In previous studies, a Trichinella spiralis serine protease (TsSP) was identified in excretion/secretion (ES) products from intestinal infective L1 larvae (IIL1) using immunoproteomics. The complete cDNA sequence of TsSP gene was 1372 bp, which encoded 429 amino acids with 47.55 kDa. The TsSP was transcribed and expressed at all T. spiralis life cycle phases, as well as mainly located at the cuticle and stichosome of the parasitic nematode. Recombinant TsSP bind to intestinal epithelial cells (IEC) and promoted larva invasion, however, its exact function in invasion, development and reproduction are still unknown. The aim of this study was to confirm the biological function of TsSP during T. spiralis invasion and growth using RNA interference (RNAi) technology. The results showed that on 1 day after electroporation using 2.5 µM siRNA156, TsSP mRNA and protein expression of muscle larvae (ML) was suppressed by 48.35 and 59.98%, respectively. Meanwhile, silencing of TsSP gene by RNAi resulted in a 61.38% decrease of serine protease activity of ML ES proteins, and a significant reduction of the in vitro and in vivo invasive capacity of IIL1 to intrude into the IEC monolayer and intestinal mucosa. When mice were infected with siRNA 156-transfected larvae, adult worm and muscle larva burdens were decreased by 58.85 and 60.48%, respectively. Moreover, intestinal worm growth and female fecundity were evidently inhibited after TsSP gene was knockdown, it was demonstrated that intestinal adults became smaller and the in vitro newborn larval yield of females obviously declined compared with the control siRNA group. The results indicated that knockdown of TsSP gene by RNAi significantly reduced the TsSP expression and enzymatic activity, impaired larvae intrusion and growth, and lowered the female reproductive capacity, further verified that TsSP might participate in diverse processes of T. spiralis life cycle, it will be a new prospective candidate molecular target of anti-Trichinella vaccines.
A T. spiralis serine protease 1.2 (TsSP1.2) was identified in the muscle larvae (ML) and intestinal larvae surface/excretory-secretory (ES) proteins by immunoproteomics. The aim of this study was to determine the TsSP1.2 function in the process of T. spiralis intrusion, growth and reproduction by using RNA interference (RNAi). RNAi was used to silence the expression of TsSP1.2 mRNA and protein in the nematode. On 2 days after the ML were electroporated with 2 µM of TsSP1.2-specific siRNA 534, TsSP1.2 mRNA and protein expression declined in 56.44 and 84.48%, respectively, compared with untreated ML. Although TsSP1.2 silencing did not impair worm viability, larval intrusion of intestinal epithelium cells (IEC) was suppressed by 57.18% (P < 0.01) and the suppression was siRNA-dose dependent (r = 0.976). Infection of mice with siRNA 534 transfected ML produced a 57.16% reduction of enteral adult burden and 71.46% reduction of muscle larva burden (P < 0.05). Moreover, silencing of TsSP1.2 gene in ML resulted in worm development impediment and reduction of female fertility. The results showed that silencing of TsSP1.2 by RNAi inhibited larval intrusion and development, and reduced female fecundity. TsSP1.2 plays a crucial role for worm invasion and development in T. spiralis life cycle, and is a potential vaccine/drug target against Trichinella infection.
The Chikungunya virus (CHIKV) is an arthropod borne virus. In the last 50 years, it has been the cause of numerous outbreaks in tropical and temperate regions, worldwide. There is limited understanding regarding the underlying molecular mechanisms involved in CHIKV replication and how the virus interacts with its host. In the present study, comparative proteomics was used to identify secreted host proteins that changed in abundance in response to early CHIKV infection. Two-dimensional gel electrophoresis was used to analyse and compare the secretome profiles of WRL-68 cells infected with CHIKV against mock control WRL-68 cells. The analysis identified 25 regulated proteins in CHIKV infected cells. STRING network analysis was then used to predict biological processes that may be affected by these proteins. The processes predicted to be affected include signal transduction, cellular component and extracellular matrix (ECM) organization, regulation of cytokine stimulus and immune response. These results provide an initial view of CHIKV may affect the secretome of infected cells during early infection. The results presented here will compliment earlier results from the study of late host response. However, functional characterization will be necessary to further enhance our understanding of the roles played by these proteins in the early stages of CHIKV infection in humans.
Tropidolaemus wagleri (temple pit viper) is a medically important snake in Southeast Asia. It displays distinct sexual dimorphism and prey specificity, however its venomics and inter-sex venom variation have not been thoroughly investigated. Applying reverse-phase HPLC, we demonstrated that the venom profiles were not significantly affected by sex and geographical locality (Peninsular Malaya, insular Penang, insular Sumatra) of the snakes. Essentially, venoms of both sexes share comparable intravenous median lethal dose (LD50) (0.56-0.63 μg/g) and cause neurotoxic envenomation in mice. LCMS/MS identified six waglerin forms as the predominant lethal principles, comprising 38.2% of total venom proteins. Fourteen other toxin-protein families identified include phospholipase A2, serine proteinase, snaclec and metalloproteinase. In mice, HPLC fractions containing these proteins showed insignificant contribution to the overall venom lethality. Besides, the unique elution pattern of approximately 34.5% of non-lethal, low molecular mass proteins (3-5 kDa) on HPLC could be potential biomarker for this primitive crotalid species. Together, the study unveiled the venom proteome of T. wagleri that is atypical among many pit vipers as it comprises abundant neurotoxic peptides (waglerins) but little hemotoxic proteinases. The findings also revealed that the venom is relatively well conserved intraspecifically despite the drastic morphological differences between sexes.
A key determinant of neuronal functionality and plasticity is the targeted delivery of select ribonucleic acids (RNAs) to synaptodendritic sites of protein synthesis. In this paper, we ask how dendritic RNA transport can be regulated in a manner that is informed by the cell's activity status. We describe a molecular mechanism in which inducible interactions of noncanonical RNA motif structures with targeting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 form the basis for activity-dependent dendritic RNA targeting. High-affinity interactions between hnRNP A2 and conditional GA-type RNA targeting motifs are critically dependent on elevated Ca(2+) levels in a narrow concentration range. Dendritic transport of messenger RNAs that carry such GA motifs is inducible by influx of Ca(2+) through voltage-dependent calcium channels upon β-adrenergic receptor activation. The combined data establish a functional correspondence between Ca(2+)-dependent RNA-protein interactions and activity-inducible RNA transport in dendrites. They also indicate a role of genomic retroposition in the phylogenetic development of RNA targeting competence.