Demethylation and microRNA differential expression regulate plasma-induced improvement of chicken sperm quality.

The sperm quality is a vital economical requisite of poultry production. Our previous study found non-thermal dielectric barrier discharge plasma exposure on fertilized eggs could increase the chicken growth and the male reproduction. However, it is unclear how plasma treatment regulates the reproductive capacity in male chickens. In this study, we used the optimal plasma treatment condition (2.81 W for 2 min) which has been applied on 3.5-day-incubated fertilized eggs in the previous work and investigated the reproductive performance in male chickens aged at 20 and 40 weeks. The results showed that plasma exposure increased sperm count, motility, fertility rate, and fertilization period of male chickens. The sperm quality-promoting effect of plasma treatment was regulated by the significant improvements of adenosine triphosphate production and testosterone level, and by the modulation of reactive oxygen species balance and adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathway in the spermatozoa. Additionally, the plasma effect suggested that DNA demethylation and microRNA differential expression (a total number of 39 microRNAs were up-regulated whereas 53 microRNAs down-regulated in the testis) regulated the increases of adenosine triphosphate synthesis and testosterone level for promoting the chicken sperm quality. This finding might be beneficial to elevate the fertilization rate and embryo quality for the next generation in poultry breeding.

BRM270 Inhibits the Proliferation of CD44 Positive Pancreatic Ductal Adenocarcinoma Cells via Downregulation of Sonic Hedgehog Signaling.

Pancreatic cancer has a poor survival rate as compared to other types of cancer. Surface marker CD44 plays important role in epithelial-mesenchymal transition and cancer stem cell phenotype. Therefore, targeting CD44 positive pancreatic cancer cells might enhance therapies effectiveness. Our previous studies indicated the antitumorigenesis effect of BRM270 in osteosarcoma, lung cancer, and glioblastoma; however there is no evidence on BRM270 impacts on pancreatic cancer growth. In this study, we investigated the effect of BRM270 on the isolated CD44 positive pancreatic ductal adenocarcinoma cells (CD44+ PDAC). Results showed that CD44 positive cells undergo apoptosis induced by BRM270. Moreover, BRM270 also inhibits stemness and metastasis traits in CD44+ PDAC via Sonic hedgehog signaling pathway and SALL4 expression. In vivo study indicated that tumor growth derived from CD44+ PDAC was suppressed as daily uptake by BRM270 5 mg/kg. These data suggest the alternative approach in antipancreatic tumorigenesis via herbal plants extract and selectively targeting CD44+ PDAC cells in tumor.

Synthesis and evaluation of the antiproliferative efficacy of BRM270 phytocomposite nanoparticles against human hepatoma cancer cell lines.

BRM270 is the most leading phytochemical extract that possesses potent anticancer properties. A major challenge associated with this drug is its low bioavailability and thus requires high dosages for cancer treatment. Here, we report the novel nano-synthesis of phyto-composite, BRM270 for the first time by mechanical milling method with specific modifications for enhanced cytotoxicity against HepG2 human hepatoma cancer cells. Unlike free BRM270 and other phytomedicines, BRM270 nanoparticles (BRM270 NPs) are well-dispersed and small sized (23 to 70 nm) which is believed to greatly enhanced cellular uptake. Furthermore, the acidic tumor microenvironment attracts BRM270 NPs enhancing targeted therapy while leaving normal cells less affected. The comparative cytotoxicity analysis using MTT assay among the three treatment groups, such as free BRM270, BRM270 NPs, and doxorubicin demonstrated that BRM270 NPs induced greater cytotoxicity against HepG2 cells with an effective drug concentration of 12 µg/ml. From FACS analysis, we observed an apoptotic cell death of 44.4% at BRM270 NPs treated cells while only 12.5% found in the free BRM270 treated cells. Further, the comparative relative expression profiling of the candidate genes were showed significant (p < 0.05) down-regulation of IL6, BCL2, p53, and MMP9 in the BRM270 NPs treated cells, compared to the free BRM270 and doxorubicin. Indeed, the genes, CASPASE 9 and BAX have shown significant (p < 0.05) upregulation in cells treated with BRM270 NPs as compared to counter treatment groups. The investigation of the signal pathways and protein-protein network associations were also carried out to elucidate the functional insights underlying anti-cancer potential of BRM270 NPs in HepG2 cells. Taken together, our findings demonstrated that these uniquely engineered BRM270 NPs effectively enter into the cancer cells due to its acidic microenvironment thereby inducing apoptosis and regulate the cell-proliferation in-vitro at extremely low dosages.

Specific Chicken Egg Yolk Antibody Improves the Protective Response against Gallibacterium anatis Infection.

Gallibacterium anatis is a pathogen associated with peritonitis and salpingitis in chickens and other avian species. Novel safety prevention strategies are urgently needed because of widespread multidrug resistance and antigenic diversity. The objective of this study was to produce a specific chicken egg yolk antibody and evaluate its protective response against a G. anatis infection model in 4-week-old chicks. Enzyme-linked immunosorbent assays showed that hens immunized with the recombinant N terminus of Gallibacterium toxin A (GtxA-N) had significantly increased antibody titers against GtxA-N in serum and egg yolk IgY. Western blotting showed that IgY antibody had specificity against GtxA-N in the egg yolks of immunized hens. The growth of G. anatis in brain heart infusion (BHI) broth and agar was significantly inhibited by the GtxA-N-specific IgY antibody. The protective effects of the specific IgY antibody were evaluated in G. anatis-infected chicks after intramuscular injection (10 mg/ml). The anti-GtxA-N antibody titers in the sera of G. anatis-challenged chicks following an injection of specific IgY antibody were significantly higher than those of the control and the nonspecific IgY groups, but lower lesion scores for the peritoneum, liver, and duodenum were found after specific IgY antibody treatment. The results from this study suggest that the GtxA-N-specific IgY antibody could potentially improve the protective response against G. anatis infection in chicks.

Innovative Approach of Non-Thermal Plasma Application for Improving the Growth Rate in Chickens.

As an innovative technology in biological applications-non-thermal plasma technique-has recently been applied to living cells and tissues. However, it is unclear whether non-thermal plasma treatment can directly regulate the growth and development of livestock. In this study, we exposed four-day-incubated fertilized eggs to plasma at 11.7 kV for 2 min, which was found to be the optimal condition in respect of highest growth rate in chickens. Interestingly, plasma-treated male chickens conspicuously grew faster than females. Plasma treatment regulated the reactive oxygen species homeostasis by controlling the mitochondrial respiratory complex activity and up-regulating the antioxidant defense system. At the same time, growth metabolism was improved due to the increase of growth hormone and insulin-like growth factor 1 and their receptors expression, and the rise of thyroid hormones and adenosine triphosphate levels through the regulation of demethylation levels of growth and hormone biosynthesis-related genes in the skeletal muscles and thyroid glands. To our knowledge, this study was the first to evaluate the effects of a non-thermal plasma treatment on the growth rate of chickens. This safe strategy might be beneficial to the livestock industry.

MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation.

Non-thermal plasma treatment is an emerging innovative technique with a wide range of biological applications. This study was conducted to investigate the effect of a non-thermal dielectric barrier discharge plasma technique on immature chicken Sertoli cell (SC) viability and the regulatory role of microRNA (miR)-7450. Results showed that plasma treatment increased SC apoptosis in a time- and dose-dependent manner. Plasma-induced SC apoptosis possibly resulted from the excess production of reactive oxygen species via the suppression of antioxidant defense systems and decreased cellular energy metabolism through the inhibition of adenosine triphosphate (ATP) release and respiratory enzyme activity in the mitochondria. In addition, plasma treatment downregulated miR-7450 expression and activated adenosine monophosphate-activated protein kinase α (AMPKα), which further inhibited mammalian target of rapamycin (mTOR) phosphorylation in SCs. A single-stranded synthetic miR-7450 antagomir disrupted mitochondrial membrane potential and decreased ATP level and mTOR phosphorylation by targeting the activation of AMPKα, which resulted in significant increases in SC lethality. A double-stranded synthetic miR-7450 agomir produced opposite effects on these parameters and ameliorated plasma-mediated apoptotic effects on SCs. Our findings suggest that miR-7450 is involved in the regulation of plasma-induced SC apoptosis through the activation of AMPKα and the further inhibition of mTOR signaling pathway.