Simple derivation of skeletal muscle from human pluripotent stem cells using temperature‐sensitive Sendai virus vector

温度感受性センダイウイルスベクターを用いて ヒトES細胞／iPS細胞から骨格筋細胞を簡便に作製する技術開発 --神経筋疾患病態モデル構築と創薬研究への利用--. 京都大学プレスリリース. 2021-09-13.Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature-sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV-Myod1), a myogenic master transcription factor. SeV-Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38℃, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre-like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell-targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human-induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future

Prediction Model of Amyotrophic Lateral Sclerosis by Deep Learning with Patient Induced Pluripotent Stem Cells

Deep LearningとALS iPS細胞を用いた疾患予測テクノロジー --人工知能のALS検知・診断への応用--. 京都大学プレスリリース. 2021-02-24.Deep learning amyotrophic lateral sclerosis by taking pictures. 京都大学プレスリリース. 2021-02-24.In amyotrophic lateral sclerosis (ALS), early diagnosis is essential for both current and potential treatments. To find a supportive approach for the diagnosis, we constructed an artificial intelligence‐based prediction model of ALS using induced pluripotent stem cells (iPSCs). Images of spinal motor neurons derived from healthy control subject and ALS patient iPSCs were analyzed by a convolutional neural network, and the algorithm achieved an area under the curve of 0.97 for classifying healthy control and ALS. This prediction model by deep learning algorithm with iPSC technology could support the diagnosis and may provide proactive treatment of ALS through future prospective research. ANN NEUROL 202

Deep Learning and ALS

In amyotrophic lateral sclerosis (ALS), early diagnosis is essential for both current and potential treatments. To find a supportive approach for the diagnosis, we constructed an artificial intelligence-based prediction model of ALS using induced pluripotent stem cells (iPSCs). Images of spinal motor neurons derived from healthy control subject and ALS patient iPSCs were analyzed by a convolutional neural network, and the algorithm achieved an area under the curve of 0.97 for classifying healthy control and ALS. This prediction model by deep learning algorithm with iPSC technology could support the diagnosis and may provide proactive treatment of ALS through future prospective research

Cellular analysis of SOD1 protein-aggregation propensity and toxicity: a case of ALS with slow progression harboring homozygous SOD1-D92G mutation

Mutations within Superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS), accounting for approximately 20% of familial cases. The pathological feature is a loss of motor neurons with enhanced formation of intracellular misfolded SOD1. Homozygous SOD1-D90A in familial ALS has been reported to show slow disease progression. Here, we reported a rare case of a slowly progressive ALS patient harboring a novel SOD1 homozygous mutation D92G (homD92G). The neuronal cell line overexpressing SOD1-D92G showed a lower ratio of the insoluble/soluble fraction of SOD1 with fine aggregates of the misfolded SOD1 and lower cellular toxicity than those overexpressing SOD1-G93A, a mutation that generally causes rapid disease progression. Next, we analyzed spinal motor neurons derived from induced pluripotent stem cells (iPSC) of a healthy control subject and ALS patients carrying SOD1-homD92G or heterozygous SOD1-L144FVX mutation. Lower levels of misfolded SOD1 and cell loss were observed in the motor neurons differentiated from patient-derived iPSCs carrying SOD1-homD92G than in those carrying SOD1-L144FVX. Taken together, SOD1-homD92G has a lower propensity to aggregate and induce cellular toxicity than SOD1-G93A or SOD1-L144FVX, and these cellular phenotypes could be associated with the clinical course of slowly progressive ALS

Degradation rate of DNA scaffolds and bone regeneration.

Scaffolds implanted into bone defect sites must achieve optimal biodegradation rates while appropriately filling the void as new bone formation progresses. We recently developed a unique biomaterial consisting of salmon deoxyribose nucleic acid (DNA) and protamine, which can be used as an osteoconductive scaffold for tissue engineering. The aim of the present study was to elucidate how the degradation rate of the scaffold affects bone regeneration. We examined the relationships between the degradation rate of salmon DNA scaffolds and new bone formation using a rat skin flank subcutaneous model and rat calvarial defect model. The degradation rates of the scaffolds were proportional to the durations of pretreatment with ultraviolet (UV) light irradiation. The biodegradation rates of the scaffolds were also dependent on the duration of UV irradiation, as tested a subcutaneous tissue implantation. Scaffolds irradiated with UV light for 0.5 h maintained gradual biodegradation of phosphate compared with scaffolds irradiated for 0 or 3 h. In the calvarial defect model, we found that new bone formation was higher in rats treated with scaffolds irradiated with UV light for 0.5 h compared with those irradiated with UV light for 0 or 3.0 h. The present results suggest that bioengineering of scaffolds for biodegradation is important to regenerate bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018.福岡歯科大学2017年

Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells

Development and practice of laboratory seismic experiment course and teaching material for understanding realistic seismic refraction survey

地球科学の現象の中でも大気海洋物理や火山分野に比べて、地震学で扱うものは視認しにくく、断層破壊や波動伝播に関する動的な実験教材は殆ど存在しない。地震の源たる断層も教科書では静的なイメージで語られがちである。我々は、実際に見て触って実感できる教材や実習が必要と考え、動的な現象としての地震像をわかりやすく伝えるための実験演習教材を開発する研究に着手した。ここでは、屈折法地震探査によって地下構造を推定する方法とその重要性について学習するための実験演習教材を紹介する。具体的には、寒天模擬地殻に力や振動を与え、波（S波）が伝播する様子を高速カメラで撮影した画像のスロー再生を実際に目で見ることで、実感をともなう理解を促しながら原理を学習する内容となっている。これまで実施した高校生向けの実習や一般公開等での展示などの実用例、およびその教育的効果について報告する。物理探査学会第132回(平成27年度春季)学術講演会（平成27年5月11日～13日, 早稲田大学西早稲田キャンパス

Working report of the combined exercise program for geological and seismological surveys

海洋研究開発機構では高校を始めとする教育機関向けに地質学実習であるSand for Students(S4S)を2005年より実施してきた。一方で、近年推進している地震発生帯研究の成果についても教育機関や中高生向けにわかりやすく伝える機会や方法について模索してきた。そして、新たに地震探査実習を考案し（桑野他、本大会地殻構造セッションにてポスター発表）、今年度S4Sとともに実施することとした。本実習は、（独）科学技術振興機構が理科教育を推進するために運営しているサイエンス・パートナーシップ・プログラムの採択校である横浜高等学校向けに実施したものである。本発表では実際の実習内容とともにその教育的効果、および課題や今後の計画についても紹介する。 　実習プログラムは7月29日（月）?8月2日（金）に実施した。それぞれ２日間の地震学実習と地質学実習を経て、最終日に生徒のとりまとめたプレゼンテーション発表までの全５日間構成となっている。地震学実習では、１日目に屈折法地震探査の実験および実習を行い、２日目に「日本列島の形成」および「海洋プレート沈み込みに伴う海溝型地震研究」をテーマとした特別セミナー、様々な海洋調査機器の見学、実際に研究で使用されている地震計の原理を紹介し、地震計や海底地震計に触れてみる体験を盛り込んだ内容とした。地質学実習は、１日目に酒匂川（丹沢）周辺で地層の観察と鉱物の採取を行い、２日目に採取した鉱物を顕微鏡で観察する内容である。 　地震探査実習では、通常、地面に震動を与えることによって生じる地下を伝わる波を地震計で計測し、記録した後、研究室などに持ち帰りデータ処理、解析をすることが一般的であり、実際の研究で実施している作業プロセスと同じ内容を行うことが多い。今回の地震探査実習では、地震波計測により地下構造が明らかにできることを直感的に理解しやすくすることをねらいとし、地殻を模した寒天２層構造を用いた実験を考えた。この実験では模擬地殻物質が透明であるため光弾性を利用することで、弾性波の伝わる様子を可視化できる。さらに寒天の横波の伝播速度は数m/sと充分遅いため観察が容易になる。この実習では、地震波が伝わる様子をその場で一目で容易に観察でき、さらに観察の様子を録画し、そのデータを画像処理することで寒天模擬地殻表面の各点での振動波形を得ることもできる。この波形データは実際の屈折法地震探査データと同様に解析できるので、これを用いて寒天２層構造の速度構造を推定する演習を行う。演習を行いながら、地震探査を実施して地殻構造を推定することが、地震の発生メカニズムの解明にとって重要であることについて理解を促す。 　地質学実習では、身近な河川の砂を採取して観察することで河川流域の地質を把握するとともに、河川流域の地質の成り立ちを学ぶことで、日本列島形成の重要なメカニズムである付加体形成についての理解を促している。これは同時に、河川を通じて深海底に運ばれる陸源物質を探す作業でもあり、いわば地殻を構成する物質循環の基礎調査に相当する。 　本実習全体のねらいは、地震および地質調査研究に関する基礎的な科学知識・技能を普及させることである。特に、地震国である日本の地で生活する上で、身につけておいてほしい知識や技能の向上、問題の認識を目的としている。今回の実習では、講義、実験、演習、地質巡検を通じて、日本列島の形成や地震研究には地質学、地震学ともに必要であること、さらには様々な学問分野の知識が地球科学にとって重要であることを実感できるような構成とした。今回2日間で実施した地震学実習全体については、今後も実習プログラムの内容の改良、発展を加える上、地震探査実習については、実験レシピを作成して公開することも目指す。P1-36ポスター要旨, 日本地震学会2013年度秋季大会（2013年10月7日～9日, 神奈川県横浜市

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Theoretical study of initial reactions of amine (CH3)(n)NH(3-n) (n=1, 2, 3) with ozone

We performed a systematic reaction path search for the initial reactions of methyl amine with ozone (O-3) in gas phase, by using a single-component artificial force induced reaction (SC-AFIR) method. The reaction rate constants calculated by UCCSD(T) energies and UM062X frequencies, agreed well with the experimental observation. This agreement could support the proposed reaction pathway for H-abstraction from amine as an initial step. The high rate constant for the reaction of trimethyl amine with O-3 could be explained by a formation of the cyclic transition state structure