Identification of microRNAs from Atlantic salmon macrophages upon Aeromonas salmonicida infection

Computational approach was used in to identify potent macrophage specific miRNAs involved in basic biological process of Salmo salar. Analysis of 1119 ESTs from macrophages of Atlantic salmon (Salmo salar) infected with Aeromonas salmonicida revealed expression of 3 miRNAs. Phylogenetic analysis of both the pre-miRNA sequence revealed its evolutionarily conserved nature among various species. Identified targets of the predicted miRNAs revealed the role of miRNA in pathogenesis, stress response and allosteric exchange of histones. Further detailed studies of these miRNAs will help in revealing its function in different biological process necessary for the action of macrophages upon pathogen infection

Metabolite Profile, Ruminal Methane Reduction, and Microbiome Modulating Potential of Seeds of Pharbitis nil

We identified metabolites in the seeds of Pharbitis nil (PA) and evaluated their effects on rumen methanogenesis, fiber digestibility, and the rumen microbiome in vitro and in sacco. Four rumen-cannulated Holstein steers (mean body weight 507 +/- 32 kg) were used as inoculum donor for in vitro trial and live continuous culture system for in sacco trial. PA was tested in vitro at doses ranging from 4.5 to 45.2% dry matter (DM) substrate. The in sacco trial was divided into three phases: a control phase of 10 days without nylon bags containing PA in the rumen, a treatment phase of 11 days in which nylon bags containing PA (180 g) were placed in the rumen, and a recovery phase of 10 days after removing the PA-containing bags from the rumen. Rumen headspace gas and rumen fluid samples were collected directly from the rumen. PA is enriched in polyunsaturated fatty acids dominated by linoleic acid (C18:2) and flavonoids such as chlorogenate, quercetin, quercetin-3-O-glucoside, and quinic acid derivatives. PA decreased (p < 0.001) methane (CH4) production linearly in vitro with a reduction of 24% at doses as low as 4.5% DM substrate. A quadratic increase (p = 0.078) in neutral detergent fiber digestibility was also noted, demonstrating that doses < 9% DM were optimal for simultaneously enhancing digestibility and CH4 reduction. In sacco, a 50% decrease (p = 0.087) in CH4 coupled with an increase in propionate suggested increased biohydrogenation in the treatment phase. A decrease (p < 0.005) in ruminal ammonia nitrogen (NH3-N) was also noted with PA in the rumen. Analysis of the rumen microbiome revealed a decrease (p < 0.001) in the Bacteroidetes-to-Firmicutes ratio, suggesting PA to have antiprotozoal potential. At the genus level, a 78% decrease in Prevotella spp. and a moderate increase in fibrolytic Ruminococcus spp. were noted in the treatment phase. In silico binding of PA metabolites to cyclic GMP-dependent protein kinase of Entodinium caudatum supported the antiprotozoal effect of PA. Overall, based on its high nutrient value and antiprotozoal activity, PA could probably replace the ionophores used for CH4 abatement in the livestock industry.N

Identification of microRNAs from Atlantic salmon macrophages upon Aeromonas salmonicida infection: DOI: 10.14800/rd.303

Computational approach was used in to identify potent macrophage specific miRNAs involved in basic biological process of Salmo salar. Analysis of 1119 ESTs from macrophages of Atlantic salmon (Salmo salar) infected with Aeromonas salmonicida revealed expression of 3 miRNAs. Phylogenetic analysis of both the pre-miRNA sequence revealed its evolutionarily conserved nature among various species. Identified targets of the predicted miRNAs revealed the role of miRNA in pathogenesis, stress response and allosteric exchange of histones. Further detailed studies of these miRNAs will help in revealing its function in different biological process necessary for the action of macrophages upon pathogen infection

Novel Aryl Hydrocarbon Receptor Agonist Suppresses Migration and Invasion of Breast Cancer Cells

<div><p>Background</p><p>Despite the remarkable progress to fight against breast cancer, metastasis remains the dominant cause of treatment failure and recurrence. Therefore, control of invasiveness potential of breast cancer cells is crucial. Accumulating evidences suggest Aryl hydrocarbon receptor (Ahr), a helix-loop-helix transcription factor, as a promising target to control migration and invasion in breast cancer cells. Thus, an Ahr-based exploration was performed to identify a new Ahr agonist with inhibitory potentials on cancer cell motility.</p><p>Methods</p><p>For prediction of potential interactions between Ahr and candidate molecules, bioinformatics analysis was carried out. The interaction of the selected ligand with Ahr and its effects on migration and invasion were examined <i>in vitro</i> using the MDA-MB-231 and T47D cell lines. The silencing RNAs were transfected into cells by electroporation. Expressions of microRNAs (miRNAs) and coding genes were quantified by real-time PCR, and the protein levels were detected by western blot.</p><p>Results</p><p>The <i>in silico</i> and <i>in vitro</i> results identified Flavipin as a novel Ahr agonist. It induces formation of Ahr/Ahr nuclear translocator (Arnt) heterodimer to promote the expression of cytochrome P450 family 1 subfamily A member 1 (Cyp1a1). Migration and invasion of MDA-MB-231 and T47D cells were inhibited with Flavipin treatment in an Ahr-dependent fashion. Interestingly, Flavipin suppressed the pro-metastatic factor SRY-related HMG-box4 (Sox4) by inducing miR-212/132 cluster. Moreover, Flavipin inhibited growth and adhesion of both cell lines by suppressing gene expressions of B-cell lymphoma 2 (Bcl2) and integrinα4 (ITGA4).</p><p>Conclusion</p><p>Taken together, the results introduce Flavipin as a novel Ahr agonist, and provide first evidences on its inhibitory effects on cancer cell motility, suggesting Flavipin as a candidate to control cell invasiveness in breast cancer patients.</p></div

Effects of Dietary Protein Concentration on Lipid Metabolism Gene Expression and Fatty Acid Composition in 18&ndash;23-Month-Old Hanwoo Steers

The present study evaluated the influence of dietary protein level on growth performance, fatty acid composition, and the expression of lipid metabolic genes in intramuscular adipose tissues from 18- to 23-month-old Hanwoo steers, representing the switching point of the lean-to-fat ratio. Forty steers with an initial live weight of 486 &plusmn; 37 kg were assigned to one of two treatment groups fed either a concentrate diet with 14.5% CP and or with 17% CP for 6 months. Biopsy samples of intramuscular tissue were collected to analyze the fatty acid composition and gene expression at 23 months of age. Throughout the entire experimental period, all steers were restrained twice daily to allow individual feeding. Growth performance, blood metabolites, and carcass traits, according to ultrasonic measurements, were not affected by the experimental diets. The high-protein diet significantly increased the expression of intramuscular PPAR&alpha; (p &lt; 0.1) and LPL (p &lt; 0.05) but did not affect genes involved in fatty acid uptake (CD36 and FABP4) nor lipogenesis (ACACA, FASN, and SCD). In addition, it downregulated intramuscular VLCAD (p &lt; 0.01) related to lipogenesis but also GPAT1 (p = 0.001), DGAT2 (p = 0.016), and SNAP23 (p = 0.057), which are involved in fatty acid esterification and adipocyte size. Hanwoo steers fed a high-protein diet at 18&ndash;23 months of age resulted in a relatively lower lipid turnover rate than steers fed a low-protein diet, which could be responsible for shortening the feeding period

Flavipin inhibits cell migration and invasion of breast cancer cell lines.

<p>(A) The IC₅₀ of Flavipin in MDA-MB-231 and T47D cells after 48 h treatment. Cell viability was quantified calorimetrically at 540 nm using CCK-8 kit. (B) Migration of MDA-MB-231 and expansion of T47D were inhibited 24 h after Flavipin treatment (200 μmol/L). (C) Cell invasion of MDA-MB-231 and T47D was suppressed 24 h after Flavipin treatment. Invasiveness of cancer cells were studied using Boyden chamber. The invaded cells were stained with Giemsa and counted in 4 different microscopic fields. Data are shown as mean ± SD from representative experiment studied in triplicates. *P<0.05.</p

The inhibitory effects of Flavipin on breast cancer cells are Ahr-dependent.

<p>(A) The inhibition of proliferation of MDA-MB-231 cells with Flavipin (200 μmol/L) was mitigated with siAhr after 48 h treatment. The cells were transfected with siAhr or siNS by electroporation. Cell proliferation was quantified calorimetrically at 540 nm using CCK-8 kit. (B) Depletion of Ahr by siAhr abrogated the inhibitory effects of Flavipin on migration of breast cancer cells. siAhr mitigated the inhibitory effects of Flavipin on invasion of (C) MDA-MB-231 and (D) T47D cells. Invasiveness of cancer cells were studied using Boyden chamber. The invaded cells were stained with Giemsa and counted in 4 different microscopic fields. Data are shown as mean ± SD from representative experiment studied in triplicates. *P<0.05.</p

The <i>in silico</i> docking of Flavipin binding potential to human Ahr Pas-A and Pas-B Ligand domain.

<p>(A) Flavipin binds to Ahr PAS-A at Phe115, Leu116 and Ala119. (B) The Flavipin interacted with the four active residues of Ahr PAS-B at Gln83, Asn85, Thr101 and Arg103. The yellow-dotted lines indicates the hydrogen bonds.</p

Flavipin induces Cyp1a1 gene expression in an Ahr-dependent fashion activity in breast cancer cell lines.

<p>(A, B) The mRNA and protein levels of Cyp1a1 in MDA-MB-231 and T47D cells were elevated after treatment with Flavipin for 48 h. Quantification was performed by real-time PCR and western blot. (C) Confirmation of siAhr efficiency in breast cancer cells. (D, E) The siAhr inhibited the increase in Cyp1a1 mRNA and protein induced by Flavipin (200 μmol/L). (F, G) Comparison of Cyp1a1 gene expression in response to TCDD or Flavipin in MDA-MB-231 and T47D cells. (H) Flavipin induced Ahr/Arnt complex. Immunoprecipitation (IP) using Ahr antibodies was performed and precipitated Ahr and Arnt were detected by western blot. Data are shown as mean ± SD from representative experiment studied in triplicates. *P<0.05.</p

Molecular docking of Flavipin with Ahr PAS-A and PAS-B domain.

<p>Molecular docking of Flavipin with Ahr PAS-A and PAS-B domain.</p