Direct electrochemistry of hemoglobin in dimethyldioctadecyl ammonium bromide film and its electrocatalysis to nitric oxide

Hemoglobin (Hb) was successfully immobilized in dimethyldioctadecyl ammonium bromide (DOAB) film at pyrolytic graphite (PG) electrode. Electrochemical experiments revealed that Hb in DOAB film exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about -0.160 V versus saturated calomel electrode (SCE) in pH 5.0 buffer, characteristic of the heme Fe(III)/Fe(II) redox couple of Hb. The electron transfer (eT) rate between Hb and the PG electrode was 0.10 s(-1). Positions of the Soret absorbance band indicated that the Hb retained its secondary structure and was similar to its native state. Furthermore, the Hb in DOAB film acted as a biological catalyst towards the reduction of nitric oxide (NO). The voltammetric response of NO at the Hb-DOAB modified electrode could be used to determine the concentration of NO in solution.     

Twin peaks of in-hospital mortality among patients with STEMI across five phases of COVID-19 outbreak in China: a nation-wide study

Lockdown and re-opening may become cyclical due to the recurrent waves of the COVID-19 epidemic. Few studies have examined temporal trends and determinants of in-hospital mortality among patients with ST-segment elevation myocardial infarction(STEMI), a life-threatening condition that requires emergency medical care. Using nation-wide data before, during and after the Wuhan lockdown, we aimed to depict temporal patterns and major determinants of STEMI in-hospital mortality in China across five t...     

MicroRNA‐29b regulates DNA methylation by targeting Dnmt3a/3b and Tet1/2/3 in porcine early embryo development

MicroRNA‐29b (miR‐29b) is a member of the miR‐29 family, which targets DNA methyltransferases (DNMTs) and ten eleven translocation enzymes (TETs), thereby regulating DNA methylation. However, the role of miR‐29b in porcine early embryo development has not been reported. In this study, we examined the effects of miR‐29b in porcine in vitro fertilization (IVF) embryos to investigate the mechanism by which miR‐29b regulated DNA methylation. The interference of miR‐29b by its special miRNA inhibitor significantly up‐regulated Dnmt3a/b and Tet1 but downregulated Tet2/3; meanwhile it increased DNA methylation levels of the global genome and Nanog promoter region but decreased global DNA demethylation levels. The inhibition of miR‐29b also resulted in a decrease in the development rate and quality of blastocysts. In addition, the pluripotency genes Nanog and Sox2 were significantly downregulated, and the apoptosis genes Bax and Casp3 were upregulated, but anti‐apoptosis gene Bcl‐2 was downregulated in blastocysts. Our study indicated that miR‐29b could regulate DNA methylation mediated by miR29b‐ Dnmt3a/b – Tet1/2/3 signaling during porcine early embryo development.     

Comparative proteomics analysis reveals roles for FADD in the regulation of energy metabolism and proteolysis pathway in mouse embryonic fibroblast

Fas‐associated death domain‐containing protein (FADD) is a classical apoptotic pathway adaptor. Further studies revealed that it also plays essential roles in nonapoptotic processes, which is assumed to be regulated by its phosphorylation. However, the exact mechanisms are still poorly understood. To study the nonapoptotic effects of FADD, a comprehensive strategy of proteomics identification combined with bioinformatic analysis was undertaken to identify proteins differentially expressed in three cell lines containing FADD and its mutant, FADD‐A and FADD‐D. The cell lines were thought to bear wild‐type FADD, unphosphorylated FADD mimic and constitutive phosphorylated FADD mimic, respectively. A total of 47 proteins were identified to be significantly changed due to FADD phosphorylation. Network analysis using MetaCore^TM identified a number of changed proteins that were involved in cellular metabolic process, including lipid metabolism, fatty acid metabolism, glycolysis, and oxidative phosphorylation. The finding that FADD‐D cell line showed an increase in fatty acid oxidation argues that it could contribute to the leaner phenotype of FADD‐D mice as reported previously. In addition, six proteins related to the ubiquitin‐proteasome pathway were also specifically overexpressed in FADD‐D cell line. Finally, the c‐Myc gene represents a convergent hub lying at the center of dysregulated pathways, and was upregulated in FADD‐D cells. Taken together, these studies allowed us to conclude that impaired mitochondrial function and proteolysis might play pivotal roles in the dysfunction associated with FADD phosphorylation‐induced disorders.     

Inhibitory effects of biochanin A on titanium particle‐induced osteoclast activation and inflammatory bone resorption via NF‐κB and MAPK pathways

Revision operations have become a new issue after successful artificial joint replacements, and periprosthetic osteolysis leading to prosthetic loosening is the main cause of why the overactivation of osteoclasts (OCs) plays an important role. The effect of biochanin A (BCA) has been examined in osteoporosis, but no study on the role of BCA in prosthetic loosening osteolysis has been conducted yet. In this study, we utilised enzyme‐linked immunosorbent assay, computed tomography imaging, and histological analysis. Results showed that BCA downregulated the secretion levels of tumor necrosis factor‐α, interleukin‐1α (IL‐1α), and IL‐1β to suppress inflammatory responses. The secretion levels of receptor‐activated nuclear factor‐κB ligand, CTX‐1, and osteoclast‐associated receptor as well as Ti‐induced osteolysis were also reduced. BCA effectively inhibited osteoclastogenesis and suppressed hydroxyapatite resorption by downregulating OC‐related genes in vitro. Analysis of mechanisms indicated that BCA inhibited the signalling pathways of mitogen‐activated protein kinase (P38, extracellular signal‐regulated kinase, and c‐JUN N‐terminal kinase) and nuclear factor‐κB (inhibitor κB‐α and P65), thereby downregulating the expression of nuclear factor of activated T cell 1 and c‐Fos. In conclusion, BCA may be an alternative choice for the prevention of prosthetic loosening caused by OCs.     

Factors affecting the electrofusion of mouse and ferret oocytes with ferret somatic cells

The domestic ferret, Mustela putorius furos, holds great promise as a genetic model for human lung disease, provided that key technologies for somatic cell nuclear transfer (SCNT) are developed. In this report, we extend our understanding of SCNT in this species by defining conditions for efficient cell fusion by electrical pulse. Two experimental systems were employed in this study. First, in vivo-matured mouse oocytes and ferret somatic cells were used to establish general parameters for fusion. One fibroblast, or cumulus cell, was agglutinated to nucleate, zona pellucida-free, mouse oocytes, and subjected to an electrical pulse. Similar electrical pulse conditions were also tested with 1 or 2 somatic cells inserted into the perivitelline space (PVS) of intact mouse oocytes. The fusion rate for a single fibroblast with a zona-free oocyte was 80.2%, significantly higher (P < 0.05) than that observed for 1, or 2, fibroblasts placed in the PVS (52.0% and 63.8%, respectively). The fusion rate (44.1%) following insertion of two cumulus cells was significantly higher (P < 0.05) than that following insertion of one cumulus cell (25.1%). Second, in vitro-matured ferret oocytes were enucleated, and one to three fibroblasts or cumulus cells were inserted into the PVS. Zona pellucida-free ferret oocytes were fragile and excluded from the study. The fusion rates with two or three fibroblasts were 71.4% and 76.8%, respectively; significantly higher (P < 0.05) than that for one fibroblast (48.6%). This cell number-dependent difference in fusion efficiency was also observed with cumulus cells. Fusion-derived (ferret-ferret) NT embryos cleaved, formed blastocysts in vitro, and underwent early-stage fetal development following embryo transfer. The rate of development was cell type-independent, in contrast to the cell type-dependent differences observed in fusion efficiency. In conclusion, fibroblasts fused more efficiently than cumulus cells and the efficiency of single cell fusions was improved when two or more cells were inserted into the PVS. These studies define conditions for efficient cell fusion with ferret oocytes and should facilitate SCNT and the development of genetically defined animal models in this species.     

The rice blast fungus MoRgs1 functioning in cAMP signaling and pathogenicity is regulated by casein kinase MoCk2 phosphorylation and modulated by membrane protein MoEmc2

GTP-binding protein (G-protein) and regulator of G-protein signaling (RGS) mediated signal transduction are critical in the growth and virulence of the rice blast pathogen Magnaporthe oryzae. We have previously reported that there are eight RGS and RGS-like proteins named MoRgs1 to MoRgs8 playing distinct and shared regulatory functions in M. oryzae and that MoRgs1 has a more prominent role compared to others in the fungus. To further explore the unique regulatory mechanism of MoRgs1, we screened a M. oryzae cDNA library for genes encoding MoRgs1-interacting proteins and identified MoCkb2, one of the two regulatory subunits of the casein kinase (CK) 2 MoCk2. We found that MoCkb2 and the sole catalytic subunit MoCka1 are required for the phosphorylation of MoRgs1 at the plasma membrane (PM) and late endosome (LE). We further found that an endoplasmic reticulum (ER) membrane protein complex (EMC) subunit, MoEmc2, modulates the phosphorylation of MoRgs1 by MoCk2. Interestingly, this phosphorylation is also essential for the GTPase-activating protein (GAP) function of MoRgs1. The balance among MoRgs1, MoCk2, and MoEmc2 ensures normal operation of the G-protein MoMagA-cAMP signaling required for appressorium formation and pathogenicity of the fungus. This has been the first report that an EMC subunit is directly linked to G-protein signaling through modulation of an RGS-casein kinase interaction.     

Leptin receptor is a key gene involved in the immunopathogenesis of thyroid-associated ophthalmopathy

Thyroid-associated ophthalmopathy (TAO), the most common and severe manifestation of Graves' disease (GD), is a disfiguring and potentially blinding autoimmune disease. The high relapse rate (up to 20%) and substantial side effects of glucocorticoid treatment further decrease the life quality of TAO patients. To develop novel therapies, we amid to explore the immunopathogenesis of TAO. To identify the key immune-related genes (IRGs) in TAO, we integrated the IRG expression profiles in thyrocytes from a GD patient set (GD vs healthy control) and a TAO patient set (TAO vs GD). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) and receiver operating characteristic (ROC) curve analyses identified the leptin receptor (LEPR) gene as the key IRG in TAO immunopathogenesis. Gene set enrichment analysis (GSEA) suggested enrichment of the antigen presentation pathway in TAO patients with higher LEPR. Increased LEPR expression was validated in TAO orbital tissues, and weighted gene co-expression network analysis (WGCNA) showed that cell adhesion processes were positively correlated with LEPR. Our study revealed that LEPR is a key gene in TAO immunopathogenesis and plays different roles in thyrocytes and orbital tissues. Our findings provide new insights into diagnostic and therapeutic biomarkers for TAO.