Transcriptome analysis of fowl adenovirus serotype 4 infection in chickens.

Fowl adenovirus serotype 4 (FAdV-4) is a causative agent of inclusion body hepatitis and hydropericardium-hepatitis syndrome. These diseases cause considerable economic losses in the global poultry industry and are significant stressors for infected chickens. However, the molecular mechanisms of FAdV-4 pathogenesis are poorly understood. In the present study, we identified differentially expressed genes from the livers of FAdV-4-infected chickens using RNA-seq at 7, 14 and 21 days after FAdV-4 infection. We identified 2395 differentially expressed genes at the three time points. These genes were enriched in variety of biological processes and pathways including PPAR and Notch signaling, cytokine-cytokine receptor interactions and Toll-like receptor signaling pathways. The transcriptional data were validated by quantitative real-time PCR. Our results will assist in the understanding of the molecular pathogenesis of FAdV-4 infection and for developing novel antiviral therapies.

Effect of miR-205 on 3T3-L1 preadipocyte differentiation through targeting to glycogen synthase kinase 3 beta.

MiR-205 plays an important role during adipogenesis by modulating the Wnt signaling pathway. Here, we report that miR-205 can regulate the differentiation of 3T3-L1 preadipocyte cells by targeting glycogen synthase kinase 3 beta (GSK-3ß), which is a negative regulatory factor of Wnt signaling. When transiently overexpressed in 3T3-L1 cells, miR-205 suppressed the translation of GSK-3ß, resulting in increased expression of ß-catenin, which can promote cell proliferation by facilitating the transcription of the Wnt target genes cyclin D1 and c-Myc. However, stable overexpression of miR-205 in 3T3-L1 cells did not show any apparent inhibitory effect on adipogenic differentiation. While endogenous miR-205 was inhibited in 3T3-L1 cells, the adipogenesis marker gene, C/EBPα, was significantly activated and more lipid droplets appeared in differentiated adipocytes. However, systemic silencing of miR-205 in mice by using a locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) did not lead to any observable increase in adipose tissue differentiation, implying that, as opposed to the findings from 3T3-L1 cells, miR-205 is dispensable for adipose tissue development in mice.

Effect of TAT-obestatin on proliferation, differentiation, apoptosis and lipolysis in 3T3-L1 preadipocytes.

It has been reported that obestatin regulates adipocyte metabolism via receptors on the cell surface. We wondered whether obestatin can interact with intracellular components that activated signalling pathways in adipocytes. Because obestatin (human) only presents one lysine (at position 10), which cannot penetrate the cell membrane, therefore, we used a cell-permeable peptide TAT (49-57) as a vector to carry obestatin across the cell membrane. The goal of this study was to further understand the function of obestatin after penetrating the cell membrane. Our results showed that TAT-obestatin could cross the 3T3-L1 cell membrane in the absence of cytotoxicity. TAT-obestatin showed no effect on the proliferation of 3T3-L1 preadipocytes. In contrast, obestatin significantly stimulated proliferation at a dose of 10(-11) M and 10(-13) M. In addition, TAT-obestatin demonstrated a more potent inhibitory effect on cell apoptosis induced by serum starvation than that of obestatin. During the progress of adipocyte differentiation, TAT-obestatin and obestatin had no effect on adipogenesis. In the lipolysis assay, TAT-obestatin significantly increased glycerol and free fatty acid release from 3T3-L1 adipocytes after 3 h treatment but showed no significant effect on lipolysis after 24 h and 48 h of treatment. In contrast, obestatin (10(-7) M) had no effect on glycerol release after 3, 24 and 48 h of treatment. The difference between the effect of TAT-obestatin and obestatin on adipocytes metabolism indicated that TAT-obestatin may trigger intracellular signalling as well as signalling at the cell membrane.

Expression patterns of insulin-like growth factor system members and their correlations with growth and carcass traits in Landrace and Lantang pigs during postnatal development.

The IGF system plays important roles in growth. Nevertheless, few data have been reported so far on the expression of IGF system members and their relationship with growth in domestic animals, especially pigs. In this study, hepatic transcript level of IGF1, IGF2, IGF binding protein 2 (IGFBP2), IGFBP3 and IGF 1 receptor (IGF1R), plasma protein level of IGF1 and IGFBP3, and eight growth or carcass traits, including chest circumference, body length, body height (BH), body weight, carcass weight, loin muscle area (LMA), back fat thickness and average daily gain, were measured in fast-growing Landrace and slow-growing Lantang pigs at the age of 1, 27, 90, 150 and 180 days. The results showed that liver mRNA level of IGF1, IGF2 and IGF1R, and blood protein level of IGF1 have a similar developmental profile in both Landrace and Lantang pigs. Their levels were higher at the early age than that at other older ages. Hepatic transcript abundances of the two growth inhibitors, IGFBP2 and IGFBP3, were mostly higher in Lantang pigs than that in Landrace pigs, at 5 examined postnatal stages. The IGF system members' liver mRNA level and/or serum protein level have significant correlation with each other in different age of Landrace or Lantang pigs. Hepatic mRNA level or serum protein level of IGF system members also has significant correlation with investigated traits, especially with BH and LMA, in different age of Lantang or Landrace pigs. Our results revealed the change profiles of porcine IGF system members' liver transcript level and plasma protein level between different pig breeds and different postnatal developmental ages. Moreover, the correlation analysis results suggest that the IGF system members act coordinately to regulate the growth performance and carcass composition in pigs. The information obtained from the present study is important for elucidation of the regulatory mechanism of IGF system underlying growth, and for genetic improvement in pigs.

MiR-143 is not essential for adipose development as revealed by in vivo antisense targeting.

MiR-143 plays an important role in promoting the adipogenic differentiation of pre-adipocytes. Here, we report that systematic silencing of miR-143 in mice by using a locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) did not lead to any obvious abnormalities in the adipose tissue differentiation. Furthermore, there were no significant differences in the expression level of several adipogenic marker genes, such as PPARγ and C/EBPα, in these animals compared with the controls. Therefore, we hypothesize that miR-143 may function as a fine tuning molecule rather than as a switch in the adipogenic regulatory network in mice. In addition, the proposed miR-143 target, ERK5, which was previously identified in human preadipocytes, was not effectively inhibited by miR-143 either in the murine preadipocyte cell line, 3T3-L1, or in primary mouse adipose tissue. However, we did fibroblast growth factor 7 (Fgf7) was identified as a target of miR-143 in murine adipogenesis.

Pathogenicity of a QX-like avian infectious bronchitis virus isolated in China.

Avian infectious bronchitis is a serious and highly contagious disease caused by infectious bronchitis virus (IBV). We isolated a highly virulent IBV strain (CK/CH/JS/TAHY) from kidneys of diseased chickens. Phylogenetic analysis based on the S1 gene revealed that CK/CH/JS/TAHY clustered with the QX-like type. The S1 gene has 1,620 nucleotides and encoded a polypeptide of 540 amino acids with typical coronavirus cleavage recognition sites of HRRR. About 1-day-old specific pathogen-free White Leghorn chickens inoculated with CK/CH/JS/TAHY at 105.5 EID50 exhibited clinical signs including coughing, sneezing, nasal discharge, and tracheal vocalization accompanied by depression with 84% mortality and 100% morbidity. The kidneys of dead birds were swollen and pale and exhibited severe urate deposition. Histopathological examination revealed kidney hemorrhages, multifocal necrosis of the renal tubules and trachea with cilia loss, sloughing of epithelial cells, and edema of the lamina propria. IBV-specific antibodies appeared at 10 D post-infection. Chickens vaccinated with a CK/CH/JS/TAHY oil-emulsion vaccine showed 26.7% morbidity and 3% mortality indicating a protective effect. In conclusion, the IBV strain is a virulent avian IBV and that exhibited severe pathogenicity in chickens and is a vaccine candidate to prevent infection by Chinese QX-like nephropathogenic IBV strains.

Pathogenicity of a fowl adenovirus serotype 4 isolated from chickens associated with hydropericardium-hepatitis syndrome in China.

Hydropericardium-hepatitis syndrome (HHS) is characterized by pericardial effusion and hepatitis and causes huge economic losses in the poultry industry in China. In this study, a strain of fowl adenoviruses (FAdV-4) (GX-1) was isolated from liver samples of diseased chickens with HHS. Phylogenetic analysis based on complete genome gene revealed that GX-1 clustered with the C-type fowl adenovirus and was serotyped as FAdV-4. Pathogenicity testing showed that the GX-1 strain caused 100% mortality in 10-day-old specific pathogen-free chickens at a dose of 104 tissue culture infective doses (TCID50) within 3 d post-infection. A viral dose of 103 TCID50 resulted in a 16% survival rate before day 9 and at 102 TCID50 an 80% rate before day 6. At necropsy, livers from infected chickens were swollen and yellow brown with necrotic foci. The hearts were flabby with amber-colored and jelly-like fluid in the pericardial sacs. The kidneys were swollen and congested. Histologically eosinophilic intranuclear inclusion body could be seen in the hepatic cell. The result of histopathological examination also revealed that heart muscle fibers were fractured with extensive congestion and hemorrhaging. Other tissues like kidney, bursa of Fabricius, thymus, and spleen were observed degeneration and necrosis. Virus-specific antibodies appeared in serum beginning at day 14 and reached statistically significant levels at 21, 28, 35, and 42 dpi (P < 0.001). In conclusion, we identified a highly virulent FAdV-4 virus as causative agent of the HHS outbreak reported here. The FAdV-4 GX-1 strain will be valuable for vaccine evaluation and development to prevent and reduce the spread of HHS in the poultry industry.

Secretory expression and purification of Bacillus licheniformis keratinase in insect cells.

The keratinase (kerA) gene from Bacillus licheniformis PWD-1 was expressed and purified in insect cells. First, the sequence encoding Ker-His-Flag was designed based on the amino acid sequence of the protein and peptide and codon optimization in order to ensure the high expression in insect cells. In the next step, the synthetic DNA was inserted into the pUC57 vector and then sub-cloned in the pFastBac™-1 donor vector by BamHI/HindIII restriction sites. The constructed vector was transformed to E. coli DH10Bac™ cell to generate recombinant bacmid carrying Ker-His-Flag. Recombinant viruses were produced by infecting insect Spodoptera frugiperda (Sf9) cells with bacmid DNA and used for proteins production. Target proteins were purified from the cell supernatants by Ni2+-NTA affinity chromatography and evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The purified product contained two peptides with molecular weights of 38 kDa and 30 kDa and had an optimal pH and temperature at 8.0 and 45°C for keratinolytic activity, respectively. The final product had a specific activity of about 635 U/mg. In summary, we have demonstrated that the open reading frame containing recombinant Ker-His-Flag was expressed and secreted by leader peptide of mellittin from Apis mellitera in insect cells and affinity purification through 8His-Flag tag. It presents an alternative technology for producing keratinases. To our knowledge, it was the first report on the expression of functional keratinase from Bacillus licheniformis in insect cells system.

Peripheral administration of TAT-obestatin can influence the expression of liporegulatory genes but fails to affect food intake in mice.

Obestatin is a 23-amino-acid peptide originally regarded as an anorexigenic factor. However, most of the subsequent studies failed to confirm the initially reported anorexigenic properties of obestatin. Obestatin is incapable of crossing the blood brain barrier (BBB), which may affect its biological function. Here, we report the physiological effects of obestatin in mice after intraperitoneal administration of obestatin conjugated to the cell-permeable peptide TAT, which is capable of delivering different types of proteins through the BBB. Acute peripheral administration of 1 µmol/kg of TAT-obestatin did not influence the 24 h cumulative food intake and body weight gain of mice that were fasted for 18 h. Fed mice were injected intraperitoneally with 100 nmol/kg of TAT-obestatin daily for 25 d. Compared with control groups, on day 3, the gain in body weight was significantly altered; on day 7, abdominal fat mass was remarkably reduced; however, on day 25, there was a surprisingly notable increase in abdominal and epididymal fat mass. In comparison with control groups, on day 25, the expression levels of adiponectin, ADD1, C/EBPα, PPARG and GLUT4 were significantly up-regulated in liver tissues; in white adipose tissue, the expression level of C/EBPα was significantly up-regulated, but adiponectin and GLUT4 were significantly down-regulated. In addition, GPR39, the suspected receptor of obestatin, was up-regulated in white adipose tissue on day 25. These findings suggest that TAT-obestatin might play a role in white adipose tissue metabolism, but its physiological effects on food intake and body weight gain regulation remain unclear.