Objective To investigate the dynamic changes in the serum inflammatory cytokines and their association with neurogenic pulmonary edema (NPE) in the patients with severe hand-foot-mouth disease (HFMD).Methods Eighty-nine patients with severe HFMD from March 2010 to December 2012 were recruited in the study.The patients were divided into NPE group and central nervous system diseases (CNSD) group according whether they had NPE.The cytokines,including interleukin (IL)-4,IL-6,IL-10,IL-17,tumor necrosis factor-α (TNF-α) and interferon-γ(IFN-γ)were evaluated by using enzyme-linked immunosorbent assay on day 1,3 and 5 after admission to hospital.Risk factors for NPE involvement during hospital stay were analyzed with multivariate Logistic regression analysis.Results (1) Compared with the CNSD group,the serum levels of IL-6 (Ftime =1.876,P =0.177,Ftime* group =2.192,P =0.145,Fgroup =7.855,P =0.007),TNF-α(Ftime =13.133,P =0.001,Ftime* group =0.291,P =0.592,Fgroup =3.644,P =0.042),IL-10 (Ftime =14.580,P =0.001,Ftime* group =2.612,P =0.078,Fgroup =16.823,P =0.000),INF-γ (Ftime =3.093,P =0.045,Ftime* group =0.513,P =0.600,Fgroup =20.141,P =0.000) were significantly higher than those in NPE group.(2)The serum levels of TNF-α,IL-10,INF-γ rose to the peak on the third day.(3) By using multivariate Logistic regression analysis,age (OR =3.383,95％ CI:1.173-4.759),days of fever (OR =4.925,95％ CI:1.758-3.794),hyperglycaemia (OR =3.465,95％ CI:1.303-5.220),leucocytosis (OR =7.579,95 ％ CI:2.530-12.704) and elevation of IL-10 (OR =1.228,95 ％ CI:1.007-1.523) were entered into equation.In the risk evaluation model,these variables remained independent predictors for NPE.Conclusions Abnormal cytokine productions appear to be responsible for the pathogenesis of NPE,and might be an effective tool for predicting NPE in infants with severe HFMD.

Mesorhizobium huakuii strain 7653R,isolated from nodules of A.sinicus L,contains two indige-nous plasmids,p7653Ra and p7653Rb,the latter being the symbiotic plasmid.We eliminated the plasmids via Tn5-sacB insertion and obtained its symbiotic plasmid-cured derivative 7653RD.Then,we transferred the symbiotic plasmid pJB5JI of Rhizobium leguminosarum bv.Viciae T83K3 into 7653R and 7653RD.The pot plant test showed an increase in competitive ability and symbiotic nitrogen fixation of transconjugant 7653R-197(pJB5JI) compared to 7653R.pJB5JI could not restore the ability of 7653RD to nodulate Astra-galus sinicus.7653RD(pJB5JI) could form ineffective nodules on peas,implying that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 7653R.We checked the stability of plasmid in transconjugants under free-living and during symbiosis.The results indi-cated pJB5JI could not be detected in some nodule isolates.We amplified kan resistance gene from all transconjugants and nodule isolates which suggested that pJB5JI might fully or partially integrated into the chromosome of recipients.

Objective To detect VP1 and 2A genes of Enterovirus type 71 (EV71) isolated from clinical specimens of patients with light or heavy symptoms and analyze the homogeneity and phylogenetic tree. Methods Fifty clinical specimens of children with hand-foot-and-mouth disease ( HFMD) were dealed with, which were tested by RT-PCR assay with specific primer pairs for EV71. EV71 isolates from patients with light or heavy clinical symptoms were tested by RT-PCR assay with two specific primer pairs for VP1 and 2A genes of EV71 respectively. All of the PCR products were sequenced and compared with that of previously isolated EV71 isolates available from GenBank by homogeneity and phylogenetic tree analyses. Results The RT-PCR results indicated that 30 isolates were EV71, 13 of 30 isolates were from clinical specimens of patients with light symptoms of hand-foot and mouth, the other were from clinical specimens of patients with heavy symptoms of complications. VP1 genes and 2A genes of 10 EV71 isolated strains including 5 light strains and 5 heavy strains were sequenced and compared with that of previously isolated 5 EV71 Chinese isolates available from GenBank (fuyangEU703814.1, xi_anHM003207. 1, shandongEU753418.1, shenzhenFJ607337.1, henanGU366191. 1) by homogeneity and phylogenetic tree analyses. The homogeneity of VP1 and 2A genes of the 10 EV71 isolated strains and 5 previously isolated strains were between 94.7% -99.4% and 93.6% -99.3% respectively, with the representative isolates of A and B genotypes was between 81.0%-84. 6% and 78. 4%-82. 2% respectively. The data suggested that all of the 10 Chinese isolates belong to EV71 genotype C. There were only 87.8% -90.2% homology among these 10 strains and the representative strains of C1, C2, C3 sub-genotypes of EV71 but 96. 8% -99.6% homology among these 10 strains and the representative strains of C4 sub-genotypes of EV71, this suggested that these 10 Chinese isolates composed the C4 sub-genotype, of the C genotype, that formed a single branch in the phylogenetic tree. Conclusion EV71 of sub-genotype C4 distributed in Mainland China, and VP1 genes have close genetic relationship between isolated strains. There is no obvious difference in 2A genes between clinical specimens of patients with light or heavy symptoms by homogeneity and phylogenetic tree analyses.

Objective To study the association of multi-drug resistance with mutations in mar gene in clinical isolates of Shigella.Methods Fifty-four clinical isolates of Shigella were collected.Susceptibility tests of tetracycline(TE),chloramphenieol(C),ampicillin(AM),cipr ofloxacin(CIP),and norfloxacin(NOR)were performed in a11 isolates.Hexane and cyclohexane were used to study the organic solvent tolerance of all clinical isolates.marOR genes of these strains were analyzed by potymerase chain reaction-single strand conformation polymorphism(PCR-SSCP)and were sequenced. Results Thirty-five multi-drug resistant strains were identified,and the rate of multi-drug resistance was 64.8%.Thirty-eight of 54 strains were tolerant to organic solvent,including 33 multi-drug resistance strains,3 drug resistant strains and 2 sensitive strains. marOR gene was found in all strains by PCR, and 5 multi-drug resistant strains,(14.29%)carrying marOR gene mutations in multi-drug resistant strains were found by SSCP analysis.DNA sequence analysis of marOR gene revealed that there was a 4 base deletion from base 1376 to 1379 in marO gene that resulted in frame-shift mutation.However,this deletion mutation didn;t exist in the standard strain S51250 and sensitive strains.There were 10 point mutations of marR gene in multi-drug resistant strains,2 of them resulting in amino acid changes:1752(G→A)Gly→ser,1854(T→c)Tyr→His.The rest mutations were all nonsense,and some of them occurred in sensitive strains or in many other strains.Conclusion The marO gene mutation may play an important role in the regulation of multi-drug resistance in Shigella spp.

In the present study, the expression, purification, toxicity detection and the adjuvant effect of the heat-labile enterotoxin in non-toxic mutant mLT63 of Escherichia coli were investigated, in which the inductive expression was performed under optimal condition for inductive expression and the toxicity of the products obtained from inductive expression were tested for toxicity after being purified and concentrated with affinity chromatography. BALB/c mice were immunized orally with the mutant mLT63 associated with Helicobacter pylori (Hp) subunit vaccine UreB, Omp11. After immunization, the specific antibody levels in serum, extract from gastric tissues and fecal extract were determind by means of ELISA assay and the results were subjected to statistical analysis. It was demonstrated that the mutant mLT63 of heat-labile entrotoxin of E.coli constructed in our laboratory devoided of any toxic effect as revealed by the rabbit ileal loop assay, but its adjuvant effect could be demonstrated in the associated immunization of mice with Hp subunit vaccine UreB and Omp11.

This study was aimed to explore the features of clustered regularly interspaced short palindromic repeats (CRISPR) structures in Shigella by using bioinformatics. We used bioinformatics methods, including BLAST, alignment and RNA structure prediction, to analyze the CRISPR structures of Shigella genomes. The results showed that the CRISPRs existed in the four groups of Shigella, and the flanking sequences of upstream CRISPRs could be classified into the same group with those of the downstream. We also found some relatively conserved palindromic motifs in the leader sequences. Repeat sequences had the same group with corresponding flanking sequences, and could be classified into two different types by their RNA secondary structures, which contain "stem" and "ring". Some spacers were found to homologize with part sequences of plasmids or phages. The study indicated that there were correlations between repeat sequences and flanking sequences, and the repeats might act as a kind of recognition mechanism to mediate the interaction between foreign genetic elements and Cas proteins.
Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; Genome, Bacterial ; Plasmids ; Shigella ; genetics

Objective To detect the influence of marOR mutations on antibiotic resistance in Shigella spp. Methods The marOR gene with four-base deletion was amplified by overlap PCR, then inserted in a T-vector and transformed into DH5α. The clone of marOR gene with four-base deletion and three point mutations was prepared from the strain having these mutations. Electrophoresis and sequencing were preformed to certify the correction of the cloned genes. Drug susceptibility tests were preformed for the strains harbouring the different clones [DH5α, DH5α (T), DH5α (marOR), DH5α (marOR-CATT), DH5α(marOR-CATT + 3m)]. Results Compared with the control strain (DH5α-T), the antibiotic resistances of marOR with four-base deletion [DH5α (marOR-CATT)] were higher to streptomycin, tobramycin, cefazolin and cefalexin, and the antibiotic resistances of marOR with four-base deletion and three point mutations [DH5α (marOR-CATT + 3m)] were higher to streptomycin and to tetracycline. The antibiotic resistances of DH5α (marOR-CATT) and DH5α (marOR-CATT +3m) to streptomycin, tetracycline, chloramphenicol, cefazolin, levofloxacin, ciprofloxacin and norfloxacin were higher than DH5α (marOR). The diameters of the antibiotics except the trimethoprim between DH5α (marOR-CATT) and DH5α (marORCATT +3m) had not significant disparity. Conclusion The four-base deletion in 1376-1379 sites of the marOR gene increased the resistance of Shigella spp to some antibiotics. The point mutations in 1411, 1417,1435 sites of the marOR gene have little influence on the antibiotic resistance of Shigella spp.

Objective To detect the mutations of the marOR gene and study the relations with the expressing level of the acrAB-tolC efflux pump in Shigella. Methods marOR genes were amplified by PCR for 100 clinical isolates and 5 reference strains of Shigella. The PCR products were digested by restriction endonuclease Taq Ⅰ , then analyzed by single strand conformation polymorphism(SSCP). The marOR genes of the mutated strains and sensitive strain were sequenced and the expressing leveLs of acrA, acrB and talC were determined by RT-PCR. Susceptibility tests of tetracycline (TE), chloramphenicol (C), ampicillin (Am) , gentamycin (GM), norfloxacin (NOR) and selectrin (SMZ-TMP) were performed in sequenced strains. Results marOR genes were found in all strains detected. SSCP analysis found the rate of mutations in marOR genes was 23%. Among 11 marOR gene-mutated strains which were sequenced, there were 9 strains having a four-base absence and three single-base mutations in different loci. The expressing levels of the acrAB-tolC efflux pump in the 11 strains were higher than those in sensitive strains and reference strain. Furthermore the 11 strains were multi-drug resistance. Conclusion The mutation rate of marOR gene in Shigella was high and the acrAB-tolC efflux pump genes were over-expressive in marOR gene-mutated strains which were multi-antibiotic resistance in the study.

Objective To construct a prokaryotic expression system of ahpC gene of Helicobacter pylori. Methods The ahpC gene was amplified from Hp chromosomal DNA by PCR technique and cloned into the expression vector pET-30a. The recombinant vector pET30a-ahpC was identified by DNA sequencing and transformed to E.coli BL21 (DE3) for expression under induction by IPTG. The expression product was analyzed by SDS-PAGE. Results PCR product showed that ahpC gene consisted of 594bp. The gene fragment that was inserted into the recombinant vector was identified to GenBank for 99%. SDS-PAGE showed that the induced protein was expressed highly in the host bacterium. Conclusion A prokaryotic high-expression system for ahpC gene has been successfully constructed. It can highly express r-AhpC protein in E.coli.

Objective To explore the ultrasound characteristics of carotid atherosclerosis in acute stroke patients with early neurological deterioration (END). Methods END was defined as a increase by at least two points in the National Institutes of Health Stroke Scale between admission and day 7. Among 128 patients with acute stroke in whom carotid ultrasound examinations were performed within 24 hours after admission, 38 patients with END and 40risk-matched patients without END were included in the END group and the non-END group,respectively. The ultrasound characteristics of carotid atherosclerosis were compared in both groups. Results Plaque score (16.7 ±4.4 mm vs. 13.3 ±3.5 mm, t=2.673, P=0.009),intima-media cross-sectional area (26. 4 ± 8. 5 mm2 vs. 20. 5 ± 6. 8 mm2, t = 3. 394, P =0. 001), arterial stiffness index (28. 94 ±4. 29 vs. 21. 22 ±5. 85, t = 6. 618, P =0. 000), and the rates of unstable plaque (66. 7% υs. 43. 3%, χ2=9. 164, P =0. 003), eccentric plaque (62. 8% vs. 45. 6%, χ2=5. 008, P =0. 025), stenosis ≥50% (71. 1% vs. 37. 5%, χ2=8. 828, P =0. 003), and negative remodeling (28. 9% vs. 7. 5%, χ2=6.087, P =0.014) in the END group were significantly higher than those in the non-END group, while the distensibility coefficient ([14. 74 ±8. 66]×10-6/P υs. [19. 16 ±9.35] × 10-6/Pa, t =2. 163, P=0. 034)and compliance coefficient ([0.49 ±0. 13] × 10-4 mm2/Pa υs. [0. 58 ±0. 11] × 10-4 mm2/Pa,t =3.307, P =0. 001) were significantly lower than those in the non-END group. Conclusions The ultrasound characteristics such as plaque score, intima-media cross-sectional area, arterial stiffness index, unstable plaque, eccentric plaque, stenosis ≥ 50%, negative remodeling,distensibility and compliance may be useful to predict END in patients with acute stroke.