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Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR （微量組織検体中の体細胞変異の検出を可能にする迅速デジタルPCR解析法の開発）
Get PDFIt is challenging to secure a cytopathologic diagnosis using minute amounts of tumor fluids and tissue fragments. Hence, we developed a rapid, accurate, low-cost method for detecting tumor cell-derived DNA from limited amounts of specimens and samples with a low tumor cellularity, to detect KRAS mutations in pancreatic ductal carcinomas (PDA) using digital PCR (dPCR). The core invention is based on the suspension of tumor samples in pure water, which causes an osmotic burst; the crude suspension could be directly subjected to emulsion PCR in the platform. We examined the feasibility of this process using needle aspirates from surgically resected pancreatic tumor specimens (n = 12). We successfully amplified and detected mutant KRAS in 11 of 12 tumor samples harboring the mutation; the positive mutation frequency was as low as 0.8%. We used residual specimens from fine-needle aspiration/biopsy and needle flush processes (n = 10) for method validation. In 9 of 10 oncogenic KRAS pancreatic tumor samples, the "water-burst" method resulted in a positive mutation call. We describe a dPCR-based, super-sensitive screening protocol for determining KRAS mutation availability using tiny needle aspirates from PDAs processed using simple steps. This method might enable pathologists to secure a more accurate, minimally invasive diagnosis using minute tissue fragments.博士（医学）旭川医科大
Pericyte-specific deletion of Ninjurin1 induces fragile vasa vasorum formation and enhances intimal hyperplasia of injured vasculature （周細胞特異的Ninjurin1欠損は傷害血管において血管外膜毛細血管を脆弱化し、内膜肥厚を増悪させる）
Get PDFAdventitial abnormalities including enhanced vasa vasorum malformation are associated with development and vulnerability of atherosclerotic plaque. However, the mechanisms of vasa vasorum malformation and its role in vascular remodeling have not been fully clarified. We recently reported that ninjurin-1 (Ninj1) is a crucial adhesion molecule for pericytes to form matured neovessels. The purpose is to examine if Ninj1 regulates adventitial angiogenesis and affects the vascular remodeling of injured vessels using pericyte-specific Ninj1 deletion mouse model. Mouse femoral arteries were injured by insertion of coiled wire. Four weeks after vascular injury, fixed arteries were decolorized. Vascular remodeling, including intimal hyperplasia and adventitial microvessel formation were estimated in a three-dimensional view. Vascular fragility, including blood leakiness was estimated by extravasation of fluorescein isothiocyanate (FITC)-lectin or FITC-dextran from microvessels. Ninj1 expression was increased in pericytes in response to vascular injury. NG2-CreER/Ninj1loxp mice were treated with tamoxifen (Tam) to induce deletion of Ninj1 in pericyte (Ninj1 KO). Tam-treated NG2-CreER or Tam-nontreated NG2-CreER/Ninj1loxp mice were used as controls. Intimal hyperplasia was significantly enhanced in Ninj1 KO compared with controls. Vascular leakiness was significantly enhanced in Ninj1 KO. In Ninj1 KO, the number of infiltrated macrophages in adventitia was increased, along with the expression of inflammatory cytokines. In conclusion, deletion of Ninj1 in pericytes induces the immature vasa vasorum formation of injured vasculature and exacerbates adventitial inflammation and intimal hyperplasia. Thus, Ninj1 contributes to the vasa vasorum maturation in response to vascular injury and to reduction of vascular remodeling.NEW & NOTEWORTHY Although abnormalities of adventitial vasa vasorum are associated with vascular remodeling such as atherosclerosis, the mechanisms of vasa vasorum malformation and its role in vascular remodeling have not been fully clarified. The present study provides a line of novel evidence that ninjurin-1 contributes to adventitial microvascular maturation during vascular injury and regulates vascular remodeling.博士（医学）旭川医科大
Sarcopenia-derived exosomal micro-RNA 16-5p disturbes cardio-repair via a pro-apoptotic mechanism in myocardial infarction in mice （サルコペニア誘導性エクソソーム マイクロRNA 16-5p はマウスの心筋梗塞におけるアポトーシス促進を介して心修復を阻害する）
Get PDFSarcopenia is a pathophysiological malfunction induced by skeletal muscle atrophy. Several studies reported an association between sarcopenia-induced cardiac cachexia and poor prognosis in heart disease. However, due to lack of an established animal models, the underlying mechanism of disturbed cardiac repair accompanied with sarcopenia remains poorly understood. Here, we developed a novel sarcopenia-induced cardiac repair disturbance mouse model induced by tail suspension (TS) after cardiac ischemia and reperfusion (I/R). Importantly, we identified a specific exosomal-microRNA marker, miR-16-5p, in the circulating exosomes of I/R-TS mice. Of note, sarcopenia after I/R disturbed cardiac repair and raised the level of circulating-exosomal-miR-16-5p secreting from both the atrophic limbs and heart of TS mice. Likewise, miR-16-5p mimic plasmid disturbed cardiac repair in I/R mice directly. Additionally, in neonatal rat ventricular myocytes (NRVMs) cultured in vitro under hypoxic conditions in the presence of a miR-16-5p mimic, we observed increased apoptosis through p53 and Caspase3 upregulation, and also clarified that autophagosomes were decreased in NRVMs via SESN1 transcript interference-mediated mTOR activation. In conclusion, we show the pro-apoptotic effect of sarcopenia-derived miR-16-5p, which may be behind the exacerbation of myocardial infarction. Therefore, miR-16-5p can be a novel therapeutic target in the context of cardiac repair disturbances in sarcopenia-cachexia.博士（医学）旭川医科大
