DNMT1 activates the canonical Wnt signaling in rheumatoid arthritis model rats via a crucial functional crosstalk between miR-152 and the DNMT1, MeCP2.

In previous study, we identified that microRNA (miR)-152 expression was down-regulated in RA model rats, and overexpression of miR-152 inhibited the canonical Wnt signaling through the DNA methyltransferase (DNMT1) inhibition. However, the exact molecular mechanisms of DNMT1 were unclear. In this work, we investigate whether DNMT1 affects the pathogenesis of RA model rats and targets the miR-152 promoter. The effects of DNMT1 on the canonical Wnt signaling, the pathogenesis of RA model rats and the SFRP1 expression were detected by the real time qPCR, Western blotting, ELISA, MTT and viable cell number assay. The interaction between miR-152 and DNMT1, methyl CpG binding protein 2 (MeCP2) was investigated by real time qPCR and chromatin immunoprecipitation (ChIP). Our results revealed that increased DNMT1 activated the canonical Wnt signaling could not only by targeting SFRP4 may also by SFRP1 in RA model rats. Furthermore, treatment of DNMT1 inhibitor, 5-aza-2'-deoxycytidine (5-azadC), or knockdown of DNMT1, or knockdown of MeCP2 led to increased miR-152 expression by reversion of its promoter hypermethylation, DNMT1 and MeCP2 binding to the CpG islands of miR-152 promoter. Interestingly, it is proved a synergistic inhibition effect of DNMT1 and MeCP2 in this process. Moreover, overexpression of miR-152 could inhibit DNMT1 expression and result in a decrease of DNMT1 and MeCP2 binding to miR-152 promoter, and inhibition of miR-152 expression would reverse it. These observations demonstrate a crucial functional crosstalk between miR-152 and the DNMT1, MeCP2 by a double-negative circuit involved in the pathogenesis of RA model rats.

Design of signal peptide bombyxin and its effect on secretory expression efficiency and levels of Helicobacter pylori urease subunit B in silkworm cells and larvae

This study employed a Bac-to-Bac/Bombyx mori bioreactor to mass-produce immunogenic urease subunit B (UreB) from Helicobacter pylori. The signal peptide bombyxin from B. mori was used to promote secretory expression to improve expression levels and was designed and integrated into the UreB gene to generate the Bacmid/BmNPV/(signal peptide)-UreB baculovirus expression system. To determine whether the bombyxin signal peptide resulted in secretory expression of recombinant UreB (rUreB) and to determine the secretory efficiency, we tested the secretory expression level of rUreB in Bm5 cells using ELISA. To further investigate whether secretory expression affected cell viability, cells were evaluated using 0.4% trypan blue staining, and Bacmid/BmNPV/UreB without the signal peptide served as a control. The above recombinant bacmid constructs were injected to silkworm larvae, and the secretory expression level of rUreB was detected using SDS-PAGE and semi-quantitative western blot analysis. The results indicated that the bombyxin signal peptide directed the secretory expression of rUreB and that this expression improved the viability of Bm5 cells. Moreover, the results showed that the expression level of rUreB was 1.5 times higher with the Bacmid/BmNPV constructs containing the bombyxin signal sequence than those without the signal sequence. These results demonstrate that secretory expression can enhance rUreB expression levels and is likely to aid in the large-scale expression and yield of rUreB in silkworm larvae.

The synthetic antimicrobial peptide pexiganan and its nanoparticles (PNPs) exhibit the anti-helicobacter pylori activity in vitro and in vivo.

The aim of this study was to probe the potential anti-H. pylori activity of the synthetic antimicrobial peptide pexiganan, which is an analog of the peptide magainin, and its nanoparticles (PNPs) that were prepared in our laboratory. To compare their antibacterial effects in vitro and in vivo, studies of H. pylori growth inhibition, kinetics and resistance assays were undertaken. The gastric mucoadhesive efficiency and H. pylori clearance efficiency of pexiganan and PNPs were evaluated in rats and mice infected with H. pylori. The eradication of H. pylori was determined using urease tests and a microbial culture method. We observed that PNPs adhered to gastric mucosa more effectively owing to a prolonged stay in the stomach, which resulted in a more effective H. pylori clearance. In addition, PNPs had greater anti-H. pylori effect than pexiganan in infected mice. The amount of pexiganan required to eradicate H. pylori was significantly less using PNPs than the corresponding pexiganan suspension. The results confirmed that PNPs improved peptide stability in the stomach and more effectively eradicated H. pylori from mice stomachs than pexiganan.