Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

ABSTRACT: BACKGROUND: Avian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited. RESULTS: Total RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6M and 3.3M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher's exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection. CONCLUSION: A comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122-1, 122-2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.     

Biological Treatment of a Pulp and Paper Industry Effluent by Pleurotus spp.

In this work, the ability of Pleurotus spp.:P. sajor-caju; P. platypus and P. citrinopileatus to treat pulp and paper mill effluent on a laboratory and pilot scale were studied. On the laboratory scale treatment, P. sajor-caju decolorized the effluent by 66.7% on day 6 of incubation. Inorganic chloride liberated by P. sajor-caju was 230.9% (814.0 mg/dl) and the COD was reduced by 61.3% (1302.0 mg/dl) on day 10 of treatment. In the pilot scale treatment maximum decolorization was obtained by P. sajor-caju (60.1%) on day 6 of the incubation. Inorganic chloride content was increased by 524.0 mg/dl (113.0%) and the COD was reduced by 1442.0 mg/dl (57.2%) by P. sajor-caju on day 7 of incubation. These results revealed that the treatment of pulp and paper mill effluent by P. sajor-caju proved as better candidate for the purpose than P. platypus and P. citrinopileatus.     

Stimulation of total phenolics, L-DOPA and antioxidant activity through proline-linked pentose phosphate pathway in response to proline and its analogue in germinating fava beans (Vicia faba)

The rationale behind the study was to enhance azetedine-2-carboxylate (A2C)-linked stress in the germinating seeds to which it responds by increased proline synthesis to overcome inhibition of proline dehydrogenase (PDH). A2C is a competitive inhibitor of proline that inhibits its transport from cytosol to the mitochondria for further metabolic recycling by binding on to the active sites of PDH. The enhanced synthesis of proline would increase the ratio of the cofactors NADP/NADPH₂. The increase in the cofactors would result in the over-expression of the phenypropanoid pathway required for the phenolic acids and L-DOPA synthesis via pentose phosphate pathway through the activity of glucose-6-phosphate dehydrogenase (G6PDH). Fava bean were chosen since it has high phenolic and L-DOPA levels and could be an important part of the diet especially for patients suffering from Parkinson's Disease. The objectives were investigated by assaying for total phenolic content, the corresponding antioxidant activity by β-carotene oxidation method, proline levels and enzymes such as G6PDH and guaiacol peroxidase (GPX) using spectrophotometric methods. L-DOPA was quantified using HPLC. The fava bean seeds were primed with water, 200 μM A2C, 0.25 mM proline and a combination of A2C and proline treatments. After the priming stage, seeds were dark germinated and grown for a period of 8 days, for biochemical analysis. L-DOPA levels did not change in comparison to the control treatments while total phenolic content, proline and G6PDH were all enhanced by the treatments. During the early stages of germination the phenolic acids were antioxidant in nature, which later was reduced as they become polymerized to lignins and lignans via the GPX activity. Total phenolic synthesis was both coupled and uncoupled to PLPPP depending on the treatments. The three treatments over-expressed PLPPP since large difference between control and the treatments were observed for all parameters, except L-DOPA content.     

A New Medium for Bacillus thuringiensis Berliner

S ummary : A new solid medium for culturing Bacillus thuringiensis consists of groundnut cake, 10%; tamarind kernel powder, 1·5% and agar, 0·5%. The quantity of agar in the medium could be decreased from 1·5 to 0·5% by adding tamarind kernel powder. A spore yield (85% sporulation) of 3·2 g/l was obtained. Bacterial spores produced on the new solid medium were pathogenic to the larvae of the almond moth, Cadra cautella.     

Standardised Extract of Bacopa monniera (CDRI-08) Improves Contextual Fear Memory by Differentially Regulating the Activity of Histone Acetylation and Protein Phosphatases (PP1α, PP2A) in Hippocampus

Contextual fear conditioning is a paradigm for investigating cellular mechanisms involved in hippocampus-dependent memory. Earlier, we showed that standardised extract of Bacopa monniera (CDRI-08) improves hippocampus-dependent learning in postnatal rats by elevating the level of serotonin (5-hydroxytryptamine, 5-HT), activate 5-HT_3A receptors, and cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein. In this study, we have further examined the molecular mechanism of CDRI-08 in hippocampus-dependent memory and compared to the histone deacetylase (HDACs) inhibitor sodium butyrate (NaB). To assess the hippocampus-dependent memory, wistar rat pups were subjected to contextual fear conditioning (CFC) following daily (postnatal days 15–29) administration of vehicle solution (0.5 % gum acacia + 0.9 % saline)/CDRI-08 (80 mg/kg, p.o.)/NaB (1.2 g/kg in PBS, i.p.). CDRI-08/NaB treated group showed enhanced freezing behavior compared to control group when re-exposed to the same context. Administration of CDRI-08/NaB resulted in activation of extracellular signal-regulated kinase ERK/CREB signaling cascade and up-regulation of p300, Ac-H3 and Ac-H4 levels, and down-regulation of HDACs (1, 2) and protein phosphatases (PP1α, PP2A) in hippocampus following CFC. This would subsequently result in an increased brain-derived neurotrophic factor (Bdnf) (exon IV) mRNA in hippocampus. Altogether, our results indicate that CDRI-08 enhances hippocampus-dependent contextual memory by differentially regulating histone acetylation and protein phosphatases in hippocampus.     

Genomic Analysis Reveals Distinct Concentration-Dependent Evolutionary Trajectories for Antibiotic Resistance in Escherichia coli

Evolution of bacteria under sublethal concentrations of antibiotics represents a trade-off between growth and resistance to the antibiotic. To understand this trade-off, we performed in vitro evolution of laboratory Escherichia coli under sublethal concentrations of the aminoglycoside kanamycin over short time durations. We report that fixation of less costly kanamycin-resistant mutants occurred earlier in populations growing at lower sublethal concentration of the antibiotic, compared with those growing at higher sublethal concentrations; in the latter, resistant mutants with a significant growth defect persisted longer. Using deep sequencing, we identified kanamycin resistance-conferring mutations, which were costly or not in terms of growth in the absence of the antibiotic. Multiple mutations in the C-terminal end of domain IV of the translation elongation factor EF-G provided low-cost resistance to kanamycin. Despite targeting the same or adjacent residues of the protein, these mutants differed from each other in the levels of resistance they provided. Analysis of one of these mutations showed that it has little defect in growth or in synthesis of green fluorescent protein (GFP) from an inducible plasmid in the absence of the antibiotic. A second class of mutations, recovered only during evolution in higher sublethal concentrations of the antibiotic, deleted the C-terminal end of the ATP synthase shaft. This mutation confers basal-level resistance to kanamycin while showing a strong growth defect in the absence of the antibiotic. In conclusion, the early dynamics of the development of resistance to an aminoglycoside antibiotic is dependent on the levels of stress (concentration) imposed by the antibiotic, with the evolution of less costly variants only a matter of time.     

ERG is required for the differentiation of embryonic stem cells along the endothelial lineage

Background

During skeletogenesis, protein levels of β-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of β-catenin turnover by down-regulating intracellular levels of β-catenin.

Results

To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of β-catenin was used, our approach resulted in the accumulation of wild type β-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells.

Conclusion

Our data indicate that a tight Apc-mediated control of β-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner.     

Thymoquinone-induced Neu4 sialidase activates NFκB in macrophage cells and pro-inflammatory cytokines <Emphasis Type="Italic">in vivo</Emphasis>

Thymoquinone (TQ) derived from the nutraceutical black cumin oil has been reported to be a novel agonist of Neu4 sialidase activity in live cells (Glycoconj J DOI 10.1007/s10719-010-9281-6). The activation of Neu4 sialidase on the cell surface by TQ was found to involve GPCR-signaling via membrane targeting of Gαi subunit proteins and matrix metalloproteinase-9 activation. Contrary to other reports, TQ had no anti-inflammatory effects in vitro. Here, we show that MyD88/TLR4 complex formation and subsequent NFκB activation are induced by the Neu4 activity associated with TQ-stimulated live primary bone marrow (BM) macrophage cells from WT and Neu1-deficient mice, HEK-TLR4/MD2 cells and BMC-2 macrophage cell line but not with primary macrophage cells from Neu4-knockout mice. Tamiflu (oseltamivir phosphate), pertussis toxin (PTX), a specific inhibitor of Gαi proteins of G-protein coupled receptor (GPCR) and the broad range inhibitor of matrix metalloproteinase (MMP) galardin applied to live primary BM macrophage cells completely block TQ-induced MyD88/TLR4 complex formation. Using immunocytochemistry and western blot analyses, Tamiflu, galardin and PTX inhibit NFκB activation induced by Neu4 activity associated with TQ-stimulated BMC-2 cells, HEK-TLR4/MD2 cells and primary BM macrophages from WT mice. EMSA analyses on HEK-TLR4/MD2 nuclear cell extracts confirm the nuclear localization and DNA binding of TQ-induced NFκB activation in a biphasic manner within 30 min. Co-immunoprecipitation experiments reveal for the first time that MMP-9 may be an important intermediate link in the TQ-induced Neu4 activity circuitously targeting TLR4 receptors. Central to this process is that Neu4 forms a complex with MMP-9, which is already bound to TLR4 receptors. Fluorescence spectrophotometer analyses of live CD14-THP1 cells treated with TQ show Neu4 sialidase activity over 5 min. Using flow cytometry analyses, CD14-THP1 cells treated with TQ express stable protein levels of Neu4, TLR4 and MMP9 on the cell surface over 30 min except for a marked diminution of MMP9 at 15 min. Using cytokine array profiling analyses of serum, Neu4-knockout mice respond poorly to TQ in producing pro-inflammatory cytokines and chemokines after 5-h treatment compared to the wild-type or hypomorphic cathepsin A mice with a secondary 90% Neu1 deficient mice. Our findings establish an unprecedented signaling paradigm for TQ-induced Neu4 sialidase activity. It signifies that MMP-9 forms an important molecular signaling platform in complex with TLR4 receptors at the ectodomain and acts as the intermediate link for TQ-induced Neu4 sialidase in generating a functional receptor with subsequent NFκB activation and pro-inflammatory cytokine production in vivo.