Efficacy of fucoxanthin extract from Sargassum horneri on 3T3-L1 pre-adipocyte differentiation.

Obesity, a chronic disease characterized by excessive body fat accumulation, is associated with significant health risks. The state of being overweight or obese leads to a number of chronic diseases, including cardiovascular disease, type 2 diabetes, cancer, and osteoarthritis. Accordingly, the regulation of adipocyte proliferation and differentiation has been the focus of many studies. The goal of the present study was to investigate the function of fucoxanthin, extracted from Sargassum horneri, in adipocyte (3T3-L1 cells) differentiation. A quantitative real-time polymerase chain reaction was conducted to investigate the mRNA expression levels of adipocyte differentiation-related genes under fucoxanthin stimulation. All adipocyte-related genes responded to PIC stimuli. Additionally, using western blotting, we confirmed that fucoxanthin reduced adipocyte differentiation. These results indicate that fucoxanthin extracted from Sargassum horneri can regulate adipogenesis. Further studies are needed to reveal the signaling pathways that lead to reduced adipocyte differentiation induced by fucoxanthin.

Research Note: Interactions among the MDA5, MAVS, and STING signaling pathways in chicken cells.

Innate immunity, as an organism's first line of defense, plays a crucial role in rapidly responding to and protecting the body against invading pathogens. As a cytosolic RNA sensor for viral infections, including infections caused by influenza virus, the innate immune system in chickens has 2 major pathogen-recognition receptors (PRRs): Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein 5 (MDA5). The signaling pathways activated by PRRs are complex, systemic processes that underlie the response to foreign molecules. In this study, we investigated the interactions among MDA5, mitochondrial antiviral signaling protein (MAVS), and stimulator of interferon genes (STING) signaling in chicken cells. To exclude the effects of TLR3, we transfected the clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) expression vector and TLR3-targeted gRNA plasmid into chicken DF-1 cells. We selected TLR3-knockout (KO) cell line and sequentially, we established 2 double-KO cell lines: TLR3-MAVS KO and TLR3-STING KO. After treatment with polyinosinic:polycytidylic acid (poly(I:C)), type I interferon (IFN), IFN-stimulated gene, and antiviral gene (IFN regulatory factor 7, IFNß, Mx1, and protein kinase R1) expression was not completely activated in TLR3-MAVS KO cells, whereas it was consistently upregulated in wild-type and TLR3-STING KO DF-1 cells. These results suggest that STING is not an intermediator between MDA5 and MAVS; moreover, it does not directly interact with MDA5 during innate immune activation in chicken DF-1 cells.

Production of chickens with green fluorescent protein-knockin in the Z chromosome and detection of green fluorescent protein-positive chicks in the embryonic stage.

OBJECTIVE: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which is the most efficient and reliable tool for precisely targeted modification of the genome of living cells, has generated considerable excitement for industrial applications as well as scientific research. In this study, we developed a gene-editing and detection system for chick embryo sexing during the embryonic stage. METHODS: By combining the CRISPR/Cas9 technical platform and germ cell-mediated germline transmission, we not only generated Z chromosome-targeted knockin chickens but also developed a detection system for fluorescence-positive male chicks in the embryonic stage. RESULTS: We targeted a green fluorescent protein (GFP) transgene into a specific locus on the Z chromosome of chicken primordial germ cells (PGCs), resulting in the production of ZGFP-knockin chickens. By mating ZGFP-knockin females (ZGFP/W) with wild males (Z/Z) and using a GFP detection system, we could identify chick sex, as the GFP transgene was expressed on the Z chromosome only in male offspring (ZGFP/Z) even before hatching. CONCLUSION: Our results demonstrate that the CRISPR/Cas9 technical platform with chicken PGCs facilitates the production of specific genome-edited chickens for basic research as well as practical applications.

Hydrogen peroxide-induced VCAM-1 expression in pancreatic islets and beta-cells through extracellular Ca2+ influx.

BACKGROUND: The use of porcine islets as alternatives to transplantable human islets is hampered by xenotransplant rejection. To identify molecular mechanisms that would allow subversion of xenoislet rejection, we investigated the role of H2O2 in vascular cell adhesion molecule-1 (VCAM-1) expression by porcine and mouse islets and beta-cell lines. METHODS: Porcine islets were treated with H2O2, tumor necrosis factor alpha, interferon-gamma, interleukin-1beta, and lipopolysaccharide, to assess the effects of inflammatory stimulators on VCAM-1 expression using flow cytometry. The role of Ca2+ in H2O2-induced VCAM-1 expression was investigated in beta-cell lines using an extracellular Ca2+ chelator and Ca2+-depleted media. Furthermore, H2O2-induced VCAM-1 expression was measured in beta-cells, pretreated with inhibitors of protein kinase C, phospholipase D, and phosphatidylinositol-3 kinase/Akt. Finally, H2O2-induced VCAM-1 expression was evaluated in porcine islets and rodent beta-cell lines infected with an adenovirus encoding catalase, a H2O2-removing enzyme. RESULTS: H2O2 was most potent inflammatory stimulator of VCAM-1 expression in porcine islets and had the greatest effect on VCAM-1 expression by beta-cells. Signaling pathway analysis demonstrated that extracellular Ca2+ influx was critical to H2O2-mediated VCAM-1 expression; however, protein kinase C, phospholipase D, and phosphatidylinositol-3 kinase/Akt activation were not required for VCAM-1 expression. Finally, catalase overexpression inhibited H2O2-induced VCAM-1 expression by islets and beta-cell lines. CONCLUSION: An extracellular calcium-dependent H2O2 pathway is the critical mediator of VCAM-1 expression by pancreatic islets and beta-cells. Inhibition of this pathway by catalase overexpression in donor islets can be exploited to protect against xenoislet immune responses.

Hydrogen peroxide increases human leukocyte adhesion to porcine aortic endothelial cells via NFkappaB-dependent up-regulation of VCAM-1.

Although a severe shortage of organs in transplantation can be overcome by using xenotransplantation of porcine donor organs, profound immune rejection to xenogeneic antigens remains a main obstacle. To elucidate the role of hydrogen peroxide (H(2)O(2)) on xenogeneic immune responses, we investigated its effects on porcine aortic endothelial cells (PAECs). We found that H(2)O(2) can specifically induce vascular cell adhesion molecule-1 (VCAM-1) expression on PAECs, but little on human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs). Furthermore, we further confirmed that H(2)O(2) induces activation of NFkappaB in PAECs, but not in HAECs. Interestingly, cell adhesion assay showed that U937, human promonocytic leukocyte, can adhere to PAECs in an H(2)O(2)-dependent manner and by using a neutralizing assay with anti-VCAM-1-specific antibodies, we also found that the interaction is mediated primarily by VCAM-1. Finally, we also demonstrated that up-regulation of VCAM-1 expression on PAECs by reactive oxygen species-producing HL-60, human leukemic neutrophil cells, could be significantly diminished by over-expressing an H(2)O(2)-removing catalase. In summary, our results suggest that NFkappaB-dependent porcine VCAM-1 expression by H(2)O(2) may promote interaction of human leukocyte to PAECs, and thus may play an important role on inducing xenogeneic immune responses.