Comparative proteomic analysis of hepatopancreas in Macrobrachium rosenbergii responded to Poly (I:C).

Gel-enhanced liquid chromatography coupled with tandem mass spectrometry (GeLC-MS/MS) was used to analyze the proteome of Macrobrachium rosenbergii hepatopancreas responded to Poly (I:C). GeLC-MS/MS analysis identified 515 differentially-expressed proteins with ≥1.5 and ≤ -0.5 log2 fold change. Of these, 195 differentially-expressed proteins were significantly matched to known proteins in the database, of which 102 proteins were up-regulated and 93 proteins were down-regulated. These proteins were classified into 21 categories, i.e. metabolic process, oxidative stress response, signaling, transcription, translation, cell cycle, transport, etc. Several immune factors were up-regulated upon Poly (I:C) injection. Protein-protein interaction network analysis of these immune factors identified three major protein clusters including RNAi, stress responses, and Toll pathway-proPO system, implying that Poly (I:C) activates immune responses in prawn through several mechanisms.

Molecular cloning and characterization of Mj-mov-10, a putative RNA helicase involved in RNAi of kuruma shrimp.

Identification and characterization of the RNAi-related genes is the key to understanding RNAi mechanism in shrimp. In this study, we have identified and characterized a novel putative RNA helicase gene, Mj-mov-10 from the kuruma shrimp, Marsupenaeus japonicus and its implication in shrimp RNAi was demonstrated. The full-length Mj-mov-10 gene contained 3536bp, including 239 bp of 5'UTR, 2895 bp of the open reading frame (ORF) and 402bp of 3'UTR, respectively. An ORF of Mj-mov-10 could be translated to a 109-kDa protein which consists of a single helicase core domain containing seven signature motifs of the RNA helicase superfamily-1. Mj-MOV-10 protein shared 47% and 40% identity with mammalian MOV-10 and plant SDE3, respectively. Expression of Mj-mov-10 gene was significantly up-regulated upon dsRNA and white spot syndrome virus (WSSV) challenge. In vivo gene knockdown of Mj-mov-10 resulted in an increase of a susceptibility of shrimp to WSSV infection. Our results implied the functional significance of Mj-MOV-10 in dsRNA-mediated gene silencing and antiviral defense mechanism in shrimp.

Penaeus monodon Tudor staphylococcal nuclease preferentially interacts with N-terminal domain of Argonaute-1.

RNA interference (RNAi) plays a crucial role as an antiviral defense in several organisms including plants and invertebrates. An understanding of RNAi machineries especially protein components of the RNA-induced silencing complex (RISC) is essential for prior to applying RNAi as a tool for viral protective immunity in shrimp. Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved protein and is one of the RISC components. In previous study, suppression of Penaeus monodon TSN (PmTSN) by double-stranded RNA (dsRNA) resulted in decreasing dsRNA-mediated gene silencing activity. To elucidate the functional significance of PmTSN in shrimp RNAi pathway, interactions between PmTSN and three Argonaute proteins (PmAgo) were characterized by yeast two-hybrid and in vitro pull-down assays. The results demonstrated that PmTSN interacted with PmAgo1, but not with PmAgo2 or PmAgo3. The interaction between PmAgo and PmTSN was mediated through the N-terminal domain of PmAgo1 and the SN1-2 domains of PmTSN. Analysis of the nuclease activity of the recombinant PmTSN indicated that PmTSN possessed calcium-dependent nuclease activity specific to single-stranded RNA (ssRNA), but not dsRNA and DNA. Knockdown of PmAgo1 and PmTSN diminished the ability of dsRNA-Rab7 to knockdown PmRab7 expression, indicating the involvement of PmAgo1 and PmTSN in shrimp RNAi pathway. Taken together, the results imply that PmTSN is one of the components of PmAgo1-RISC, thus providing new insights in the RNAi-based mechanism in shrimp.

Molecular cloning and functional characterization of Argonaute-3 gene from Penaeus monodon.

Argonaute (Ago) proteins play a crucial role in the shrimp RNA interference pathway. In this study, we identified and characterized a novel Ago gene from black tiger shrimp, Penaeus monodon. The complete open reading frame of P. monodon Ago3 (PmAgo3) consisted of 2559 nucleotides encoding a polypeptide of 852 amino acids with a predicted molecular weight of 97 kDa and an isoelectric point of 9.42. Analysis of the deduced amino acid sequence of PmAgo3 revealed the presence of two signature domains of the proteins in Argonaute family including PAZ and PIWI. Phylogenetic analysis indicated that PmAgo3 is classified into Ago subfamily and shared the highest amino acid sequence identity (83%) with Litopenaeus vannamei Ago2. Monitoring of the PmAgo3 expression by quantitative real-time PCR revealed that this gene was significantly up-regulated following dsRNA administration, while no significant difference in its expression was observed following yellow head virus (YHV) challenge. In contrast, inhibition of YHV mRNA expression was observed in PmAgo3-knockdown shrimp. These data imply that PmAgo3 is involved in the dsRNA-mediated gene silencing mechanism and plays an important role in YHV replication in the black tiger shrimp.

Naringin Ameliorates Skeletal Muscle Atrophy and Improves Insulin Resistance in High-Fat-Diet-Induced Insulin Resistance in Obese Rats.

Obesity causes progressive lipid accumulation and insulin resistance within muscle cells and affects skeletal muscle fibres and muscle mass that demonstrates atrophy and dysfunction. This study investigated the effects of naringin on the metabolic processes of skeletal muscle in obese rats. Male Sprague Dawley rats were divided into five groups: the control group with normal diet and the obese groups, which were induced with a high-fat diet (HFD) for the first 4 weeks and then treated with 40 mg/kg of simvastatin and 50 and 100 mg/kg of naringin from week 4 to 8. The naringin-treated group showed reduced body weight, biochemical parameters, and the mRNA expressions of protein degradation. Moreover, increased levels of antioxidant enzymes, glycogen, glucose uptake, the expression of the insulin receptor substrate 1 (IRS-1), the glucose transporter type 4 (GLUT4), and the mRNA expressions of protein synthesis led to improved muscle mass in the naringin-treated groups. The in vitro part showed the inhibitory effects of naringin on digestive enzymes related to lipid and glucose homeostasis. This study demonstrates the potential benefits of naringin as a supplement for treating muscle abnormalities in obese rats by modulating the antioxidative status, regulating protein metabolism, and improved insulin resistance in skeletal muscle of HFD-induced insulin resistance in obese rats.

A Tudor staphylococcal nuclease from Penaeus monodon: cDNA cloning and its involvement in RNA interference.

RNA interference (RNAi) plays an important role in an antiviral defense in shrimp. RNAi technology has been extensively used for inhibition of viral replication and studying gene function. However, the mechanism of shrimp RNAi pathway is still poorly understood. In this study, we identified and characterized an additional protein in the RNAi pathway, Tudor staphylococcal nuclease from Penaeus monodon (PmTSN). The full-length cDNA of PmTSN is 2897 bp, with an open reading frame encoding a putative protein of 889 amino acids. Phylogenetic analysis and domain structure comparison revealed that PmTSN is more closely related to vertebrate TSN by sharing the amino acid sequence identity of 57% with TSN of zebrafish. This represents a new type of TSN proteins by exhibiting the four tandem repeat of staphylococcal nuclease-like domain (SN), followed by a Tudor and a partially truncated C-terminal SN domain. Knockdown of PmTSN by dsRNA targeting SN3 domain resulted in the impairment of dsRNA targeting PmRab7 gene to silence PmRab7 expression. In addition, the efficiency of dsRNA targeting YHV-protease gene inhibiting yellow head virus replication was decreased in the PmTSN-knockdown shrimps. Our results imply that PmTSN is involved in dsRNA-mediated gene silencing in shrimp and thus we identified the additional protein involved in shrimp RNAi pathway.