Objective To construct the eukaryotic expression plasmid of recombinant human hypoxia-inducible factor-1?(HIF-1?) and get ready for the coloning and expressing of HIF-1? gene. Methods The total RNA was isolated from blood cells,and cDNA library was constructed by reverse transcription PCR method.PIREGFP containing EGFP fragment and cDNA were used as templet,the three designed primers (EGFP-linker,HIF-1? upstream and HIF-? downstream)were put into,after amplification the target gene fragment was inserted into the shuttle vector T,and transfected with E.coli DH5?,the bacterial colonies containing recombinant plasmids were identified by LB/KANA-agar plate,and the recombinant plasmids were extracted and purified.All sequences amplified by PCR were confirmed by complete sequencing.The correct sequences were cloned into the pVAX1 vector.Results The amplified fragments were about 870,1 199,672 bp by PCR,they were inserted into the shuttle vector T and sequenced. The result of sequencing was identical with that provided by GenBank.The final plasmid about 1 800 bp was obtained by the identification of enzyme digestion and it had same molecular size as expected.Conclusion The eukaryotic expression plasmid pVAX1-EGFP-linker-HIF-1? of recombinant human HIF-1? is successfully constructed.

Objective To investigate the distribution of 16S rRNA methylase genes in Klebsiella pneumoniae strains producing Klebsiella pneumoniae ealbapenenase type 2(KPC-2).Methods A total of 37 Klebsiella pneumoniae isolates producing KPC-2 were collected.The minimal inhibitory concentrations (MICs)of these strains to amikacin,gentamyein and netilmicin were determinated by agal dilution method.Six 16S rRNA methylase genes(armA,rmtA,rmtB,rmtC,rmtD and npmA)were detected by PCR.Results The resistant rates to amikacin,gentamycin and netilmicin were 97.3%(MIC50≥1024μg/mL).Among those resistant strains,8 were armr/A positive,25 were rmtB positive,4 were both armA and rmtB positive.and no other 16S rRNA methylase genes were found.The total positive rate of 16S rRNA methylase genes was 78.4%(29/37).Conclusion 16S rRNA methylase genes armA and rmtB ale prevalent in Klebsiella pneumoniae strains producing KPC-2.

Objective To investigate the antibiotic resistance of extended-spectrum-β-1actamases (ESBLs)-producing Escherichia coli (E.coli) and Klebsiella pneumoniae (K.pneumoniae) isolates and the risk factors of bloodstream infections caused by these strains.Methods Clinical data of 131 patients with E.coli or K.pneumoniae-induced bloodstream infections admitted in the Second Affiliated Hospital of Zhejiang Chinese Medical University during September 2009 and June 2014 were retrospectively analyzed.Species identification and antimicrobial susceptibility test were performed by Vitek 2 system,and ESBLs production was tested by standard disk diffusion method.Logistic regression analysis was performed to identify the risk factors of bloodstream infections induced by ESBLs-producing strains.Results Among 131 patients,65 were infected with ESBLs-producing strains,and 66 were infected with non-ESBLs-producing strains.The resistance rates of ESBLs-producing strains were above 50％ for penicillin,aztreonam and third/fourth generation cephalosporins,which were significantly higher than those of non-ESBLs producing strains.The resistance rates of ESBLs-producing E.coli and K.pneumoniae to carbapenems and piperacillin/tazobactam were 0-2.0％,2.3％ and 0-14.3％,26.7％,respectively.The univariate analysis revealed that patients with exposure to cephalosporins in recent 3 months (x2 =18.322,P ＜ 0.01),prior infection with ESBLs-producing strains (x2=14.610,P＜0.01),indwelling catheter in recent 3 months (x2 =13.016,P ＜ 0.01),history of hospitalization in recent 3 months (x2 =11.269,P ＜ 0.01),exposure to quinolones in recent 3 months (x2 =10.638,P ＜ 0.01),nosocomial infection (x2 =8.205,P ＜ 0.01),history of indwelling deep venous catheter or percutaneous central catheter in recent 3 months (x2 =4.817,P ＜ 0.05) and exposure to glucocorticoid hormone in recent 3 months (x2 =4.265,P ＜ 0.05) were associated with infection of ESBLs-producing strains.Multivariate Logistic regression analysis revealed that exposure to quinolones in recent 3 months (OR =6.851,P ＜ 0.01),prior infection with ESBLs-producing strains (OR =6.344,P ＜ 0.01),exposure to cephalosporins in recent 3 months (OR =3.719,P ＜ 0.01),and indwelling catheter in recent 3 months (OR =3.180,P ＜ 0.05) were independent risk factors for ESBLs-producing E.coli or K.pneumoniae infection.Conclusions ESBLs-producing E.coli or K.pneumoniae isolates are highly resistant to most antibiotics,and multidrug-resistance is common.Carbapenems were still the most effective antibiotics against ESBLs-producing E.coli or K.pneumoniae infection.Rational use of cephalosporins and quinolones,strictly following aseptic technique in operation,strict use of indications for indwelling catheterization,and completely eradicating ESBLs-producing strains in previous infections may be helpful in reducing bloodstream infections by ESBLs-producing E.coli or K.pneumoniae.

Objective To study the epidemiology and genotypes of extended-spectrum beta-lactamases (ESBL)-producing Escherichia coli (EC) and Klebsiella pneumoniae (KP) that caused community-onset bloodstream infections (COBSI) in 9 county hospitals of Zhejiang Province.Methods This is a multi-center, prospective, observational study.The cases and isolates with COBSI caused by EC and KP were consecutively collected from 9 county hospitals in Zhejiang Province between 1st March 2014 and 30th April 2015.The double disk diffusion method was used to confirm the production of ESBL.The ESBL genotypes were determined by polymerase chain reaction(PCR) amplification and sequence analysis.Multi-locus Sequence Typing (MLST) was used to analyze the homology of ESBL-producing isolates.Minimal inhibitory concentration (MIC) of frequently used drugs for ESBL-producing isolates was determined by in-vitro antimicrobial susceptibility tests.Results During the study period, a total of 172 cases with COBSI were collected and 171 cases were eligible, among which 126 were caused by EC and 45 were caused by KP.The overall prevalence of ESBL was 34.5% (59/171),and the prevalence of ESBL-EC and-KP was 41.3% (52/126) and 15.6% (7/45), respectively.CTX-M-type ESBL accounted for 96.6% (57/59) of all the ESBLs-producing isolates, and the most common type was CTX-M-14 (27.1%, 16/59), followed by CTX-M-55 (22%, 13/59).MLST analyses revealed significant genetic diversity among ESBL-EC and-KP.The most prevalent ST of ESBL-EC was ST131 (23.1%).In addition to carbapenems, cefoperazone/sulbactam, piperacillin/tazobactam, moxalactam, amikacin and fosfomycin also showed good in-vitro activity against ESBL-EC and-KP.Conclusions The prevalence of ESBL in EC and KP is high in 9 county hospitals of Zhejiang Province, and the most common genotypes are CTX-M-14 and CTX-M-55.The detection rate of ESBL in EC is higher than in KP.It could be considered adequate empirical therapy according to the results of antimicrobial susceptibility tests.Carbapenems, cefoperazone/sulbactam, piperacillin/tazobactam, moxalactam, amikacin and fosfomycin have good in-vitro activity against ESBL-EC and-KP.

Objective To study the epidemiologic characteristics of trauma in the prehospital first-aid in megapolis. Methods The epidemiologic data of 10 654 traumatic patients,including treated by prehospital treatment and emergency treatment from January 2000 to January 2005,were analyzed.Results The proportion of male was 70.96%,adult patients(21～50 years old)79.23%,suburb 62.86%,downtown 37.14%.The patients whose ISS scores surpassed 16 accounted for 37.98%,which caused by traffic accident was 37.74%,by public order 24.39%,by industrial trauma 21.71%.The trauma in the limbs accounted for 67.51%,cephalic and cervical wounds accounted for 58.64%,multiple wounds 41.77%,thoracic and abdominal wounds 39.41%.Three hundred and sixty one were killed on the spot,which caused by traffic accident were 46.81%,by public order 28.81%,by industrial trauma 14.40%.Forty-two percent point six six patients died of multiple trauma,54.07% died of cephalic and cervical trauma,15.79% died of thoracic and abdominal wounds.Conclusion Suburban area gradually became the frequently-occurred areas of trauma in megapolis. The wounded were mainly young adults and had a tendency of juvenility.The majority of damaging and lethal factors were traffic accident,public order and industrial trauma.Some pertinet measures and professional first-aid models may improve the traumatic first-aid level.

Objective: To observe the effect of cinobufacin on the expression of phosphorylated PTEN protein in human breast cancer MCF-7 and MDA-MB-231 cells. Methods: The MCF-7 and MDA-MB-231 cells were divided into control group, high dose group and low dose group according to random number table method. Low and high dose groups were added with 0.5, 5.0 mg/ml cinobufacin, 100 μl/well for intervention, respectively, while the control group was added with equal volume of RPMI-1640 medium. Cell Counting Kit-8 was used to detect cell proliferation assay at 24, 48, and 72 h following intervention. Immunoblotting was used to detect the expression of AKT/mTOR pathway protein expression and p-PTEN. Results: At 24, 48, and 72 h after intervention, the proliferation of MCF-7 and MDA-MB-231 cells in high dose group were significantly decreased (P<0.05). Compared with the control group, the expression of p-AKT (MCF-7: 0.357 ± 0.064, 0.215 ± 0.056 vs. 0.924 ± 0.085; MDA-MB-231: 0.310 ± 0.022, 0.194 ± 0.019 vs. 0.811 ± 0.089), p-mTOR (MCF-7: 0.476 ± 0.039, 0.217 ± 0.038 vs. 0.838 ± 0.058; MDA-MB-231: 0.300 ± 0.031, 0.223 ± 0.025 vs. 0.896 ± 0.096), p-S6 (MCF-7: 0.551 ± 0.068, 0.428 ± 0.041 vs. 1.254 ± 0.264; MDA-MB-231: 0.281 ± 0.014, 0.197 ± 0.012 vs. 0.748 ± 0.022), p-PTEN (MCF-7: 0.487 ± 0.170, 0.184 ± 0.135 vs. 1.003 ± 0.284, P<0.05; MDA-MB-231: 0.261 ± 0.184, 0.170 ± 0.105 vs. 1.014 ± 0.206) in the low and high dose group significantly decreased (P<0.05). Conclusions Cinobufacin inhibited the proliferation of MCF-7 and MDA-MB-231 cells, which may be mediated by down-regulation of the expression of the upstream of the AKT/mTOR signaling pathway p-PTEN.

Objective To investigate antimicrobial resistance among nosocomial gram-negative bacilli in 2016 across China.Methods About 1 394 consecutive and non-repetitive gram-negative bacilli were isolated from 14 teaching hospitals from March to August in 2016 across China.All of these isolates were sent to the central laboratory for reidentification and susceptibility testing.The minimal inhibitory concentration(MICs)of meropenem and other antibacterial agents were determined by agar dilution method.The data were analyzed by using WHONET-5.6 software.Results The activity of antimicrobial agents against Enterobacteriaceae was as follows in descending order of susceptible rate: meropenem (95.2%,891/936), amikacin (94.6%,885/936), ertapenem (92.1%,862/936), piperacillin/tazobactam (88.1%,825/936), imipenem (88.0%,823/936), cefoperazone-sulbactam (83.1%,778/936), cefepime (72.2%,676/936), cefiazidime (72.2%,676/936), levofloxacin(68.8%,644/936), ciprofloxacin (63.2%,592/936), minocyline (62.9%,589/936), cefiriaxone (54.9%,514/936), cefotaxime (54.0%,505/936), cefoxitin (44.3%,415/936).The sensitivities of E.coli to carbapenems, amikacin, piperacillin-tazobactam, polymyxin B and cefoperazone-sulbactam were over 80%.The more sensitive antibiotic to Klebsiella pneumoniae was polymyxin B (99.0%), followed by amikacin (84.9%), meropenem (84.4%) and imipenem (82.0%).The prevalence of extended-spectrum β-lactamase was 62.8%(137/218)in Escherichia coli and 28.3%(58/205)in Klebsiella pneumonia.The activity of antimicrobial agents against E.cloacae, E.aerogenes and Citrobacter freundii was as follows in descending order of susceptible rate: meropenem (97.0%-98.5%), amikacin (95.8%-98.3%), imipenem (94.5%-97.5%), polymyxin B (96.4%-100%), cefoperazone-sulbactam (76.5%-90.0%), ertapenem (73.3%-90.1%), piperacillin/tazobactam (82.4%-88.3%).The most active agents against Pseudomonas aeruginosa were polymyxinB (100%), followed by amikacin (89.3%) and ciprofloxacin (82.4%).The most active agents against Acinetobacter baumannii were polymyxinB (100%).The sensitivities of Acinetobacter baumannii to meropenem, imipenem, minocycline and cefoperazone-sulbactam were 20.3%(39/202), 19.3%(41/202), 66.3%(134/202) and 24.8%(50/202), respectively.Conclusions Carbapenems remain high sensitive against Enterobacteriaceae.Controlling carbapenem resistant Klebsiella pneumoniae is urgent.Drug antimicrobial resistance in A.baumanni is a still serious problem.

Objective To investigate the effect of salidroside on xenograft tumor of human pancreatic cancer cells BxPC-3 in nude mice.Methods BxPC-3 subcutaneous xenograft tumor nude mice model was established,and the mice were randomly divided into control group,salidroside 25 mg group and salidroside 50 mg group using random number method (6 mice in each group).25 mg/kg and 50 mg/kg salidroside by intragastric administration was performed per day for 3 weeks and the tumor was resected.The tumor was weighed and the diameter was measured.Cell apoptosis was detected by Tunel staining.Results As result,tumor weight was (1.561 ± 0.416)g in the control group,(0.742 ± 0.272)g in the 25 mg group and (0.276 ± 0.064) g in the 50 mg group respectively;tumor volume was (2354.35 ± 523.11) mm3,(991.43 ± 348.91) mm3 and (403.60 ± 130.98) mm3,respectively;and the apoptosis rate of the transplanted tumor cells was (23.74 ± 5.88) ％,(49.30 ± 6.75) ％ and (64.97 ± 6.99) ％,respectively,with significant difference among three groups (all P ＜ 0.01).The effect of salidroside on inhibiting proliferation and promoting apoptosis was dose dependent.Conclusions Salidroside can significantly inhibit the proliferation and growth of pancreatic cancer BxPC-3 cells via promoting cell apoptosis and inhibiting cell proliferation.

Cryptococcal meningitis is a common and refractory central nervous system infection,with high rates of mortality and disability.The experts of the Society of Infectious Diseases of Chinese Medical Association have reached this consensus after a thorough discussion.Based on the current situation of cryptococcal meningitis in China,the management of cryptococcal meningitis includes 6 aspects:introduction,microorganism identification,clinical manifestations and diagnosis,principles of antifungal therapy,treatment of refractory and recurrent meningitis,treatment of intracranial hypertension.There is not a separate consensus on human immunodeficiency virus (HIV) infection in patients with cryptococcal meningitis.This article focuses on different antifungal regimens and reducing intracranial pressure by reference to Infectious Disease Society of America (IDSA) guidelines.The importance of early diagnosis,combined long-term antifungal therapy,control of intracranial hypertension are emphasized.

Objective : This study aimed to explore the influencing factors of dynamic changes in traditional Chinese medicine (TCM) constitution based on general statistics, Apriori-DEMATEL algorithm, and DoWhy causal inference framework methods. Methods: Dynamic collection of TCM constitution identification data was conducted from the population aged 18 − 60, containing collection time and constitution type, and 11 constitution influencing factors including dietary habit, sleeping habit, sleeping duration, exercise habit, emotion state, stress level, living environment, work/life calamity, family atmosphere, business trip frequency, and overtime situation. General statistical analysis was used to analyze the relative percentage of corresponding influencing factors of different types of constitution changes, the Apriori-DEMATEL algorithm was used to analyze the correlation between 11 constitution influencing factors such as dietary habit and constitution changes, and the DoWhy causal inference framework was used to analyze the causality between dietary habit,sleeping habit, sleeping duration, exercise habit, emotion state, and stress level, explore the frequency of constitution type transformation-change factors, and determine the key influencing factors causing dynamic changes in constitution type. Result: After preprocessing, 13536 valid data points were obtained. Based on the Apriori-DEMATEL algorithm, the factors were divided into six original factors including dietary habit, sleeping habit, sleeping duration, exercise habit, emotion state, and stress level, and five result factors includ ing living environment, work/life calamity, family atmosphere, business trip frequency, and overtime situation. Combining with general statistics, we found that among the original factors, changes in dietary habit, sleeping habit, sleeping duration, and stress level had a greater impact on other factors. In the process of constitution conditioning, attention should be paid to these four factors to maintain constitution balance. Among the five resultfactors, the absolute values of work/life calamity and family atmosphere were relatively large, indicating that these two factors were easily influenced by other factors. The dietary habit,sleeping habit, sleeping duration, exercise habit, emotion state, and stress level have higher centrality in changes, indicating that these six factors had important in constitution changes. According to the statistical frequency of constitution changes corresponding to each factor, we found that the changes of these six factors accounted for a large proportion of the constitution transformation frequency among Qi deficiency constitution, balanced constitution, and allergic constitution, indicating that the changes of these six factors played an important role in the changes of the three constitution types. Combined with the results of the Apriori-DEMATEL algorithm, and DoWhy causal inference framework analysis, it was inferred that dietary habit and sleeping duration indirectly lead to constitution changes by affecting the changes of other factors. Conclusion This study explored the influencing factors of dynamic changes in TCM constitution from the perspective of dynamic data and multiple analysis methods, and the results showed that the changes of dietary habit, sleeping habit, sleeping duration, exercise habit, emotion state, and stress level had a great impact on the changes of Qi deficiency constitution, balanced constitution and allergic constitution. Attention should be paid to the changesof these six factors in daily life, and corresponding improvement plans should be formulated to reduce the probability of transforming into biased constitution. Our study also provided data support and objective analysis reference for the analysis of influencing factors of dynamic changes in TCM constitution types.