Background: MajorClostridium difficilevirulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce.Results: The toxin genestcdAandtcdBwere amplified by PCR using chromosomal DNA from a toxigenic strain as a template, and cloned into a shuttle vector pHis1522. The sequences of bothtcdAandtcdBgenes in the vector have been verified by DNA sequencing. The constructs were transformed intoB. megateriumprotoplasts and the protein expression was controlled under a xylose promoter. The recombinant toxins (rTcdA and rTcdB) were purified from bacterial crude extracts. Approximately 5 – 10 mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. The resulting rTcdA and rTcdB had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination.Conclusion: We have generated the full length and active recombinant TcdA and TcdB inBacillus megaterium.

Background: Differences in 5-hydroxymethylcytosine, 5hmC, distributions may complicate previous observations of abnormal cytosine methylation statuses that are used for the identification of new tumor suppressor gene candidates that are relevant to human hepatocarcinogenesis. The simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine is likely to stimulate the discovery of aberrantly methylated genes with increased accuracy in human hepatocellular carcinoma. Results: Here, we performed ultra-performance liquid chromatography/tandem mass spectrometry and single-base high-throughput sequencing, Hydroxymethylation and Methylation Sensitive Tag sequencing, HMST-seq, to synchronously measure these two modifications in human hepatocellular carcinoma samples. After identification of differentially methylated and hydroxymethylated genes in human hepatocellular carcinoma, we integrate DNA copy-number alterations, as determined using array-based comparative genomic hybridization data, with gene expression to identify genes that are potentially silenced by promoter hypermethylation. Conclusions: We report a high enrichment of genes with epigenetic aberrations in cancer signaling pathways. Six genes were selected as tumor suppressor gene candidates, among which, ECM1, ATF5 and EOMES are confirmed via siRNA experiments to have potential anti-cancer functions.

Background: Host genetic variations may contribute to disease susceptibility of influenza. IL-1A and IL-1B are important inflammatory cytokines that mediate the inflammation and initiate the immune response against virus infection. In this study, we investigated the relationship between single-nucleotide polymorphisms (SNPs) of Interleukin-1A (IL-1A) and Interleukin-1B (IL-1B) and the susceptibility to 2009 pandemic A/H1N1 influenza (A(H1N1)pdm09). 167 patients whom were confirmed with A(H1N1)pdm09 and 192 healthy controls were included in this study. Four SNPs (rs1304037, rs16347, rs17561, rs2071373) in IL1A gene and three SNPs (rs1143623, rs3917345, rs1143627) in IL1B gene were genotyped by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry platform, and the associations of the genetic variants of IL-1 with susceptibility to A(H1N1)pdm09 were then assessed. Results: The polymorphisms of rs17561 in IL1A gene and rs1143627 in IL1B gene were found to be associated with susceptibility to A(H1N1)pdm09 with P values of 0.003 (OR 2.08, 95% CI 1.27-3.41) and 0.002 (OR 1.62 , 95% CI 1.20-2.18), respectively. However, no significant difference in allelic frequency was observed for other SNPs between cases and controls. Conclusions: This study provides a new insight into pathogenesis of A(H1N1)pdm09, suggesting that genetic variants of IL-1A and IL-1B may exert a substantial impact on the susceptibility of A(H1N1)pdm09 virus infection.

Gamma interferon (IFN-γ) release assays have been proven to be useful in the diagnosis of Mycobacterium tuberculosis infection. Nevertheless, their specificity and sensitivity vary among the different populations studied. Here, we evaluate the value of an in-house IFN-γ enzyme-linked immunospot (ELISPOT) assay in the diagnosis of active tuberculosis (TB) in Shenzhen, China, where the prevalence of tuberculosis is severe and Mycobacterium bovis BCG vaccination is mandatory at birth. A total of 305 patients with active tuberculosis, 18 patients with nontuberculosis lung diseases, and 202 healthy controls were recruited in this study. Among them, 156 individuals were simultaneously tested for IFN-γ responses by the commercial QuantiFERON-TB Gold in-tube (QFT-IT) assay. Tuberculin skin tests (TST) were performed with 202 healthy controls. The overall sensitivities of the ELISPOT and QFT-IT assays for active tuberculosis were 83.60% and 80.85%, respectively; the specificities were 76.6% and 73.26%, respectively. The IFN-γ ELISPOT responses, but not those of the TST, were significantly correlated with TB exposure ( r = −0.6040, P < 0.0001). The sensitivities of the ELISPOT assay varied for patients with different forms of tuberculosis, with the highest sensitivity for patients with sputum-positive pulmonary tuberculosis (89.89%) and the lowest for those with tuberculous meningitis (62.5%). In conclusion, the IFN-γ ELISPOT assay is a useful adjunct to current tests for diagnosis of active TB in China. The ELISPOT assay is more accurate than TST in identifying TB infections.

HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus.