Objective To study the genetic basis for biovar conversion of Y. pestis. Methods In silico comparative genomic analysis was conducted and some critical genetic variations of Yersinia pestis were comparatively analyzed by means of PCR and DNA sequencing. Results A 93bp in-frame deletion in glpD gene results in the glycerol negative characteristic of Orientalis strains. A point mutation in the napA gene may cause the negative characteristic of nitrate reduction in Mediaevalis and Microus strains. A 122-bp frameshift deletion in the araC gene may lead to the arabinose negative phenotype of Microus strains. Conclusion In this study, Microtus strains with their unique pathogenic, biochemical and molecular features, were proposed as a novel biovar Microtus. In the light of its differential ability to ferment glycerol and arabinose and to reduce nitrate, Y. pestis can be classified into four biovars-Antiqua(glycerol positive, arabinose positive and nitrate positive), Mediaevalis(glycerol positive, arabinose positive and nitrate negative), Orientalis(glycerol negative, arabinose positive and nitrate positive), and Microtus(glycerol positive, arabinose negative and nitrate negative).

Objective To study the genome dynamics of Y. pestis and look for the relationship between its genome microevolution and niche adaption.Methods The DNA microarray combined with PCR was used to perform comparative genomic analysis of natural populations of Y.pestis.Results It was revealed that considerable genome dynamics of Y. pestis were the result of gene acquisition and loss in genome. We established a genomotyping system to group homologous isolates of Y. pestis, drew an outline of parallel microevolution of the Y. pestis genome, and established the link between the bacterial niche adaptation and genome microevolution.Conclusion The transmission, colonization and expansion of Y. pestis in natural foci are the results of its parallel, directional and gradual adaptation to the complex interactions among the environment, the hosts, and the pathogen itself.

Objective To better understand the pathogenicity and evolution of Yersinia pestis, we carried out the whole genome sequencing of human-avirulent Yersinia pestis strain 91001, which was isolated from a species of rodent-Microtus brandti. Methods We utilized “whole genome shotgun” approach to get the genome sequence of 91001. Based on the finished and annotated genome sequence of 91001, as well as the previously published genome sequences of CO92 and KIM, we performed detailed comparative genomics analysis on their chromosomes and plasmids. Results The genome of 91001 consisted of one chromosome and four plasmids (pPCP1, pCD1, pMT1 and pCRY). The pPCP1 plasmid of 9 609bp was almost identical with its counterparts from reference strains, which possessed 10 CDS. Plasmid pCD1 was found to be a plasmid of the type III secretory apparatus, and its length was 70 159bp. Although its CDS are quite similar to those of the reference plasmids, there were obvious rearrangements which produced certain differences in structure among them. Another plasmid was pMT1, a 106 642bp plasmid, which showed slightly different architecture compared with the reference ones. There was no mutation in virulent-related genes of pMT1 and pMT1 of 91001, which seemed to have retained more fragments of an ancestor plasmid. pCRY was a novel plasmid discovered in this work. It was 21 742bp long and harbored a group of gene encoding type IV secretory system. pCRY seemed to be able to replicate. The length of chromosome of 91001 was 4 595 065bp, and among its 4 037 predicted CDS (coding sequences), 141 were possibly pseudogenes. There were many IS in the chromosome. Due to the rearrangments mediated by IS, the structure of 91001 chromosome showed significant differences compared with CO92 and KIM. According to the results of comparative genomics analysis, we deduced the genetic mechanisms of nitrate reduction, glycerol fermentation, arabinose and milibiose utilization in 91001. Conclusion According to the analysis of plasmids structure, pseudogenes distribution, nitrate reduction negative mechanism, gene comparison and chromosome architecture, we conclude that 91001 and other strains isolated from Microtus brandti and Microtus fuscus evolved from ancestor Y. pestis and then developed into a different lineage. The deletion of large genome fragments from 91001 chromosome and pseuogenes might contribute to its unqiue pathogenicity and host-specificity.

Objective To establish reliable proteomics analysis models for Yersinia pestis and obtain the basic proteomics data of this pathogen. Methods The human-avirulent Y. pestis strain 91001 was cultured as an experimental strain, and the proteins of which were extracted and divided into three parts fractionally according to their solubility. Then three different methods (Shotgun-LC-MS-MS, 1D-LC-MS-MS and 2D-MS) were used to analyze the extracted proteins. The obtained data were compared with the theoretical protein database of strain 91001 so as to identify the expressed protein of Y.pestis strain 91001 in this study. Results 971 proteins were identified by shotgun-LC-MS-MS method, accounting for 23.4% of the predicted proteins of strain 91001 (971/4 143). 915 proteins were identified by 1D-LC-MS-MS method, accounting for 22.1% of the predicted proteins of strain 91001 (915/4 143). However, with 2D-MS only 5.62% of the predicted proteins (233/4 143) were identified. Altogether 1 193 proteins were identified when the results of the 3 methods added together, accounting for 28.7% of all the predicted CDS in 91001. Conclusion The kind and quantity of proteins identified by various proteomics methods differ from each other dramatically, therefore it is necessary to utilize multiple methods to get more reliable protein profiles of Y. pestis.

Objective To identify the DNA tag sequences with the purpose of rapid and specific characterization of Y. pestis. Methods DNA microarray hybridization combined with PCR was used to perform genomic comparison between strains of Y. pestis and Y. pseudotuberculosis in order to screen and identify Y. pestis-specific genes. Results Twenty eight signature genes of Y. pestis were discovered. Three pairs of Y. pestis-specific primers were designed according to tag genes and proved to amplify the specific sequences of the target bacterium, showing no cross-reaction with the closely related Y. pseudotuberculosis and a large collection of genomic DNAs from other organisms. Conclusion DNA tag sequence is an ideal target for the rapid detection and identification of Y. pestis by PCR method.

Objective To study genetic variations of pgm locus and its flanking regions in Yersinia pestis isolated from Chinese natural focus so as to understand differences in virulence between different strains and to improve the prevention of plague. Methods We analysed the sequence variations of pgm locus and its flanking regions by using PCR-sequencing and allele-specific PCR among 260 strains of Y. pestis isolated from different foci and 7 strains of Yersinia pseudotuberculosis. Results For YP1666, only the strains of Xilin Gol grassland type and Microtus fuscus type had intact transmembrane helix, and the T-deletion at base 1406 was unique in strains of Orientalis. Same as Y. pseudotuberculosis, there was no IS100-insertion at the 3′ end of pgm locus of strains from Eastern section of North Tianshan Mountain type, Western section of North Tianshan Mountain type A and B, Xilin Gol grassland type and M. fuscus type. Only the strains of Xilin Gol grassland type and M. fuscus type had IS285-insertion in pigmentation segment and IS100-insertion at its downstream flanking region. pgm locus was deleted entirely from 36 strains, most of which came from Ordos plateau type, Song-liao plain type B , Kunlun Mountain type A and B. Conclusion Strains of Eastern section of North Tianshan Mountain type, Western section of North Tianshan Mountain type A and B, Xilin Gol grassland type and M. fuscus type are the oldest lineage of Chinese isolates. The pgm locus of the strains of these three types is very stable because there is no IS100-insertion at its 3′ terminal. We suggest that the strains of Xilin Gol grassland type and M. fuscus type should be grouped into a fourth biovar: Microtus. pgm locus is highly conserved among strains of different ecotypes, and its variations are well correlated with biovar, focus and ecotype, which indicates that pgm locus has played a role in strains′ adaptation to their environment.

In order to investigate the affinity of aptamers to Bacillus anthracis spore, a custom synthesized 78 mer random DNA library was subjected to 15 rounds of selection against spores of vaccine strain A.16R by using SELEX method. The selected aptamers were cloned and sequenced. Macaw 2.05 and DNAsis 2.5 package were employed to analyze the conserved sequences and second structure of the aptamers, respectively. Affinities of aptamers to the spores were visualized by biotin streptavidin horseradish peroxidase system. The results showed that affinities of the aptamers were different. The highest OD at 450nm was 1.2, and the lowest was 0.25. The second structure analysis revealed possible stem loops for binding to the spores. The conserved sequences, AGGGG, CCCCG, GGGTT and ACACT, were found and the aptamers having same conserved sequence demonstrated similar affinity to the spores.

Bacteriophages are abundantly distributed viruses , which are able to infect bacteria .Bacteriophages are becoming a focus of attention for microbiologists , as they can cause pollution to the fermentation industry and might serve as alternative therapies for antibiotic-resistant pathogenic bacteria .Effective bacteriophage infection normally involves adsorp-tion, injection, replication, assembly and release , against which bacteria have developed various resistance strategies .The research progress in the bacteriophage resistance mechanisms is briefly reviewed in this paper .

Objective To investigate the effects of single and combined exposure to ozone and nitrogen dioxide on airway injury and inflammatory levels in mice. Methods Eighty BALB/C mice were randomly divided into eight groups:four were sensitized model groups(the control group,the O3 group,the NO2 group and the O3+NO2 group based on the way of exposure) ,the other four were non-sensitized groups. The concentration of O3 was 0.16 mg/m3,NO2 was 0.30 mg/m3. Mice were exposed 2 hours every day for 7 consecutive days. Mice were sacrificed in the exposure end and cell counting and cytokine measurement from bronchoalveolar lavage fluid(BALF) were performed. Results In the sensitized groups,neutrophil proportion and IL-6 were significantly increased except the control group and IL-2 and MDA were also significantly increased in the O3 group and the O3+NO2 group(P

Objective To analyze the correlation between chronic diseases,depression and activities of daily living (ADL) in the elderly,and to explore the risk factors of depressive symptoms and ADL.Methods Randomized multi-stage sampling method was used to select 504 elderly (aged ≥65 years old) in Jining City.The participants were then investigated and assessed by using Geriatric Depression Scale15,Katz-Activity of Daily Living (Katz-ADL) and Lawton instrumental ADL (Lawton-IADL).Results The prevalence of chronic diseases was 74.01％ among the elderly who lived in Jining City.The positive rate of depressive symptom was 7.14％ and the rate of ADL or IADL limitation was 19.05％ and 66.67％,respectively.The positive rate of depressive symptom and the rate of ADL or IADL limitation of the male were lower than those of female (x2 values were 18.15,12.31 and 30.18,respectively; all P＜ 0.05).The number of chronic diseases was a risk factor of depressive symptom and ADL limitation (odds ratio (OR) =1.71 or 1.53,respectively).ADL and IADL limitations were also the risk factors of depressive symptom (OR =3.15 or 9.36,respectively).Over adjustment of gender and age,the impact of the number of chronic diseases on depressive symptom,ADL and IADL and the influence of ADL and IADL limitations on depressive symptom showed no statistical significance.Conclusions Gender and age may be fundamental risk factors of depressive symptom and ADL and IADL limitations in the elderly.Family members and society workers should improve ADL and positive emotion and reduce the likelihood of depressive symptom among aging population.