A Language Support for Exhaustive Fault-Injection in Message-Passing System Models

This paper presents an approach towards specifying and verifying adaptive distributed systems. We here take fault-handling as an example of adaptive behavior and propose a modeling language Sandal for describing fault-prone message-passing systems. One of the unique mechanisms of the language is a linguistic support for abstracting typical faults such as unexpected termination of processes and random loss of messages. The Sandal compiler translates a model into a set of NuSMV modules. During the compilation process, faults specified in the model will be woven into the output. One can thus enjoy full-automatic exhaustive fault-injection without writing faulty behaviors explicitly. We demonstrate the advantage of the language by verifying a model of the two-phase commit protocol under faulty environment.Comment: In Proceedings MOD* 2014, arXiv:1411.345

細胞膜受容体の分子挙動と細胞応答の統合的測定技術の開発

金沢大学医薬保健研究域医学系1.細胞表面受容体のオリゴマー化モニタリング系の開発細胞膜受容体であるRAGE (receptor for advanced glycation endproducts)をモデルとし、2分子FRET法により受容体オリゴマー化をモニタリングする技術を確立した。これにより、受容体の蛍光タンパク融合組換え体を作製することなくFRETでオリゴマー化をモニタリングすることが可能となった。(1)RAGE細胞外ドメインに結合し、かつRAGEとリガンドとの結合を阻害しないモノクローナル抗体を作製し、FRETのドナーとアクセプターの対をなす2種の蛍光色素で標識した。(2)RAGE過剰発現細胞株の培養液中に上記の2種の蛍光標識抗体を加え、生細胞表面のRAGEを蛍光標識し、RAGEのオリゴマー化をFRETシグナルの変化を測定することによりモニタリングした。(3)その結果、リガンド刺激後20分をピークとするFRETシグナルの上昇が検出され、本技術の有効性が示された。2.1分子FRET法による細胞内シグナルモニタリングと受容体発現量の相関関係の解析細胞内シグナル強度のモニタリングと、受容体の発現量解析を同時に行うことにより、受容体発現量とシグナル強度の相関解析が可能な系を開発した。(1)NFκBの活性化によりβ-ラクタマーゼを発現するレポーター遺伝子を組み込んだ細胞株に、RAGE発現ベクターを導入した。(2)上記細胞にβ-ラクタマーゼによる分解で分子内FRETが消失する蛍光色素分子を取り込ませ、FRETシグナルによりNFκB活性化を検出すると同時に、蛍光抗体でRAGEを標識し発現量を測定した。(3)その結果、RAGE発現量が中程度の細胞において最も強くNFκBが活性化されており、発現量が過剰な細胞ではむしろ活性化の程度が低いことが明らかとなった。研究課題/領域番号:18038017, 研究期間(年度):2006出典：「細胞膜受容体の分子挙動と細胞応答の統合的測定技術の開発」研究成果報告書　課題番号18038017（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-18038017/)を加工して作

多機能受容体RAGEによるシグナリングネットワークの解明

金沢大学医学系研究科本研究は、糖尿病血管症などの病態に重要な細胞表面受容体RAGE(receptor for advanced glycation endproducts)により活性化される細胞内シグナルの解析を通してRAGEによるシグナル生成機構を明らかにすることを目指し、当初計画に沿って遂行され、以下の成果を得た。(1)全長型RAGE発現細胞と、細胞内ドメイン欠失RAGE発現細胞をAGEによって刺激した所、全長型RAGE発現細胞でのみチロシンリン酸化が亢進するタンパク質が複数同定された。その一部は、新たなRAGE依存性チロシンリン酸化タンパク質と考えられたため、質量分析により数個の候補タンパク質を同定した。(2)各種の酵素阻害剤を用い、これらのタンパク質のチロシンリン酸化はERKの上流もしくは別個のシグナル伝達経路に位置し、チロシンホスファターゼによる制御を受けているものがあることを明らかにした。(3)RAGE発現細胞をAGEで刺激後、クロスリンカーにより細胞表面上で隣接しているタンパク質同士を架橋し、抗RAGE抗体で免疫沈降を行った。次に還元剤処理によりクロスリンカー分子内のジスルフィド結合を切断した後、ウェスタンブロットによりチロシンリン酸化タンパク質を検出した。その結果、複数のチロシンリン酸化タンパク質がRAGEと複合体を形成してることが示された。(4)上記チロシンリン酸化タンパク質の一部はAGE刺激によりチロシンリン酸化が亢進し、刺激時間依存性およびAGE濃度依存性が認められた。また、(1)で同定されたタンパク質と一致する分子量を示すものもあった。(5)全長型RAGE、またはを細胞内ドメイン欠失RAGEを発現している細胞をAGEで刺激後、既知チロシンキナーゼ型受容体のチロシンリン酸化をアッセイする抗体アレイで解析したところ、AGE刺激依存性にチロシンリン酸化が亢進する受容体が見出された。RAGE (receptor for advanced glycation endproducts) is a cell surface receptor that bind several ligands, such as AGE (advanced glycation endproducts), HMGB-1 (high molecular group box-1 protein), amyloid beta. The intracellular signals evoked by RAGE-ligand interaction were thought to play pivotal roles in development of various diseases. However, the mechanism of generation of intracellular signals by RAGE was poorly understood.In this research we revealed the presence of novel signaling pathway that is activated by the association of RAGE (receptor for advanced glycation endproducts) with its ligands. The pathway appears to involve tyrosine phosphorylation. We also found tyrosine-phosphorylated proteins that associate with RAGE on cell surface.(1) We found increased tyrosine-phosphorylation of several proteins after ligand stimulation. Their phosphorylation requires cytoplasmic domain of RAGE because the increase of tyrosine-phosphorylation was not observed in a cell line that expres s cytoplasmic domain-deleted RAGE. We identified several candidates of those proteins by mass-spectrometry.(2) We revealed that the stimulation-dependent tyrosine phosphorylation event was located upstream of ERK activation or on ERK-independent signaling pathway, using kinase inhibitors. Furthermore, tyrosine phosphorylation of a few proteins was regulated by tyrosine phosphatase.(3) We found that several tyrosine-phosphorylated proteins associated with RAGE on cell surface using cross-linking followed by immunoprecipitation.(4) We found that the tyrosine phosphorylation of the RAGE associated proteins was enhanced by ligand stimulation in dose-dependent and time-dependent manner. These proteins might be co-receptors of RAGE.(5) We identified a known whose tyrosine-phosphorylation was enhanced by RAGE ligands using antibody array that evaluate tyrosine phosphorylation of known receptor tyrosine. A few known receptor tyrosine kinases were constitutively activated by full-length RAGE or cytoplasmic domain-deleted RAGE. These receptors might be involved in RAGE dependent-signaling pathways.研究課題/領域番号:16570113, 研究期間(年度):2004-2005出典：「多機能受容体RAGEによるシグナリングネットワークの解明」研究成果報告書　課題番号16570113（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） 本文データは著者版報告書より作

新規糖化蛋白分子種による血管障害機序の解明

金沢大学医学系研究科本研究は、糖尿病性血管合併症の発生に重要と考えられている終末糖化産物(AGE, advanced glycation endproducts)とその受容体RAGE(Receptor for AGE)の結合による血管細胞障害のメカニズムについて解明することを目指し、当初計画通りに遂行され、以下の成果を得た。1.ウシ血清アルブミンをグリセルアルデヒドまたはグリコールアルデヒドとインキュベートして得られた新規AGE分子種-AGE2とAGE3-が細胞表面RAGEと強く結合することを明らかにした。2.表面プラスモン共鳴法を用いた解析により、AGE2・AGE3結合部位は、いずれもRAGEのアミノ端の免疫グロブリンV領域様ドメイン上に存在することを明らかにした。3.培養血管内皮細胞を用いた解析により、AGE2・AGE3いずれもERK1・ERK2などのMAPキナーゼ経路を活性化するとともにVCAM-1発現量を増加させることを明らかにした。4.RAGEノツクアウト糖尿病マウスでは対照糖尿病マウスに比して糖尿病性腎症の進行が有意に抑制されてることを明らかにし、RAGEが糖尿病性腎症に促進的に働いていることを示した。5.ヒト血管内皮細胞から分泌型RAGE蛋白をコードするcDNAを単離し、esRAGE(endogenous secretory RAGE)と命名した。6.esRAGEの血管細胞での役割を解析し、esRAGEは細胞外でAGE2・AGE3などのRAGEリガンドを補足することにより、血管保護作用を有することを明らかにした。7.esRAGE ELISA定量系を開発し、血中esRAGEと生活習慣病罹患との相関を解析することにより、esRAGEが生活習慣病の罹患リスクマーカーとなる可能性を示した。血中esRAGE量の生活習慣病の罹患リスクマーカーとしての臨床応用に関する特許出願を平成16年5月に予定している。In this research, we revealed that the novel species of advance glycation endproducts (AGE), whose level in serum is elevated in diabetic patients, do bind to their cellular receptor RAGE and induce intracellular signals.We also identified a novel splicing variant of RAGE-endogenous secretory RAGE (esRAGE)-and found that this variant has protective effect against vascular injury induced by the novel AGE species. Moreover, we found the significant coirelation between serwn esRAGE level and susceptibility of some life-style related diseases.(1) We demonstrated that the novel AGE species, which is generated by incubating bovine serum albumin with glyceraldehyde or glycolaldehyde, strongly binds to cellular RAGE at comparable dissociation constants calculated by the suiface plasmon resonance method.(2) We mapped the binding region of the novel AGEs on RAGE at an inununoglobulin V-domain-like region on its N-terminal end.(3) We demonstrated that the interaction of the new AGEs to RAGE activated MAP kinase signaling pathway and induced V CAM-i expression in cultured human endothelialcells.(4) We created RAGE knockout mice and demonstrated that the development of diabetic nephropa thy was dramatically suppressed in the absence of RAGE..(5) We isolated a novel splicing variant of RAGE from human endothelial cells that lacks transmembrane domain and is secreted from cells, and termed this novel variant esRAGE (~ndogenous secretory RAGE).(6) Furthennore we found that esRAGE has a protective effect against the vascular injuly induced by the novel AGEs, by extracellular capture of the RAGE lingads.(7) We established esRAGE ELISA system and analyzed serum level of esRAGE in patients with life-style related deseases. Then we found significant correlation between serum esRAGE level and susceptibility of some life-style related diseases, suggesting that. the serum esRAGE level could be a useful risk marker for some life-style related deseases.研究課題/領域番号:14580645, 研究期間(年度):2002-2003出典：「新規糖化蛋白分子種による血管障害機序の解明」研究成果報告書　課題番号14580645 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

マルチリガンド受容体RAGEによる細胞内シグナル生成機構の解明

金沢大学医学系研究科細胞表面受容体RAGEの細胞内シグナル生成に関わる分子を同定することを目的とし、研究実施計画に基づいて研究を遂行した結果、以下の成果を得た。1.RAGE分子のアスパラギン酸結合型糖鎖修飾部位に変異を導入し、糖鎖を持たないRAGE分子を細胞に発現させることにより、糖鎖のリガンド結合およびシグナル生成への関与を検討した。その結果、糖鎖の付加によって一部のリガンドとの親和性が低下し、細胞内シグナル生成も減弱していることが明らかとなった。2.RAGE分子の細胞外ドメインに対するモノクローナル抗体を蛍光色素で標識し、生細胞表面RAGEを蛍光標識することによりRAGE分子の挙動を観察した。その結果、RAGEはpinocytosisの関わる機構により恒常的に細胞内へ取り込まれていることが明らかとなった。この細胞内取り込みはリガンド刺激による影響を受けなかった。3.上記モノクローナル抗体をFRETのドナーとアクセプターの対をなす2種の蛍光色素で標識し、生細胞表面RAGE分子同士の2分子FRETを観察することにより、高分子リガンド依存性のRAGEのオリゴマー化の検出に成功した。4.RAGE分子のオリゴマー化を引き起こさないと予想される低分子量ヘパリンなどの低分子RAGEリガンドは、RAGEを介する細胞内シグナルを惹起せず、むしろ高分子リガンドによるシグナル生成を阻害する。これらの新知見から、高分子リガンドによるRAGEのオリゴマー化が細胞内シグナル生成の最初のステップであることが強く示唆され、臨床的応用が期待されるRAGE拮抗薬開発の基本原理が明らかになった。RAGE (receptor for advanced glycation endproducts) is a cell surface receptor that bind several ligands, such as AGE (advanced glycation endproducts), HMGB-1 (high mobility group box-I protein), amyloid beta. The intracellular signals evoked by RAGE-ligand interaction were thought to play pivotal roles in development of various diseases. However, the mechanism of generation of intracellular signals by RAGE was poorly understood.This study shed lights on several new aspects of RAGE signaling mechanism as follows.I. Recombinant wild-type, de-N-glycosylation and G82S RAGE proteins were produced in COS-7 cells, purified and assayed for ligand-binding abilities. De-N-glycosylation at N81 and G82S mutation decreased Kd for glycolaldehyde-derived AGE to three orders of magnitude lower levels compared with wild-type. AGE-induced upregulation of VEGF mRNA was significantly augmented in endothelial cell-derived ECV304 cells expressing de-N-glycosylated and G82S RAGE when compared with wild-type expresser.2. RAGE molecules of living cell surface were labeled by fluorescent dye-conjugated monoclonal antibody that binds to extracellular domain of RAGE (mAbl3G4), and the dynamic state of RAGE was analyzed by confocal fluorescence laser microscopy. The results indicated that RAGE molecules were constitutively internalized, and pinocytotic mechanism was, at least partly, involved in RAGE internalization. The rate of RAGE internalization was not altered by addition of RAGE ligand.3. RAGE molecules of living cell surface were labeled by mixture of mAbl3G4 conjugated with Alexa 488 and mAbl3G4 with Alexa 594, which are a pair of donor and acceptor for FRET (fluorescence resonance energy transfer), to detect oligomerization of RAGE molecule by FRET. The results indicated that the RAGE molecule oligomerized upon binding its ligand.4. Low molecular weight RAGE ligands such as low molecular weight heparin, which are speculated not to induce the RAGE oligomerization, did not evoke RAGE-mediated intracellular signal, and inhibited the signaling by high molecular weight ligands.These new findings strongly suggested that the oligomerization is the first step of the signal generation, and revealed the principle of the development of RAGE antagonists that should be promising candidates of the preventive medicine for RAGE-related diseases.研究課題/領域番号:18590260, 研究期間(年度):2006-2007出典：「マルチリガンド受容体RAGEによる細胞内シグナル生成機構の解明」研究成果報告書　課題番号18590260（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-18590260/185902602007kenkyu_seika_hokoku_gaiyo/)を加工して作

OstemiR: A Novel Panel of MicroRNA Biomarkers in Osteoblastic and Osteocytic Differentiation from Mesencymal Stem Cells

MicroRNAs (miRNAs) are small RNA molecules of 21–25 nucleotides that regulate cell behavior through inhibition of translation from mRNA to protein, promotion of mRNA degradation and control of gene transcription. In this study, we investigated the miRNA expression signatures of cell cultures undergoing osteoblastic and osteocytic differentiation from mesenchymal stem cells (MSC) using mouse MSC line KUSA-A1 and human MSCs. Ninety types of miRNA were quantified during osteoblastic/osteocytic differentiation in KUSA-A1 cells utilizing miRNA PCR arrays. Coincidently with mRNA induction of the osteoblastic and osteocytic markers, the expression levels of several dozen miRNAs including miR-30 family, let-7 family, miR-21, miR-16, miR-155, miR-322 and Snord85 were changed during the differentiation process. These miRNAs were predicted to recognize osteogenic differentiation-, stemness-, epinegetics-, and cell cycle-related mRNAs, and were thus designated OstemiR. Among those OstemiR, the miR-30 family was classified into miR-30b/c and miR-30a/d/e groups on the basis of expression patterns during osteogenesis as well as mature miRNA structures. In silico prediction and subsequent qRT-PCR in stable miR-30d transfectants clarified that context-dependent targeting of miR-30d on known regulators of bone formation including osteopontin/spp1, lifr, ccn2/ctgf, ccn1/cyr61, runx2, sox9 as well as novel key factors including lin28a, hnrnpa3, hspa5/grp78, eed and pcgf5. In addition, knockdown of human OstemiR miR-541 increased Osteopontin/SPP1 expression and calcification in hMSC osteoblastic differentiation, indicating that miR-541 is a negative regulator of osteoblastic differentiation. These observations indicate stage-specific roles of OstemiR especially miR-541 and the miR-30 family on novel targets in osteogenesis

OstemiR: A Novel Panel of MicroRNA Biomarkers in Osteoblastic and Osteocytic Differentiation from Mesencymal Stem Cells

　MicroRNAs (miRNAs) are small RNA molecules of 21–25 nucleotides that regulate cell behavior through inhibition of translation from mRNA to protein, promotion of mRNA degradation and control of gene transcription. In this study, we investigated the miRNA expression signatures of cell cultures undergoing osteoblastic and osteocytic differentiation from mesenchymal stem cells (MSC) using mouse MSC line KUSA-A1 and human MSCs. Ninety types of miRNA were quantified during osteoblastic/osteocytic differentiation in KUSA-A1 cells utilizing miRNA PCR arrays. Coincidently with mRNA induction of the osteoblastic and osteocytic markers, the expression levels of several dozen miRNAs including miR-30 family, let-7 family, miR-21, miR-16, miR-155, miR-322 and Snord85 were changed during the differentiation process. These miRNAs were predicted to recognize osteogenic differentiation-, stemness-, epinegetics-, and cell cycle-related mRNAs, and were thus designated OstemiR. Among those OstemiR, the miR-30 family was classified into miR-30b/c and miR-30a/d/e groups on the basis of expression patterns during osteogenesis as well as mature miRNA structures. In silico prediction and subsequent qRT-PCR in stable miR-30d transfectants clarified that context-dependent targeting of miR-30d on known regulators of bone formation including osteopontin/spp1, lifr, ccn2/ctgf, ccn1/cyr61, runx2, sox9 as well as novel key factors including lin28a, hnrnpa3, hspa5/grp78, eed and pcgf5. In addition, knockdown of human OstemiR miR-541 increased Osteopontin/SPP1 expression and calcification in hMSC osteoblastic differentiation, indicating that miR-541 is a negative regulator of osteoblastic differentiation. These observations indicate stage-specific roles of OstemiR especially miR-541 and the miR-30 family on novel targets in osteogenesis

Plasma Nitridation of 4H-SiC by Glow Discharge of N2/H2 Mixed Gases

The mixed gas of nitrogen and hydrogen was used for the plasma nitridation of SiC surface.A small amount of hydrogen was effective to activate the nitridation reaction and suppress the oxidationreaction. The interface properties were improved by using nitride layer as an interfacial bufferlayer of SiC MIS structure.ArticleMaterials Science Forum, Vols. 821-823, pp. 504-507 (2015)journal articl

Preparation and Characterization of Nitridation Layer on 4H SiC (0001) Surface by Direct Plasma Nitridation

A nitride layer was formed on a SiC surface by plasma nitridation using pure nitrogen as the reaction gas at the temperature from 800°C to 1400°C. The surface was characterized by XPS. The XPS measurement showed that an oxinitride layer was formed on the SiC surface by the plasma nitridation. The high process temperature seemed to be effective to activate the niridation reaction. A SiO2 film was deposited on the nitridation layer to form SiO2/nitride/SiC structure. The interface state density of the SiO2/nitride/SiC structure was lower than that of the SiO2/SiC structure. This suggested that the nitridation was effective to improve the interface property.ArticleMaterials Science Forum, Vols. 778-780, pp. 631-634 (2014)journal articl

Epidemiology of plasmid-mediated quinolone resistance in salmonella enterica serovar typhimurium isolates from food-producing animals in Japan

A total of 225 isolates of Salmonella enterica serovar Typhimurium from food-producing animals collected between 2003 and 2007 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants, namely qnrA, qnrB, qnrC, qnrD, qnrS, qepA and aac(6')Ib-cr, in Japan. Two isolates (0.8%) of S. Typhimurium DT104 from different dairy cows on a single farm in 2006 and 2007 were found to have qnrS1 on a plasmid of approximately 9.6-kbp. None of the S. Typhimurium isolates had qnrA, qnrB, qnrC, qnrD, qepA and acc(6')-Ib-cr. Currently in Japan, the prevalence of the PMQR genes among S. Typhimurium isolates from food animals may remain low or restricted. The PFGE profile of two S. Typhimurium DT104 isolates without qnrS1 on the farm in 2005 had an identical PFGE profile to those of two S. Typhimurium DT104 isolates with qnrS1. The PFGE analysis suggested that the already existing S. Typhimurium DT104 on the farm fortuitously acquired the qnrS1 plasmid