Object\ To develop a convenient and practical method for the identification of Carapax Trionycis Methods\ Based on the sequence variations of 12S rRNA gene between Pelodiscus sinensis and other softshell turtles, a pair of allele specific primers was designed to distinguish P. sinensis from other species of Trionychidae. DNA were extracted and anplified and Carapax Trionycis could be identified accurately by polymerase chain reaction (PCR) using the primers Results\ Ten samples of turtle shell from different sources were indentified by the allele specific PCR with the primers The result indicated that three samples were substitutes of Carapax Trionycis, consilient with the result from DNA sequence analysis The mitochondrial 12S rRNA gene fragment of P. maculatus and a faked imitation had also been sequenced Conclusion\ The primers could be used as key components in Carapax Trionycis identification kit

Objective To study prognosis and grading of somatosensory evoked potential(SEP)in long-term unconscious patients after severe traumatic brain injury(TBI).Methods Five prognostic factors including age,sex,injury mechanism,history of temporal craniotomy and SEP grading were selected and analyzed in 47 patients after severe TBI with a duration of unconsciousness longer than two weeks.The prognosis was judged by Glasgow Outcome Scale.Results Prognosis was closely associated with SEP grading(P=0.024).The accuracy of SEP in assessing the prognosis was 91.5%.About 95%-100% of patients with SEP at grade Ⅲ-Ⅲ ended up with severe disability,persistent vegetative state or death.However,43.75% of patients with SEP at grade Ⅰ had good prognoses.Conclusions The SEP grading can objectively and accurately evaluate patients' prognosis and demonstrate the brain function.

Objective To investigate the effect of salinity and temperature on motility of Vibrio parahaemolyticus. Methods V.parahaemolyticus was inoculated on swarming or swimming agar plates containing different amounts of salinity (0.5%, 1.0%, 2.0%, and 4.0% NaCl, respectively), followed by incubation at 26 or 37℃, before the diameters of bacterial lawns were measured .Results and Conclusion The swarming motility was not affected by salinity , while the swimming motility was positively correlated with salinity .Maximum swimming occurred in 2.0% NaCl, and displayed a slight decline in salinity of 4.0%.Both swimming and swarming were affected by temperature , and the motility was signifi-cantly enhanced in 37℃vs 26℃.These results indicate that both salinity and temperature can modulate the motility of V. parahaemolyticus.

AIM　It is difficult to identify the Chinese crude drug snake gallbladder accurately by morphological and microscopical characteristics or chemical components only. In order to solve the problem, the technique based on DNA molecular marker was introduced into the authentication of snake gallbladder. METHODS　DNA templates were extracted from the membrane or the bile of snake gallbladder, and also from the muscle of the original animal Elaphe schrenckii. About 400 bp DNA fragments of 12S rRNA gene were amplified from the templates and sequenced subsequently. RESULTS　Enough amounts of DNA templates could be extracted from a bit of membrane or bile of snake gallbladder. The sequence of amplicons from the membrane, bile and muscle of the same individual were identical completely. CONCLUSION　The technique of DNA molecular marker could be used for the authentication of snake gallbladder and bile. The results indicate that the technique could be used for the identification of crude drugs from other animal secretion. DNA sequence analysis also demonstrated that the origins of commercial snake gallbladder were complicated and more efficient quality control was necessary for supervising the crude drug in the market.

Objective To investigate the regulation of swimming motility by H-NS in Vibrio parahaemolyticus(VP). Methods VP was inoculated into the semi-solid swimming agar plate containing 1% Oxoid tryptone, 2% NaCl, 0.5%Difco Noble Agar, and 0.1% arabinose followed by incubation at 37℃ for 4.5 h before the diameters of bacterial lawns were measured.Total RNAs were extracted from the wild-type (WT) strains and the hns null mutant (Δhns), and the quantitative real-time( RT)-PCR( qRT-PCR) was carried out to calculate the transcriptional variation of flaA between WT andΔhns strains.The entire promoter DNA region of flaA was amplified and cloned into the lacZ fusion vector pHRP309 containing a promoterless lacZ gene. The recombinant lacZ reporter plasmid was transformed into WT and Δhns, respectively, to measure the β-galactosidase activities in cellular extracts using the β-galactosidase enzyme assay system. Results and Conclusion The phenotype results showed that swimming motility of VP was enhanced by H-NS.The qRT-PCR and LacZ fusion results indicated that the transcription of flaA was positively regulated by H-NS.Collectively, H-NS promotes the swimming motility of VP, at least partly, by activating the transcription of flaA.

Objective To investigate the relationship between serum ferritin and nonalcoholic fatty liver diseases in obese children.Methods Obese children aged 6 to 14 years old were enrolled.Duration of obesity, anthropometric parameters (height, body weight, waist circumference, hip circumference), bioelectrical impedance analysis (body fat), serological parameters (liver transaminases, lipid metabolism, fasting blood glucose, fasting insulin, serum ferritin) and liver ultrasonography were recorded.Insulin resistance (IR) index was calculated by homeostasis model assessment (HOMA).All subjects were divided into 3 groups according to liver ultrasound and liver transaminases : simple obese children (SOC) group, obese children with nonalcoholic simple fatty liver (NAFL) group and obese children with nonalcoholic steatohepatitis (NASH) group.Results 86 obese children entered the study, with a mean age of (10.4 ± 1.9) years, including 26 in the SOC group, 28 in the NAFL group and 32 in the NASH group.Waist circumference standard deviation score (SDS or Z-score), waist-to-hip ratio, HOMA-IR index and serum ferritin in the NASH group were obviously higher than those in the NAFL group [2.3 ± 0.3 vs.2.1 ± 0.3, P =0.020;1.0 ± 0.0 vs.0.9 ± 0.1,P=0.014;4.0±1.7 vs.2.9±1.8, P=0.006;(104.1 ±49.6) μg/Lvs.(68.4 ±22.7) μg/L, P=0.004] and the SOC group [2.3 ±0.3 vs.1.9 ±0.3, P=0.000;1.0±0.0vs.0.9 ±0.1, P=0.012;4.0 ±1.7 vs.2.5 ±1.6, P=0.001;(104.1 ±49.6) μg/Lvs.(59.2 ±28.9) μg/L, P=0.001], while there was no significant difference in body mass index Z-score [2.8 ± 0.5 vs.2.7 ± 0.6, P =0.524;2.8 ± 0.5 vs.2.7 ± 0.6, P =0.662].There were no significant differences between the NAFL group and the SOC group in the above indicators [2.1 ±0.3 vs.1.9 ±0.3, P =0.260;0.9 ±0.1 vs.0.9 ±0.1, P =0.952;2.9 ± 1.8vs.2.5±1.6, P=0.283;(68.4±22.7) μg/Lvs.(59.2±28.9) μg/L, P=0.161].Mter controlling age, body mass index, waist circumference, waist-to-hip ratio, triglyceride, and HOMA-IR index, serum ferritin was still positively correlated with the magnitude of nonalcoholic fatty liver diseases in obese children (r =0.335, P =0.002).Conclusion Serum ferritin is probably an independent risk factor for NASH in obese children.

Objective To investigate the risk factors of central venous catheters (CVC) colonization.Methods A retrospective study was performed on adult patients with CVCs placement in Renji Hospital,Shanghai Jiaotong University from January 2006 to March 2010.Clinical data,catheter-related information (including duration of catheter placement,position and purpose of catheterization,and whether or not out-of-ward catheterization),catheter culture results,and prevalence of catheter-related blood stream infection (CRBSI)was collected.Results A total of 651 patients aged 18 to 97 years (median:63 years) were enrolled in the study,in whom 762 CVC were placed.The median duration of catheter placement was 1 1 days (2 to 122 days)and the total duration of CVC placement was 10 725 days.The prevalence of catheter colonization was 16％(122/762),and 134 germs were cultured.Gram-positive cocci was the most common colonized bactera (52.2％,70/122),followed by gram-negative bacilli (33.6％,45/122) and fungi (14.2％,19/122).Overall 13 CRBSI were confirmed and the rate of CRBSI was 1.21/1000 catheter-days.Logistic regression analysis demonstrated that the risk factors for CVC colonization included mechanical ventilation [odds ratio (OR) =1.783,95％ confidence interval (Cl) =1.108 ～2.870],serum albumin concentration less than 25 g/L before catheterization (OR =1.783,95％ Cl =1.357 ～ 6.757),prolonged duration of catheter placement (OR =1.105,95％ Cl =1.009 ～ 1.111),and out-of-ward catheterization (OR =2.837,95％ Cl =1.010 ～7.969).Conclusion Patients with prolonged duration of catheter placement and out-of-ward catheterization are inclined to CVC colonization.

Objective To study the transcriptional regulation of vp1667 by H-NS in Vibrio parahaemolyticus.Methods Total RNAs were extracted from Δhns and WT strains.Quantitative RT-PCR was carried out to calculate the transcriptional variation of vp1667 between Δhns and WT.Primer extension assay was also employed to detect the transcription start site and the promoter activity (i.e.the amount of primer extension products) of vp1667 in Δhns and that in WT.The promoter DNA region of vp1667 was amplified, purified, and cloned into the corresponding restriction endonuclease sites of pHRP309 that harbors a gentamicin resistance gene and a promoterless lacZ reporter gene.The recombinant pHRP309 plasmid was transformed into Δhns and WT, respectively, while β-galactosidase activity in cellular extracts was measured using a β-galactosidase enzyme assay system.The over-expressed His-H-NS was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns.The electrophoretic mobility shift assay (EMSA) and DNaseⅠ footprinting were then applied to analyze the DNA-binding activity of His-H-NS to vp1667 promoter region in vitro.Results and Conclusion The primer extension assay detected one transcription start site for vp1667, which was located at 28 bp upstream of vp1667, and its transcribed activity was under the negative control of the H-NS.The EMSA and DNaseⅠ footprinting assay results showed that His-H-NS was unable to bind to the promoter-proximal DNA region of vp1667, suggesting that H-NS indirectly inhibits the transcription of vp1667.

Objective To investigate the transcriptional autoregulation of PhoP/PhoQ under different growth conditions in Yersinia pestis.Methods The entire promoter region of YPO1635 was amplified and cloned into the pRW50 vector containing a promoterless lacZ reporter gene.The recombinant LacZ reporter plasmid was transformed into the wild-type strain (WT) and the phoP mutant strain (ΔphoP),respectively,to measure the promoter activity (the β-galactosidase activity) of the target gene in WT and ΔphoP by using the β-galactosidase enzyme assay system.Total RNAs were extracted from WT and ΔphoP strains,and primer extension assay was employed to detect the promoter activity by examining the amount of primer extension products of YPO1635 in WT and ΔphoP.Results The LacZ fusion results showed that the transcription of YPO1635 was positively regulated by PhoP under L-TMH and brain-heart infusion(BHI) conditions,but it was not regulated in H-TMH medium.The primer extension assay detected two transcriptional start sites located at 90 and 118 bp upstream of the translation initiation site of phoP,named P1 and P2,respectively.Under low Mg2+ TMH conditions,the promoter activity of P1 rather than P2 was positively regulated by PhoP.Under high Mg2+ TMH conditions,the promoter activities of both P1 and P2 showed no obvious difference in the WT and ΔphoP strains.Under rich BHI conditions,both promoters were under negative control of PhoP.Conclusion Different autoregualtion patterns of PhoP/PhoQ under different growth conditions would help Y.pestis to quickly adapt to the changing living environment.

Objective To construct a new recombinant immunotoxin expression vector by using human VEGF165 and a truncated pseudomonas exotoxin A ramification (PE38) gene, and explore the expression of the VEGF165-PE38 fusion protein in HEK293 cells. Methods VEGF165 was cloned by polymerase chain reaction (PCR). PE38 gene was gained from an vector plasmid pRB391 by restriction endonuclease digestion, and then inserted to the eukaryotic expression vector pIRES2-EGFP. After the eukaryotic recombinant vector pIRES2-VEGF165-PE38-EGFP was identified by restriction endonuclease digestion and sequence analysis, the vector was transfected into HEK293 cells by liposome protocol. RT-PCR and ELISA method was used to confirm the expression of the fusion gene in the HEK293 cells. Results Restriction endonuclease digestion and sequence analysis revealed the VEGF165-PE38 fusion gene was cloned into the eukaryotic expression plasmid vector pIRES2-EGFP successfully. The pIRES2-VEGF165-PE38-EGFP fusion gene could express in the HEK293 cells. Conclusion The result provide the basis for search of the targeted cytotoxic activity to tumor vascular endothelial cells and may have some potential value in clinical application.