Skin proteomic screening and functional analysis of differential proteins associated with coat color in sheep (Ovis aries).

Objective: Coat color is an important characteristic and economic trait in domestic sheep. In this study, we explored the potential mechanisms and the signaling pathways involved in coat color regulation for sheep. Methods: Isobaric tags for relative and absolute quantification (iTRAQ) technology was used to catalog global protein expression profiles in skin of sheep with black versus white coat color. Immunofluorescence was used to observe the expression localization of differential protein. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used to evaluate their role in the coat color formation of sheep. Results: A total of 136 differential proteins were obtained in different coat colors, including 101 up-regulated and 35 down-regulated. Pigmentation function entries were enriched through GO annotation. Tyrosine metabolism and platelet activation signaling pathway were extracted by KEGG analysis. APOA1 (Apolipoprotein A-1) and FGA (Fibrinogen alpha chain) were found to be critical differential proteins by the interaction of differential proteins in the direct-interaction network diagram. Strikingly, twenty candidate differential proteins were screened, from which ACTB (Beta-actin) protein showed higher expression in white sheep skin, while ALB (albumin), APOA1 MAOA (Amine oxidase) and FGA proteins showed higher expression in black sheep skin, which validated by immunofluorescence, western blot and qRT-PCR. Conclusion: Our studies identified several novel proteins that may involved in the coat color formation of sheep. The white and black sheep skin proteome profiles obtained provide a valuable resource for future research to understand the network of protein expression controlling skin physiology and melanogenesis in sheep.

Bisphenol A exposure induces apoptosis and impairs early embryonic development in Xenopus laevis.

Bisphenol A (BPA), an endocrine-disrupting chemical that is largely produced and used in the plastics industry, causes environmental pollution and is absorbed by humans through consumption of food and liquids in polycarbonate containers. BPA exerts developmental and genetic toxicities to embryos and offsprings, but the embryotoxicity mechanism of this chemical is unclear. This study aimed to explore the toxic effect of BPA on embryonic development and elucidate its toxicity mechanism. Embryos of Xenopus laevis as a model were treated with different concentrations (0.1, 1, 10, and 20 µM) of BPA at the two-cell stage to investigate the developmental toxicity of BPA. Embryonic development and behaviors were monitored 24 h-96 h of BPA exposure. BPA concentrations greater than 1 µM exerted significant teratogenic effects on the Xenopus embryos, which showed short tail axis, miscoiled guts, and bent notochord as the main malformations. The 20 µM BPA-treated embryos were seriously damaged in all aspects and exhibited deformity, impaired behavioral ability, and tissue damage. The DNA integrity and apoptosis of the Xenopus embryos were also investigated. Exposure to BPA concentrations higher than 0.1 µM significantly induced DNA damage (p < 0.05). The 10 and 20 µM BPA-treated embryos exhibited higher levels of cleaved caspase-3 protein than the control. The ratios of bax/bcl-2 mRNA were significantly higher in the 10 µM and 20 µM-treated embryos than the ratio in the control group. Overall, data indicated that BPA can delay the early development, induce DNA damage and apoptosis, and eventually cause multiple malformations in Xenopus embryos.

Growth and cardiovascular development are repressed by florfenicol exposure in early chicken embryos.

Florfenicol (FLO) is one of the most popular antibacterial drugs used in veterinary clinics and aquaculture. The drug was found to decrease the hatchability of eggs laid by treated hens in veterinary clinics and research work. However, the pathological changes in developing embryos and their cardiovascular system and the mechanism underlying FLO-induced embryonic death remain unclear. In the present study, fertilized eggs laid by hens treated with a therapeutic dose of FLO were collected and incubated. Results showed that FLO exposure repressed embryonic development and induced early embryonic death. As a result, FLO decreased the hatchability and increased the proportion of weak chicks. Moreover, FLO exposure led to embryonic lethality and inhibited the development of chick embryos as characterized by decreased weights, lagging distribution of Hamburger-Hamilton stages, and dysplastic eyes. Pathological examination indicated that FLO exposure affected the normal development of the heart in 4.5-day-old chick embryos, as characterized by shorter transverse cardiac diameter, disordered arrangement of trabecular muscles in ventricles, and reduced thickness of ventricular walls. Furthermore, FLO decreased blood vascular densities and downregulated the expression levels of key angiogenesis-related genes, including the vascular endothelial growth factor and fibroblast growth factor, in the yolk sac membrane. These findings indicated that FLO exposure restricted vascular development during early embryonic development. In summary, our data suggest that the restricted growth and abnormal cardiovascular development may be responsible for FLO-induced early embryonic death. Thus, these findings can be useful for guiding the proper use of FLO and in laying a foundation for further studies on the mechanism of FLO-induced embryonic toxicity.

iTRAQ-based proteomic analysis reveals key proteins affecting cardiac function in broilers that died of sudden death syndrome.

Sudden death syndrome (SDS), which is a cardiac-related condition commonly observed in chickens selected for rapid growth, causes significant economic losses to the global poultry industry. Its pathogenesis in broilers is poorly understood, and little is known about the proteome of the heart tissue of SDS broilers. A quantitative proteomic approach using isobaric tags for relative and absolute quantification labeling of peptides was used to characterize the protein expression profiles in the left ventricle of SDS broilers. These proteins were further analyzed by bioinformatics, and two proteins were validated by western blot analysis. We identified 186 differentially expressed proteins (DEPs), of which 72 were upregulated, and 114 were downregulated in the SDS group. Functional annotation suggested that 7 DEPs were related to cardiac muscle contraction, and another 7 DEPs were related to cardiac energy metabolism. Protein interaction network predictions indicated that differences in cardiac muscle contraction between SDS and healthy groups were regulated by troponin T, tropomyosin alpha-1 chain, fast myosin heavy chain HCIII, myosin-1B, coronin, and myoglobin, whereas differences in cardiac energy metabolism and biosynthesis of amino acids were regulated by gamma-enolase, phosphoglycerate mutase, NADH-ubiquinone oxidoreductase chain 2, serine/threonine-protein kinase, myoglobin, and alpha-amylase. Our expression profiles provide useful information and new insights into key proteins to elucidate SDS for further studies.

Cadmium induces actin cytoskeleton alterations and dysfunction in Neuro-2a cells.

Cadmium (Cd) is considered a possible etiological factor in neurodegenerative diseases. However, the exact mechanism by which Cd induces neurotoxicity is not well elucidated. In this study, Neuro-2a cells were treated with 0, 10, 20, and 40 µM cadmium chloride for 24 hours to investigate the effects of Cd on the cytoskeleton of nerve cells. MTT assay and ELISA assay were used to examine cell viability and release of lactate dehydrogenase (LDH) from cells, respectively. Results showed that Cd reduced cell viability and increased the release of LDH in a dose-dependent manner (P < 0.05). The morphology of treated cell was damaged as indicated by cell collapse and dimensionality reduction. Moreover, the axonal spines and normal features of Cd-treated neurons disappeared. We checked the ultrastructure of Neuro-2a cells and found that Cd-induced swelling, membrane damage, overflow of cytoplasm contents, and cell fragmentation. Damaged mitochondria, expanded endoplasmic reticulum, and abnormal microfilaments were found in Cd-treated cells rather than in untreated cells. Compared with the control group, the relative release of glutamate in the supernatant after Cd treatment was reduced, indicating that Cd exposure could reduce the release of glutamate by inhibiting the function of nerve-2a cells. Cd decreased the mRNA and protein expression levels of cytoskeletal proteins including DBN, SYP, and TAU, which might promote cytoskeleton alterations in Cd-treated cells. In conclusion, Cd-induced actin cytoskeleton alterations and dysfunction of cultured neurons. The results of the present study provide new insights for the investigation of Cd-induced neurotoxicity.