methodological quality were grade C.The results of Metaanalysis showed that compared with esophageal stents along,esophageal stents combined with radiotherapy improve 1-year survival rates and reduce the local recurrence rates.Gastrointestinal bleeding rates,chest pain rates,gastro-esophageal reflux rates remained similarily.Conclusion Compared with esophageal stents along,esophageal stents combined with radiotherapy can improve 1-year survival rates and reduce the local recurrence rates.

Objective To compare and analyze the phylogenetic tree and sequence variant characteristics of Klebsiella species between 16S rDNA and rpoB. Methods Eighteen isolates identified as genus Klebsiella (with 15 of K. Pneumoniae and 3 of K. Oxytoca) by automated biochemical tests were selected. DNA were extracted, 16S rDNA and rpoB genes were amplified and sequenced with Klebsiella 16S rDNA and rpoB primers. Together with already published 8 species of Klebsiella and 9 species of Enterobacteriaceae 16S rDNA and rpoB sequences from GenBank, totally 35 sequences of 16S rDNA and rpoB respectively, phylogenetic trees were constructed with MEGA 4.0 to the analysis of groups. DNAStar/MegAlign was used for comparison of variable regions of 16S rDNA and rpoB, with analysis of degree of divergent at the same time. Results As for all 35 sequences, both 16S rDNA and rpoB phylogenetic trees divided Klebsiella species into three groups, 15 of K. Pneumoniae in this study and 6 of K. Pneumoniae from GenBank (except for K. Oxytoca and K. Mobilis) cluster to group Ⅰ, K. Oxytoca and K. Mobilis were cluster to group Ⅱ and Ⅲ, respectively. In rpoB phylogenetic tree, no matter group Ⅰ and group Ⅱ, or subgroup within group Ⅰ, the bootstrap values in each node of rpoB phylogenetic tree is obviously higher than that of 16S rDNA. Moreover, as for cluster to K. Oxytoca, rpoB is better than 16S rDNA. Analysis nucleic acid sequences of Klebsiella species, with 41 variable regions and 4 most significant regions were found within the Klebsiella 16S rDNA, while rpoB with 63 variable regions, and 1 most significant region. The similarity of 16S rDNA and rpoB within Klebsiella were 95.9%-100% and 90.2%-100% respectively. Further analysis divergent degree of 16S rDNA and rpoB within Klebsiella, the divergent value of rpoB (0-10.6) is higher than that of the 16S rDNA(0-4.0). Conclusion As for molecular classification and identification within KlebsieUa species, rpoB has more advantages than 16S rDNA.

Objective To characterize Hfq-dependent phenotypes in stress response and to dissect Hfq-dependent transcription of virulence genes and stress-responsive genes in Vibrio cholera.Methods The hfq null mutant strain (△hfq) and the complemented mutant strain (△hfq/pUC18-hfq) were constructed from the wild-type Vibrio cholera.Comparisons on the motility,biofilm formation,growth under various oxygen-supplying conditions,outer membrane resistance,and sensitivity to oxidative stress were analyzed between the wild type strain and the mutant strains.Reverse-transcript fluorescence quantitative PCR (RT-qPCR) was used to determine the transcriptional levels of target genes in the above mentioned strains.Results △hfq and △hfq/pUC18-hfq strains were successfully constructed.The motility,outer membrane resistance and sensitivity to oxidative stress were reduced,but biofilm formation was enhanced in the hfq null mutant strain.RT-qPCR testified that Hfq had regulation effects on gene transcription for forming falagellum,extracellular polysaccharide,outer membrane protein and oxidative stress in Vibrio cholera.Conclusion As a RNA chaperone,Hfq could affect Vibrio cholera in its biofilm formation,resistance to oxidative stress and antibiotics resistance through regulating the transcription of multiple metabolic genes and virulence genes,which indicates that Hfq,combined with other regulators,may play a key role in the complex regulation of metabolic genes and virulence genes.

Background and purpose:It has a signiifcant effect for erlotinib on treatment of patients with epidermal growth factor receptor mutation in advanced non-small cell lung cancer (NSCLC). But almost all patients will eventually progress for the resistance of drug. This study was to evaluate the efifcacy and safety of retreatment of erlotinib in patients with advanced NSCLC. Methods:It was a retrospective analysis of the 46 advanced NSCLC patients who previously treated with erlotinib and had clinical beneift. The patients were given erlotinib 150 mg orally once daily after failure to other medications until disease progression or the occurrence of intolerable toxicity. The clinical features, therapeutic effect and survival were analyzed. Results: The objective response rate of retreatment with erlotinib was 28.3%. The disease control rate was 60.9%. The rate of symptom relief was 45.7%. The median progression-free survival was 3.6 months. The median overall survival was 7.3 months. One-year survival rate was 8.7%. The median progression-free survival was signiifcant longer in the patients who stopped taking erlotinib more than 6 months than those less than 6 months (P=0.002). The median overall survival was signiifcant longer in the patients whose ECOG ≤2 than those ECOG >2 (P=0.038). The most common drug-related adverse events were rash and diarrhea. Conclusion:The retreatment of erlotinib could possibly prolong the survival time of patients who previously treated with erlotinib and had clinical beneift.

OBJECTIVETo analyses the genetic diversity and relationship of Salmonella typhi strains isolated from different years and districts in Guizhou province.

METHODSRibotyping with 16s rDNA probe was used to describe the diversity of the 209 strains which were isolated in 26 counties of Guizhou province, from 1959 to 1999. The antibiotics resistance was also studied.

RESULTSTwenty-six ribotypes were found in all 209 strains, with two dominant types. The strains isolated from local typhoid epidemics belonged to the unique Ribotypes. The major ribotypes of the resistant strains were RT7 and RT1.

CONCLUSIONThe Salmonella typhi isolates from Guizhou diverged obviously. The abundant clones and multi-resistance of the strains might serve the major reasons of the high morbidity of typhoid in Guizhou.

Blotting, Southern ; China ; DNA, Bacterial ; genetics ; DNA, Ribosomal ; genetics ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Ribotyping ; Salmonella typhi ; classification ; genetics ; isolation & purification

Objective To understand the elonal expansion and genetic diversity of Salmonella en-terica semtype Paratyphi A (SPA) and to construct a typing method to determine the epidemic clones of the isolates. Methods Antimicrobial susceptibility testing was performed with 3980 SPA isolates by the cen-trolled Kirby-Bauer disc diffusion technique on Muller-Hinton agar plates. A total of 15 SPA with nalidixie acid resistance for mutations in gyrA, gyrB, gyrC and gyrE genes within the quinolone-resistant determina-tion region (QRDR) were examined. Subtyping of 121 isolates of SPA from seven counties in Yuxi were studied using pulsed-field gel eleetrophoresis (PFGE) analysis following digestion of chromosomal DNA with restriction endanucleases Spe Ⅰ and Xba Ⅰ. PFGE patterns were analyzed by duster analysis. Results The nalidixic acid-susceptible isolates predominated in 1999 but was replaced by nalidixic acid -resistant (NAR) isolates after 2000. Amplification by PCR and sequencing of the genes with subsets of 15 NAR strains re-vealed that the resistance mechanisms had resulted from single point mutations in the gyrA gene. Spe Ⅰ and Xba Ⅰ digestion of 121 isolates gave five and four different PFGE patterns with the predominance of the Spe Ⅰ 01 and Spe Ⅰ 02 (or the Xba Ⅰ 01) epidemic patterns, respectively. Spe Ⅰ 01 and Spe Ⅰ 02 consisted of 37.2% and 57.9% of isolates, respectively, or Xba Ⅰ 01 consisted of 95.0% of isolates. Conclusion The incidence of resistance to nalidixic acid of the isolates increased during the study period. PFGE patterns Spe Ⅰ 01 and Spe Ⅰ 02 (or Xba Ⅰ 01), the main clones of the epidemics, are highly prevalent in Yuxi. PFGE with Spe Ⅰ and Xba Ⅰ is a useful technique to differentiate SPA.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Diarrhea ; virology ; Female ; Humans ; Male ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; methods ; Young Adult