SCFAs-Induced GLP-1 Secretion Links the Regulation of Gut Microbiome on Hepatic Lipogenesis in Chickens.

The impact of gut microbiota and its metabolites on fat metabolism have been widely reported in human and animals. However, the critical mediators and the signal transductions are not well demonstrated. As ovipara, chicken represents a specific case in lipid metabolism that liver is the main site of lipid synthesis. The aim of this study is to elucidate the linkage of gut microbiota and fat synthesis in broiler chickens. The broilers were subjected to dietary treatments of combined probiotics (Animal bifidobacterium: 4 × 108 cfu/kg; Lactobacillus plantarum: 2 × 108 cfu/kg; Enterococcus faecalis: 2 × 108 cfu/kg; Clostridium butyrate: 2 × 108 cfu/kg, PB) and guar gum (1 g/kg, GG), respectively. Results showed that dietary supplementation of PB and GG changed the cecal microbiota diversity, altered short chain fatty acids (SCFAs) contents, and suppressed lipogenesis. In intestinal epithelial cells (IECs), SCFAs (acetate, propionate, and butyrate) up-regulated the expression of glucagon-like peptide-1 (GLP-1) via mitogen-activated protein kinase (MAPK) pathways, mainly via the phospho - extracellular regulated protein kinase (ERK) and phospho-p38 mitogen activated protein kinase (p38 MAPK) pathways. GLP-1 suppressed lipid accumulation in primary hepatocytes with the involvement of (AMP)-activated protein kinase/Acetyl CoA carboxylase (AMPK/ACC) signaling. In conclusion, the result suggests that SCFAs-induced GLP-1 secretion via MAPK pathway, which links the regulation of gut microbiota on hepatic lipogenesis in chickens.

Pentapeptide-insertion scanning mutational analysis of turkey herpesvirus HVT063 reveals residues important for its RNA silencing suppression activity.

HVT063, an RNA-binding protein encoded by turkey herpesvirus, has been shown previously to suppress RNA silencing. Here, a scanning library produced by pentapeptide-insertion scanning mutagenesis was used to identify key residues associated with its RNA silencing suppressor (RSS) activity. Forty-two in-frame insertion mutants of HVT063 protein were evaluated for their RSS activity using the dual-luciferase transient expressing assay system. Sixteen mutations resulted in a loss of RSS activity, 20 mutations resulted in decreased RSS activity, and six mutations exhibited high RSS activity similar to wild-type HVT063. Based on a three-dimensional structure prediction, most of the loss-of-function mutations were located around a predominantly α-helical region at the C-terminal end of HVT063. In particular, a conserved domain in this region, named herpes_UL69, showed low tolerance for five-amino-acid insertions. Combined with the results of our previous studies, basic amino acids could play a key role in RSS activity. These results also demonstrate that pentapeptide-insertion scanning mutagenesis combined with dual-luciferase assays is an effective method to functionally characterize RSSs.

Mutation analysis of the RNA silencing suppressor NS1 encoded by avian influenza virus H9N2.

Non-structural protein 1 (NS1) binds small interfering RNA and suppresses RNA silencing in plants, but the underlying mechanism of this suppression is not well understood. Therefore, here we characterized NS1 encoded by the avian influenza virus H9N2. The NS1 protein was able to suppress RNA silencing induced by either sense RNA or double-stranded RNA (dsRNA). Using deletion and point mutants, we discovered that the first 70 residues of NS1 could suppress RNA silencing triggered by sense transgene, but this sequence was not sufficient to block dsRNA-induced silencing. Any mutations of two arginine residues (35R and 46R) of NS1, which contribute to its homodimeric structure, caused the loss of its silencing suppression activity. These results indicate that the region after residue 70 of NS1 is essential for the repression activity on dsRNA-induced RNA silencing, and that the dimeric structure of NS1 plays a critical role in its RNA silencing suppression function.

A multifunctional protein encoded by turkey herpesvirus suppresses RNA silencing in Nicotiana benthamiana.

Many plant and animal viruses counteract RNA silencing-mediated defense by encoding diverse RNA silencing suppressors. We characterized HVT063, a multifunctional protein encoded by turkey herpesvirus (HVT), as a silencing suppressor in coinfiltration assays with green fluorescent protein transgenic Nicotiana benthamiana line 16c. Our results indicated that HVT063 could strongly suppress both local and systemic RNA silencing induced by either sense RNA or double-stranded RNA (dsRNA). HVT063 could reverse local silencing, but not systemic silencing, in newly emerging leaves. The local silencing suppression activity of HVT063 was also verified using the heterologous vector PVX. Further, single alanine substitution of arginine or lysine residues of the HVT063 protein showed that each selected single amino acid contributed to the suppression activity of HVT063 and region 1 (residues 138 to 141) was more important, because three of four single amino acid mutations in this region could abolish the silencing suppressor activity of HVT063. Moreover, HVT063 seemed to induce a cell death phenotype in the infiltrated leaf region, and the HVT063 dilutions could decrease the silencing suppressor activity and alleviate the cell death phenotype. Collectively, these results suggest that HVT063 functions as a viral suppressor of RNA silencing that targets a downstream step of the dsRNA formation in the RNA silencing process. Positively charged amino acids in HVT063, such as arginine and lysine, might contribute to the suppressor activity by boosting the interaction between HVT063 and RNA, since HVT063 has been demonstrated to be an RNA binding protein.