<b>Establishment of Pancreatic Beta Cell-specific Gene Knockout System Based on CRISPR-Cas9 Technology with AAV8-mediated gRNA Delivery</b>

The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout, including pancreatic beta cells associated with the pathogenesis of diabetes mellitus. However, it is expensive and time-consuming to generate transgenic mice harboring floxed genes of interest and cross them with cell-specific Cre-expression mice. We establish a βCas9 system with mice expressing Cas9 in pancreatic beta cells and adeno-associated virus 8 (AAV8) -mediated gRNA delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-LoxP-Stop-LoxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucose-tolerant βCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in beta cells. We also show significant beta cell-specific gene knockout efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administer AAV8 to βCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4 (MODY4). As reported previously, we demonstrate that those mice show glucose intolerance with trans-differentiation of Pdx1 knockout beta cells into glucagon-expressing cells. We successfully generate a convenient beta cell-specific gene knockout system with βCas9 mice and AAV8-mediated gRNA delivery.ARTICLE HIGHLIGHTS・ Generating pancreatic beta cell-specific gene knockout mice without time- and money-consuming breeding could be beneficial.・ We establish a convenient βCas9 system, which enables the beta cell-specific gene knockout with mice expressing Cas9 in pancreatic beta cells and adeno-associated virus 8-mediated gRNA delivery by simple intraperitoneal injection.・ A fluorescent reporter-based estimation shows the high efficiency of gene knockout in the βCas9 system.・ Beta cell-specific Pdx1 knockout in the βCas9 system validates the concept of our procedure by demonstrating glucose intolerance and trans-differentiation.</p

Effects of Interfacial Interactions on Electrocatalytic Activity of Cytochrome <i>c</i> Oxidase in Biomimetic Lipid Membranes on Gold Electrodes

Effects of interfacial interactions on the electrocatalytic activity of protein-tethered bilayer lipid membranes (ptBLMs) containing cytochrome c oxidase (CcO) for the oxygen reduction reaction are studied by using protein film electrochemistry and surface-enhanced infrared absorption (SEIRA) spectroscopy. Mammalian CcO was immobilized on a gold electrode via self-assembled monolayers (SAMs) of mixed alkanethiols. The protein orientation on the electrode is controlled by SAM–CcO interactions and is critical to the cytochrome c (cyt c) binding. The CcO–phospholipid and CcO–cyt c interactions modulate the electrocatalytic activity of CcO, and more densely packed ptBLMs show higher electrocatalytic activity. Our study indicates that spectroscopic and electrochemical studies of ptBLMs can provide insights into the effects of relatively weak protein–protein and protein–lipid interactions on the enzymatic activity of transmembrane enzymes

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

<p>Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene <i>atg7</i> (<i>Atg7cKO</i>) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from <i>Atg7cKO</i> mice. In addition, <i>Atg7cKO</i> mice crossed with <i>Apoe</i> (apolipoprotein E)-deficient mice (<i>apoeKO; Atg7cKO:apoeKO</i>) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, <i>Atg7cKO: apoeKO</i> mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in <i>Atg7cKO:apoeKO</i> mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.</p

<b>Low Handgrip Strength (Possible Sarcopenia) with Insulin Resistance Is Associated with Type 2 Diabetes Mellitus, </b>Supplementary Tables.

Supplementary Table 1. The number of individuals with lipid-lowering and diabetes drugs in groups.Supplementary Table 2. Associations between the prevalence of type 2 diabetes and IR-Possible sarcopenia in subjects with IR.Supplementary Table 3. Associations between the prevalence of type 2 diabetes and IR-Sarcopenia.</p

Additional file 1 of Increased risk of cardiovascular mortality by strict glycemic control (pre-procedural HbA1c < 6.5%) in Japanese medically-treated diabetic patients following percutaneous coronary intervention: a 10-year follow-up study

Additional file 1. Additional figures and tables