<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

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

One year follow-up after a randomized controlled trial of a 130 g/day low-carbohydrate diet in patients with type 2 diabetes mellitus and poor glycemic control

<div><p>Background & aims</p><p>Recently, we conducted a prospective randomized controlled trial (RCT) showing that a 6-month 130g/day low-carbohydrate diet (LCD) reduced HbA1c and BMI more than a calorie restricted diet (CRD). [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188892#pone.0188892.ref001" target="_blank">1</a>] To assess whether the benefits of the LCD persisted after the intensive intervention, we compared HbA1c and BMI between the LCD and CRD groups at 1 year after the end of the 6-month RCT.</p><p>Methods</p><p>Following the end of the 6-month RCT, patients were allowed to manage their own diets with periodic outpatient visits. One year later, we analyzed clinical and nutrition data.</p><p>Results</p><p>Of the 66 participants in the original study, 27 in the CRD group and 22 in the LCD group completed this trial. One year after the end of the original RCT, the carbohydrate intake was comparable between the groups (215 [189–243]/day in the CRD group and 214 (176–262) g/day in the LCD group). Compared with the baseline data, HbA1c and BMI were decreased in both groups (CRD: HbA1c -0.4 [-0.9 to 0.3] % and BMI -0.63 [-1.20 to 0.18] kg/m²; LCD: HbA1c -0.35 [-1.0 to 0.35] % and BMI -0.77 [-1.15 to -0.12] kg/m²). There were no significant differences in HbA1c and BMI between the groups.</p><p>Conclusions</p><p>One year after the diet therapy intervention, the beneficial effect of the LCD on reduction of HbA1c and BMI did not persist in comparison with CRD. However, combining the data of both groups, significant improvements in HbA1c and BMI from baseline were observed. Although the superiority of the LCD disappeared 1 year after the intensive intervention, these data suggest that well-constructed nutrition therapy programs, both CRD and LCD, were equally effective in improving HbA1c for at least 1 year.</p><p>Trial registration</p><p>University Hospital Medical Information Network (UMIN) <a href="https://clinicaltrials.gov/ct2/show/ID000010663" target="_blank">ID000010663</a></p></div

Data_Sheet_1_Efficacy and safety of single-dose ivermectin in mild-to-moderate COVID-19: the double-blind, randomized, placebo-controlled CORVETTE-01 trial.docx

BackgroundTo investigate whether ivermectin inhibits SARS-CoV-2 proliferation in patients with mild-to-moderate COVID-19 using time to a negative COVID-19 reverse transcription-polymerase chain reaction (RT-PCR) test.MethodsCORVETTE-01 was a double-blind, randomized, placebo-controlled study (August 2020–October 2021) conducted in Japan. Overall, 248 patients diagnosed with COVID-19 using RT-PCR were assessed for eligibility. A single oral dose of ivermectin (200  μg/kg) or placebo was administered under fasting. The primary outcome was time to a negative COVID-19 RT-PCR test result for SARS-CoV-2 nucleic acid, assessed using stratified log-rank test and Cox regression models.ResultsOverall, 112 and 109 patients were randomized to ivermectin and placebo, respectively; 106 patients from each group were included in the full analysis set (male [%], mean age: 68.9%, 47.9 years [ivermectin]; 62.3%, 47.5 years [placebo]). No significant difference was observed in the occurrence of negative RT-PCR tests between the groups (hazard ratio, 0.96; 95% confidence interval [CI] 0.70–1.32; p = 0.785). Median (95% CI) time to a negative RT-PCR test was 14.0 (13.0–16.0) and 14.0 (12.0–16.0) days for ivermectin and placebo, respectively; 82.1% and 84% of patients achieved negative RT-PCR tests, respectively.ConclusionIn patients with COVID-19, single-dose ivermectin was ineffective in decreasing the time to a negative RT-PCR test.Clinical Trial RegistrationClinicalTrials.gov, NCT04703205.</p