Mechanism of Escherichia coli Lethality Caused by Overexpression of flhDC, the Flagellar Master Regulator Genes, as Revealed by Transcriptome Analysis.

The flhDC operon of Escherichia coli encodes a transcription factor that initiates flagella synthesis, elevates flagella construction and enhances cell motility, which all are energetically costly and highly regulated processes. In this study, we found that overexpression of flhDC genes from a strong regulatable pN15E6 plasmid could inhibit the growth of E. coli host cells and even eventually cause death. We used transcriptome analysis to investigate the mechanism of flhDC overexpression lethal to host bacteria. The results showed that a total of 568 differentially expressed genes (DEGs), including 378 up-regulated genes and 190 down-regulated genes were detected when the flhDC genes were over-expressed. Functional enrichment analysis results showed that the DEGs are related to a series of crucial biomolecular processes, including flagella synthesis, oxidative phosphorylation and pentose phosphate pathways, etc. We then examined, using RT-qPCR, the expression of key genes of the oxidative phosphorylation pathway at different time points after induction. Results showed that their expression increased in the early stage and decreased afterward, which was suggested to be the result of feedback on the overproduction of ROS, a strong side effect product of the elevated oxidative phosphorylation process. To further verify the level of ROS output, flhDC over-expressed bacteria cells were stained with DCHF-DA and a fluorescence signal was detected using flow cytometry. Results showed that the level of ROS output was higher in cells with over-expressed flhDC than in normal controls. Besides, we found upregulation of other genes (recN and zwf) that respond to ROS damage. This leads to the conclusion that the bacterial death led by the overexpression of flhDC genes is caused by damage from ROS overproduction, which leaked from the oxidative phosphorylation pathway.

Effector responses of bovine blood neutrophils against Escherichia coli: Role of NOD1/NF-κB signalling pathway.

Neutrophils use a broad array of pattern recognition receptors to sense and respond to invading pathogens and are important in the early control of acute bacterial infections. Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan. Reduced neutrophil NOD1 expression has been reported in periparturient dairy cows. The aim of this study was to investigate the role of NOD1 signalling in the early responses of bovine neutrophils to bacterial infections. Blood neutrophils from healthy heifers were preincubated for 2h with ML130, a selective inhibitor of NOD1-dependent nuclear factor-κB (NF-κB) activation. Thereafter, cells were cultured with live Escherichia coli for additional 30 min or subjected to Boyden chamber cell migration assay with E. coli in the lower chamber. Results showed that ML130 inhibited E. coli-induced NF-κB nuclear translocation. There was an indication, although not significant, that ML130 down-regulated gene expression of proinflammatory cytokines interleukin (IL)-1ß and tumour necrosis factor (TNF)-α, chemokines IL-8 and C-X-C motif ligand 2 (CXCL2), and adhesion molecule CD62L, in E. coli-challenged neutrophils. Flow cytometry-based Annexin V staining revealed a considerable increase in neutrophil survival upon E. coli infection, an effect that was attenuated in the presence of ML130. Additionally, inhibition of NOD1/NF-κB signalling resulted in reduced migration of neutrophils to E. coli, and impaired phagocytosis, intracellular bacterial killing and reactive oxygen species production by neutrophils. These results indicate that NOD1/NF-κB pathway plays a crucial role in modulating neutrophil responses that are important for early control of infections. Approaches aiming at restoring neutrophil NOD1 function could be beneficial for prevention or treatment of coliform mastitis.

[Study on method of paeonia radix alba producing and concocting integration processing].

OBJECTIVE: To study the origin pre-treating and processing integration techniques of Paeonia Radix Alba. METHOD: Different processing integration techniques were adopted and compared with traditional processing techniques to determine drying rate, aqueous extracts and peoniflori content. RESULT: Half-dry slices baked at 100 degrees C for 20 min and steamed at 100 degrees C for 10 min had the highest peoniflori contents. Half-dry slices baked at 100 degrees C for 20 min had the highest content of aqueous extracts. Products processed with conventional method and sulfur-fumigation had the lowest content of aqueous extracts. CONCLUSION: The origin processing integration techniques of Paeonia Radix Alba lose less active ingredients than conventional processing methods.

Construction and application of the vectors to identify genes encoding exported proteins of Escherichia coli.

In order to clone genes having signal sequences of Escherichia coli, four vectors with or without Lac or Ara promoter were constructed using a leaderless ß-lactamase as reporter. Fragments of tetracycline resistance gene (Tet) with or without promoter were used to confirm the vectors' ability to clone and report signal sequences. The minimum inhibitory concentration of ampicillin of the transformants was measured to detect the expression and secretion efficiency of the vectors. The results showed that the ß-lactamase could be co-expressed and secreted with Tet protein. The Lac or Ara promoter in the vectors could be regulated by different inducers, and the Ara promoter showed higher regulative efficiency than the Lac. The best induction dose of L-arabinose for the Ara promoter is 1.25 %. All the four vectors were stably maintained in host after being inoculated for 20 passages in antibiotics-free media. Genomic library of an avian pathogenic strain, E. coli O2, was constructed using the pMB-Ara-T vector we developed. 318 clones were obtained from the genomic library of E. coli strain O2, and the inserts in these clones represented 276 genes based on sequence analysis. Among the 276 cloned fragments, only 128 had complete promoter sequence. For the 128 fragments with promoter, only 27 could be expressed under LB culture condition without inducer, the other 101 were only expressed under induction. The results showed our constructed vectors could efficiently capture all kinds of exported protein genes in vitro, including the ones without promoter or with inactive promoter.

[Cloning and function identification of gene 'admA' and up-stream regulatory sequence related to antagonistic activity of Enterobacter cloacae B8].

To reveal the antagonistic mechanism of B8 strain to Xanthomonas oryzae pv. oryzae, transposon tagging method and chromosome walking were deployed to clone antagonistic related fragments around Tn5 insertion site in the mutant strain B8B. The function of up-stream regulatory sequence of gene 'admA' involved in the antagonistic activity was further identified by gene knocking out technique. An antagonistic related left fragment of Tn5 insertion site, 2 608 bp in length, was obtained by tagging with Kan resistance gene of Tn5. A 2 354 bp right fragment of Tn5 insertion site was amplified with 2 rounds of chromosome walking. The length of the B contig around the Tn5 insertion site was 4 611 bp, containing 7 open reading frames (ORFs). Bioinformatic analysis revealed that these ORFs corresponded to the partial coding regions of glyceraldehyde-3-phosphate dehydrogenase, two LysR family transcriptional regulators, hypothetical protein VSWAT3-20465 of Vibrionales and admA, admB, and partial sequence of admC gene of Pantoea agglomerans biosynthetic gene cluster, respectively. Tn5 was inserted in the up-stream of 200 bp or 894 bp of the sequence corresponding to anrP ORF or admA gene on B8B, respectively. The B-1 and B-2 mutants that lost antagonistic activity were selected by homeologuous recombination technology in association with knocking out plasmid pMB-BG. These results suggested that the transcription and expression of anrP gene might be disrupted as a result of the knocking out of up-stream regulatory sequence by Tn5 in B8B strain, further causing biosythesis regulation of the antagonistic related gene cluster. Thus, the antagonistic related genes in B8 strain is a gene family similar as andrimid biosynthetic gene cluster, and the upstream regulatory region appears to be critical for the antibiotics biosynthesis.

[Construction and transfection experiment of a goose circovirus infectious clone].

A pair of primers with BamH I restriction site were designed to amplify the complete genome of goose circovirus. Two copies of the genome were ligated in tandem and cloned into pGEM-T Easy vector to construct an infectious clone named as pGEMT-2GoCV. The pGEMT-2GoCV linearized with EcoR I was transfected to negative embryos and gosling with Lipfectamine. PCR detection verified the proliferation of GoCV in geese. Some sera of the embryo transfected group were detected to be positive at 2 and 4 weeks after hatching and one bursa was detected to be positive at 4 weeks. Some sera of the gosling transfected group were also detected to be positive at 2 weeks after transfection. Furthermore, the mark in the PCR products were identified by BamH I digestion and the GoCV in positive tissue and sera were quantitated by Real-time PCR. The results showed that the virus load in positive bursa was 1.57 x 10(6) copies/mg, the virus load in positive sera were 3.52 x 10(4)-5.92 x 10(5) copies/microL. In conclusion, the infectious DNA clone constructed with two copies of full-length GoCV genome in tandem can transfect embryo and gosling and propagate the marked goose circovirus.

[Construction of a temperature-sensitive and replication-defective T-vector and its application for gene knockout in Salmonella pullorum].

Knockout is an important method for gene function study, while vector is the core of gene knockout. In order to obtain an effective vector for rapid construction of mutant and essentiality identification of the corresponding gene, we constructed a recombinant plasmid named plDM-T based on the temperature-sensitive and replication- defective plasmid plDM1 by inserting an Xcm I adapter into the EcoR I and Pst I sites of pIDM1. Digesting with Xcm I, pIDM-T can be prepared as a linear T-vector for PCR products cloning and be used to knockout the corresponding gene in the genome with insertion duplication mutagenesis. After the verification of temperature sensitivity of the replication of the plasmid, we cloned two Salmonella pullorum genes eno and ybdr into the constructed pIDM-T, and two recombinant plasmids pIDM-T_eno and pIDM-T_ybdr were identified. The recombinant plasmids were then transformed into S. pullorum strain CVCC527 and the IPC (Integration rate per cell) values were calculated. As a result, we identified the eno gene as an essential gene and the ybdr as a non-essential gene in CVCC527. We verified the correctness of recombination site in ybdr recombinant 527 clones (Sal delta ybdr) by PCR and sequencing. The pIDM-T vector can be used for gene knockout in S. pullorum, as well as the identification of essentiality of the corresponding genes, which offers an effective and rapid tool for gene function study in Salmonella.

[Study on the initial processing of Corydalis yanhusuo].

OBJECTIVE: To establish initial processing technology of Corydalis yanhusuo. METHODS: Investigated the effect of the factors such as slice method, dry method, drying temperature on the content of water-extract, ethanol-extract, effective component in Corydalis yanhusuo pieces. Compared the quality with that of the traditional initial processing samples. RESULTS: The best initial process method was: cut fresh Corydalis yanhusuo into 4 - 5 mm thick slices, dry at 70 - 80 degrees C or microwave dry. CONCLUSION: The study provides theoretical base for modifying the initial processing.

Whole-genome sequences of four Mycobacterium bovis BCG vaccine strains.

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only vaccine available against tuberculosis (TB). A number of BCG strains are in use, and they exhibit biochemical and genetic differences. We report the genome sequences of four BCG strains representing different lineages, which will help to design more effective TB vaccines.

Pathological observations of an experimental infection of geese with goose circovirus.

There is so far no report describing the pathogenicity of goose circovirus (GoCV) following experimental infection. We report here an experimental inoculation of 21-day-old geese with a GoCV polymerase chain reaction (PCR)-positive sample of bursa of Fabricius (BF) homogenate, and the results of clinicopathological changes. Forty geese were randomly assigned to two groups: the inoculated group (n=30) receiving tissue homogenate and the uninoculated group (n=10) receiving a placebo (physiological saline). Tissue samples were collected for histopathological examination and GoCV detection. The main clinical signs in the GoCV-inoculated geese included diarrhoea at 17 to 30 days post inoculation, marked differences of growth rate and feather disorders in three of the geese. Viraemia was detected by PCR only at 14 days post inoculation. The BF, thymus, spleen, duodenum and liver of some inoculated geese were also PCR-positive. Real-time PCR showed that there was relatively more viral load in the BF. Histological examination revealed lymphocytic depletion and histiocytosis in the BF and inflammation characterized by lymphatic infiltration in the lung, liver and kidney. Immunohistochemistry applied to PCR-positive samples showed that in only three BF samples was GoCV antigen detected in lymphocytes in both the cortex and medulla. In situ apoptosis detection of all BF samples indicated that GoCV inoculation was associated with an increase in bursal lymphocytic apoptotic events.

[Factors influence volatile oil content in Ocimun basilicum introduced from Xinjiang province to south-east China].

OBJECTIVE: To obtain the information on ecological adaptation of Ocimun basilicum introduced from Xinjiang to Hangzhou and study the effect of different harvesting times, drying methods, and different organs of Ocimun basilicum on Volatile oil content METHODS: Extraction was undertaken according to The Pharmacopoeia of China, 2010 edition. RESULTS: Sun-drying was the most efficient way to obtain Volatile oil compared with other methods. The largest biomass was harvested at 3rd, September. Furthermore, Volatile oil was found to accumulate mostly in the flowers and little in the stems. CONCLUSION: Ocimun basilicum can readily inhabit in Hangzhou and its economic value can be significant improved by growing two seasons per year. Only harvest leaves and flowers can significantly reduce the cost for transport and also increase oil extract rate of Volatile oil.

[Study on method of Corydalis yanhusuo producing and concocting integration processing].

OBJECTIVE: To establish integration processing method for pretreating and vinegar producing Corydalis yanhusuo. METHOD: Different processing methods were contrasted with the traditional processing technology, and contents of corydalis B, water extract and ethanol extract in samples of different processing products were determined. RESULT: The content of corydalis B were best in the samples of vacuumizing C. yanhusuo chips scaked in rice vinegar for twice or soaked in rice vinegar after chip drying. The water extract was highest in the samples of chip soaked in rice vinegar after drying, followed with chip vacuumizing twice, and there were no remarkable difference between the other samples and the traditional process. The difference of ethanol extract was not remarkable in all the samples. CONCLUSION: The study provide the feasibility of C. yanhusuo producing and concocting integration processing.

Novel parvovirus sublineage in the family of Parvoviridae.

Parvoviridae, which are classified into two subfamilies Parvovirinae and Densovirinae, can infect both vertebrate and insects and are related to a wide range of diseases in insects, animals, and humans. In this report, several new parvoviruses were identified in swine sera collected in southeastern China. The sequence analyses showed that the parvoviruses detected in southeastern China formed a distinct sublineage within the subfamily Parvovirinae. Based on these results, we propose a novel parvovirus sublineage, Cnvirus, to describe these parvoviruses.

[Cloning and analysis of the complete genomes of pigeon circovirus from Zhejiang Province].

Pigeon circovrius (PiCV) is a member of circovirus, which is usually regarded as an immunosuppression agent. There were reports that pigeons infected by PiCV showed symptoms of lethargy, weight loss, vomiting, diarrhea, respiratory distress, etc. In this study, we established a PCR method for the detection of PiCV DNA. Samples from 5 different farms in Zhejiang Province were examined and samples from a farm in Hangzhou were positive. Furthermore, the genomic segments of 2 strains of PiCV were amplified, cloned and sequenced using designed primers and the complete genomes of the strains were then assembled and named as PiCV-zj1 and PiCV-zj2, respectively. The sequences were deposited in GenBank under the GenBank Accession number of DQ090945 and DQ090944, respectively. Sequence Analysis had shown that the complete genomes of 2 strains of PiCV from Zhejiang Province had 2 039 nucleotides totally in length and common characters of circovirus such as a stem-loop structure and conserved motifs for Rep protein, which were supposed to be related to the replication of the virus. Pairwise comparisons showed that the nucleotide sequence of the genome of PiCV strains from Zhejiang Province had 86%-89.1% identities to that of 11 published PiCV strains, and that the amino acid identities of the replication-associated protein (Rep) and capsid protein (Cap) displayed 92.1%-94.7% and 76.6%-81.4%, respectively. A phylogenic tree was built using PHYLIP with bootstrap support for 1 000 replicates. The result showed that 10 strains from Europe and America formed one big branch and the others from Zhejiang Province and Australia formed the other two, respectively. This was the first report on the detection and full genome sequencing of PiCV in China.

[Study on the influence on Rhizoma Corydalis by different harvest time and processing methods].

OBJECTIVE: To choose the best harvest time and initial processing method of Rhizoma Corydalis. METHODS: Taking the content of tetrahydropalmatine and dry rates as major indices, different samples from different harvest time and different processing methods were investigated by HPLC. The C18 column (250 mm x 4.6 mm, 5 microm) was used with mobile phase of methanol-0.1% phosphoric acid solution (pH = 6.0, adjusted with triethylamine) (58: 42). The mobile phase flow rate was 1.0 mL/min. The detection wavelength was 280 nm. RESULTS: The contents of tetrahydropalmatine were similar although the harvest time was different. The dry rate on May 21 was higher than the others. The content of tetrahydropalmatine was the highest by steaming and reached 0.11%. CONCLUSION: The best harvest time for Rhizoma Corydalis is around 10 days after the above-ground plant wilted, and the best initial processing method for Rhizoma Corydalis is steaming.

[AFLP analysis of genetic diversity of Prunella].

OBJECTIVE: To explore the variety of the genetic polymorphism of eight Prunella germplasm resources by AFLP analysis. METHOD: The amplified fragment length polymorphism (AFLP) tags were applied to screen out 32 selective amplification primer pairs, the amplified bands as original matrix were analyzed with NTSYS-PC software for the similarity between the Prunella germplasm and the construction of genetic phylogenetic tree. RESULT: SDS extraction of genomic Prunella DNA showed a good quality, could meet the requirements of AFLP analysis. From 32 selective amplification primer pairs, 10 pairs with strong polymorphism, better band and higher resolution were used for the construction of the AFLP Prunella fingerprint, all eight Prunella germplasms were separated, they were divided into 3 categories. CONCLUSION: Prunella germplasm resources are rich in genetic diversity, certain morphological characteristics and differences are associate with genotype.

MyBASE: a database for genome polymorphism and gene function studies of Mycobacterium.

BACKGROUND: Mycobacterial pathogens are a major threat to humans. With the increasing availability of functional genomic data, research on mycobacterial pathogenesis and subsequent control strategies will be greatly accelerated. It has been suggested that genome polymorphisms, namely large sequence polymorphisms, can influence the pathogenicity of different mycobacterial strains. However, there is currently no database dedicated to mycobacterial genome polymorphisms with functional interpretations. DESCRIPTION: We have developed a mycobacterial database (MyBASE) housing genome polymorphism data and gene functions to provide the mycobacterial research community with a useful information resource and analysis platform. Whole genome comparison data produced by our lab and the novel genome polymorphisms identified were deposited into MyBASE. Extensive literature review of genome polymorphism data, mainly large sequence polymorphisms (LSPs), operon predictions and curated annotations of virulence and essentiality of mycobacterial genes are unique features of MyBASE. Large-scale genomic data integration from public resources makes MyBASE a comprehensive data warehouse useful for current research. All data is cross-linked and can be graphically viewed via a toolbox in MyBASE. CONCLUSION: As an integrated platform focused on the collection of experimental data from our own lab and published literature, MyBASE will facilitate analysis of genome structure and polymorphisms, which will provide insight into genome evolution. Importantly, the database will also facilitate the comparison of virulence factors among various mycobacterial strains. MyBASE is freely accessible via http://mybase.psych.ac.cn.

Novel genome polymorphisms in BCG vaccine strains and impact on efficacy.

Bacille Calmette-Guérin (BCG) is an attenuated strain of Mycobacterium bovis currently used as a vaccine against tuberculosis. Global distribution and propagation of BCG has contributed to the in vitro evolution of the vaccine strain and is thought to partially account for the different outcomes of BCG vaccine trials. Previous efforts by several molecular techniques effectively identified large sequence polymorphisms among BCG daughter strains, but lacked the resolution to identify smaller changes. In this study, we have used a NimbleGen tiling array for whole genome comparison of 13 BCG strains. Using this approach, in tandem with DNA resequencing, we have identified six novel large sequence polymorphisms including four deletions and two duplications in specific BCG strains. Moreover, we have uncovered various polymorphisms in the phoP-phoR locus. Importantly, these polymorphisms affect genes encoding established virulence factors including cell wall complex lipids, ESX secretion systems, and the PhoP-PhoR two-component system. Our study demonstrates that major virulence factors are different among BCG strains, which provide molecular mechanisms for important vaccine phenotypes including adverse effect profile, tuberculin reactivity and protective efficacy. These findings have important implications for the development of a new generation of vaccines.

[Study on correlation between Atractylodes macrocephala viability and germination rate].

OBJECTIVE: To screen the optimized methods for detection seed viability and germination rate determination of Atractylodes macrocephala, and determine the relationship between seed viability and germination rate. METHOD: There were four methods, which including 2,3,5-triphynel tetrazolilum chloride (TTC) staining, red ink staining, BTB staining and Nongjia method, to evaluate the 12 A. macrocephala local varieties'seed viability and measure their germination rate. RESULT: Seed viability of A. macrocephala using TTC staining ranked the first compared to that of other three methods. Seed viability was significantly related with germination rate using TTC method. Their correlation coefficient reached 0.915 and regression equation was also found out between seed viability (X) and germination rate (Y), which was Y = -0.083 4 + 0.995 4X. CONCLUSION: TTC staining was the optimal method to determine A. macrocephala seed vitality. Furthermore, seed viability was significant related with germination rate of A. macrocephala.

Reassortant infectious bursal disease virus isolated in China.

Infectious bursal disease virus (IBDV) is a bi-segmented, double-stranded RNA virus which belongs to the genus Avibirnavirus of the family Birnavirideae. In this study, we determined the complete nucleotide sequences of a reassortment IBDV strain TL2004 with segments A and B derived from attenuated and very virulent strains of IBDV. This strain is pathogenic to SPF-embryonated eggs and chickens, although it is not as virulent as very virulent strain. Genomic sequence in GenBank analysis showed that both types of natural genetic reassortment of infectious bursal disease virus emerged in China. Our findings, which strongly suggest genetic exchange between attenuated and very virulent strains of IBDV, emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines.