Construction of sigF deletion mutant of Bacillus anthracis and its effect on formation of spores / 军事医学

Objective To construct sigF deletion mutant of Bacillus anthracis and the complementary strain of sigF deletion mutant in order to analyze the effect of losing sigF on formation of spores.Methods The spectinomycinadenyltransferase gene(spc) was inserted to replace sigF of B.anthracis by homologous recombination.A plasmid which contained sigF and sigF promotor was constructed and then transferred to the mutant to get a complementary strain of sigF deletion mutant.The characters of the mutant were analyzed by measuring growth curves, the ability of carbohydrate metabolism was compared, and spore formation was observed under a microscope.Results The sigF deletion strain A16D2△sigF was constructed from A16D2,which had a similar growth rate to the wild type A16D2 in logarithmic phase, but was not significantly different from the initial strain in the ability to use carbohydrates,although unable to form spores.The strain was found to maintain the state of asymmetric division by microfluidics experiment.Conclusion It is showed by this study that sigF is the essential gene of B.anthracis for spore formation, but not essential for vegetative growth.

Arabinose-controlled expression of htrA in Shigella flexneri through suicide plasmid-mediated homologous recombination / 军事医学

Objective To achieve arabinose-controlled expression of HtrA strain and detect the expression of HtrA protein.Methods Arabinose promoter with htrA100 was amplified from pACD-htrA vector by PCR and cloned into pGP704 vector.Then, Shigella flexneri 2a strain 301 was transferred with the recombinant plasmid pGD-htrA and an AraC-expression vector .The expressions of HtrA in whole-cell and periplasmic space were detected by Western blotting .Results The suicide plasmid-mediated homologous recombinant vector and the inducible HtrA expression strain were successfully constructed.Without arabinose,HtrA protein was hardly detected ,but in the presense of arabinose , HtrA protein could be detected in whole-cell lysate and in periplasmic space lysate by Western blot .Conclusion Homologous recombination using suicide plasmid can significantly knock down the expression of HtrA protein .After being induced with arabinose , HtrA protein can be expressed normally .

Clinical features and prognostic risk factor analysis for female patients with acute myocardial infarc-tion / 心血管康复医学杂志

Objective:To analyze the clinical features and prognostic risk factors of female patients with acute myo‐cardial infarction (AMI) .Methods :From Apr 2011 to Oct 2014 ,a total of 97 female AMI patients treated in our hospital were enrolled as female group ,meanwhile 120 male AMI patients were regarded as control group (male group) .All patients received three‐month follow‐up after hospitalization .Baseline data ,clinical features and prog‐nosis were compared between two groups ,and prognostic risk factors of female patients were analyzed at the same time .Results:Compared with male group ,there were significant rise in percentages of cardio‐and cerebrovascular diseases ,diabetes mellitus and hyperlipidemia ,and significant reductions in red blood cell count ,hemoglobin level , and percentages of smoking&drinking history , digestive&urinary system diseases in female group , P<0.05 or <0.01. Compared with male group , there were significant rise in percentages of Killip class Ⅳ (14.17% vs . 24.74% ) ,three‐vessel coronary disease (3.33% vs . 12.37% ) , therapeutic rate of reperfusion (51.67% vs . 73.20% ) ,mortality (2.50% vs .9.28% ) and incidence of complications (19.17% vs .31.96% ) in female group ,P<0.05 all .Logistic analysis of regression showed that age ,previous cardio‐ and cerebrovascular diseases ,diabetes mellitus ,hyperlipidemia ,Killip cardiac function class and number of diseased coronary vessels were significantly cor‐related with prognosis (OR=0.176~4.931 , P<0.05 all) .Conclusion:Prognosis of female AMI patients is poorer than that of males .Prognosis is related to age ,cardio‐ and cerebrovascular diseases ,diabetes mellitus ,hyperlipi‐demia ,Killip cardiac function class ,and number of diseased coronary vessels .

Comparative proteomic analysis of Yersinia pestis 201 and 201△pCD1 strains / 军事医学

Objective To explore the potential pathogenesis of Yersinia pestis and provide new clues for vaccine development through comparative proteomic analysis of wild-type and pCD1 cured strain of Yersinia pestis 201.Methods Differentially expressed proteins at 26℃ and 37℃ were separated and identified using two-dimensional electrophoresis coupled with mass spectrometry .Results A total of 24 differently expressed proteins were successfully identified from the samples of bacteria grown at 26℃ and 25 proteins at 37℃.Among these, 7 proteins were encoded by pCD 1 plasmid. Conclusion Through comparative proteomic research, we have found that the abundance of several proteins can be dramatically changed when the large plasmid pCD 1 is missing,suggesting that the plasmid can regulate the expression of many genes located in the chromosome .

Comparison of two protein preparation methods by two-dimensional gel electrophoresis / 军事医学

Objective To compare the separation effects of protein samples extracted by two different methods with two -dimensional gel electrophoresis (2-DE) .Methods Ultrasonic disruption and glass-beads grounding were used to prepare protein samples of Gram-negative bacteria , Gram-positive bacteria and animal tissues .The actual results of the two sample preparation methods were compared by 2-DE.Results The 2-DE maps of samples extracted by the two methods were obtained.Conclusion The 2-DE maps of glass-beads grounding samples are better than those of ultrasonic disruption thanks to their lower backgrounds , which are beneficial for further image analyses .

Germination of Bacillus anthracis spores:research advances / 军事医学

A spore is another life cycle form of Bacillus anthracis for resisting starvation.When conditions are favorable for growth, the dormant spore will germinate,go through outgrowth, and are ultimately converted back into a growing cell. As the first step back to vegetative growth, germination could be induced by nutrients and a variety of non-nutrient agents. Nutrient germinants trigger cation release and water absorption by binding to receptors in the spore′s inner membrane.Then the spore′s peptidoglycan cortex is hydrolyzed and the spore core rehydrates, which allows the resumption of spore metabo-lism and macromolecular synthesis.This paper reviews the nutrient germinant receptor and cortex lytic enzymes in the spore germination process of B.anthracis.

Improvement on simulation algorithm of excitation propagation in heart modeling / 生物医学工程学杂志

It is important to simulate the excitation propagation process of cardiac bio-electricity in the research of ECG forward problem. Traditional methods describe them with wave simulation algorithm such as LFX simulation algorithm and vector propagation algorithm etc, these methods have some problems to certain extent, due to the presence of discreteness of space and time and asymmetry of the myocardium. This paper discussed the simulation algorithm in 2-dimension space under the circumstance of layered and non-layered structure of myocardium. By calculating the theoretic values of simulating time based on Huygen's principle, we found that there were errors in LFX algorithm and no errors in vector propagation algorithm under the circumstance of non-layered structure of the myocardium, no mater what myocardium is isotropic or anisotropic. However, there exist errors from both algorithms when the myocardium has the layered structure. An improved algorithm is proposed and the simulations have been performed to examine the efficacy of the new algorithm, and the errors are reduced obviously. By increasing the number of myocardial blocks in the model, we also analyzed its influence on the error of simulation algorithm.

Construction of cDNA Expression Library from Lung Cancer Cells / 中国肿瘤生物治疗杂志

Messenger RNA was isolated directly from lung cancer cell line A549 using magnetic particles. First strand synthesis from mRNA was driven by M-MLV(Moloney Murine Leukemia Virus) reverse transcriptase and random hexam-eric primer, followed by second strand synthesis using RNase H and DNA polymerase I After treatment with T4 DNA polymerase to flush the ends, the double-stranded cDNA was cloned into the plasmid expression vector digested with EcoRI and followed by removing cohesive end. The number of independent clones of the resulting cDNA library was about 9.0 x 105. The estimated percentage of colonies with inserts was about 85 % . The insert size ranges from 0.5 kb ~ 4 kb. The CPP32 gene coding for death protease was obtained by PCR with the cDNA library from lung cancer cells as a template for the first time. Construction of the cDNA library laid a foundation for screening other genes regulating death of lung cancer cells.

THE REMOVAL OF LARGE INVASIVE PLASMIDS FROM SHIGELLA SPECIES / 微生物学通报

In order to remove invasive plasmids from Shigella flexneri 2a 2457T and Shigella sonnei S7 based on the principle of plasmid incompatibility Ori and inc genes were amplified by PCR from S. flexneri 2a invasive plasmids, and then they were cloned into plasmid pMD18 T, resulting recombinant plasmids pMDori and pMDinc After S flexneri 2a 2457T and S sonnei S7 were transformed with pMDori or pMDinc respectively Invasive plasmids were removed from S flexneri 2a 2457T and S sonnei S7