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application/pdf, 世界自動車製造企業175社の2014年度の株価成長について時系列分析する。株価上昇率の高い企業を発見するため，我々は従来の時系列データクラスタリング手法ではなく，上昇率の違いを抽出可能なAmplitude-based クラスタリング手法を用いた。これにより，2014年度の１年間では，インドの自動車製造業において株価上昇が顕著であることを発見した。次に，企業の規模を含めて2013年度から2014年度への業界全体の変動と会社固有の局所的な変動を解析するため，収益額PLBT 及び，売上高を用いて統計的形状分析shape analysis の手法による分析を行った。2013年度から2014年度の変動においては，PLBT よりも売上高の変化率が大きく，業界全体として成長をしており，上位と下位の企業の差は縮まる傾向にあることが分かった。また，株価上昇率が高いインドの企業は，全体のフォーメーションの中での位置は2014年度においては業界平均よりも下位に属し，MULTI SUZUKI INDIA を除いては相対的に見て成長率は平均に届いていないことが分かった。2014年度の動きの特徴として，インド自動車製造企業は規模は小さいが，株価上昇率で顕著な成長変動を示したことが抽出できた。

Supplementary Figure S4: (A,B) Viable AXT cells after the treatment of chemotherapeutic agents. (C) Representative flow cytometric analysis of γ-H2AX abundance in AXT cells as described in Fig.4E. (D) Immunofluorescence analysis of γ-H2AX in AXT cells.

Supplementary Table S1. Sequences of primers and predicted product sizes for real-time RT-PCR analysis

Supplementary Information. Supplementary Materials and Methods and Supplementary Figure Legends

Supplementary movie S1

Summary Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, we developed a three-dimensional culture protocol to differentiate hypertrophic chondrocytes from induced pluripotent stem cells (iPSCs) and examine the phenotype caused by MATN3 and COL10A1 mutations. Intracellular MATN3 or COL10 retention resulted in increased ER stress markers and ER size in most mutants, but activation of the UPR was dependent on the mutation. Transcriptome analysis confirmed a UPR with wide-ranging changes in bone homeostasis, extracellular matrix composition, and lipid metabolism in the MATN3 T120M mutant, which further showed altered cellular morphology in iPSC-derived growth-plate-like structures in vivo. We then applied our in vitro model to drug testing, whereby trimethylamine N-oxide led to a reduction of ER stress and intracellular MATN3., Highlights • A new induction method enables hypertrophic chondrocyte differentiation from iPSCs • Chondrodysplasia mutants show intracellular MATN3 or COL10 accumulation • Some, but not all, mutants have ER stress with an unfolded protein response • This induction system is applicable to transcriptomic analysis and drug development, Chondrodysplasias are highly heterogeneous genetic cartilage diseases. Here, Toguchida and colleagues establish a new induction system to differentiate hypertrophic chondrocytes from iPSCs and analyze these disorders in vitro. They found that different chondrodysplasia mutants, despite mutations being in the same gene, showed varying phenotypes and transcriptomic changes. This system provides an initial platform for further investigation and drug development.

The recapitulation of bone formation via the in vitro generation of bone-like nodules is frequently used to understand bone development. However, current bone-induction techniques are slow and difficult to reproduce. Here, we report the formation of bone-like nodules within ten days, via the use of retinoic acid (RA) to induce the osteogenic differentiation of human induced pluripotent stem cells (hiPSCs) into osteoblast-like and osteocyte-like cells that create human bone tissue when implanted in calvarial defects in mice. We also show that the induction of bone formation depends on cell signalling through the RA receptors RARα and RARβ, which simultaneously activate the BMP (bone morphogenetic protein) and Wnt signalling pathways. Moreover, by using patient-derived hiPSCs, the bone-like nodules recapitulated the osteogenesis-imperfecta phenotype, which was rescued via the correction of disease-causing mutations and partially by an mTOR (mechanistic target of rapamycin) inhibitor. The method of inducing bone nodules may serve as a fast and reproducible model for the study of the formation of both healthy and pathological bone. A fast in vitro model of the formation of bone-like nodules, enabled by the retinoic-acid-mediated induction of the osteogenic differentiation of patient-derived induced pluripotent stem cells, recapitulates the osteogenesis-imperfecta phenotype.

Aim Pre-administration screening of active infections is imperative for the safe use of biological disease-modifying antirheumatic drugs (bDMARDs) in patients with rheumatoid arthritis (RA). However, a standardized screening method is lacking. We therefore implemented a novel systematic screening method with a simple predetermined questionnaire on infections and assessed its effectiveness. Methods We retrospectively reviewed medical records of individuals for whom intravenous bDMARDs were administered for RA from January 2016 to April 2019. We evaluated the performance of the new screening method based on physicians' assessments. In addition, a survey was administered to nurses, regarding their assessment of the usefulness of this new screening. The incidence of infections was also assessed. Results A total of 1636 cases underwent this new screening. The new screening method showed high sensitivity (0.97) and specificity (0.89) with a negative predictive value of 99.9%, as determined based on the physician's decision. Administration of bDMARDs was postponed in 37 (2.5%) patients, and there was only one case in which the screening failed to note an active infection. The nurses' survey demonstrated high agreement (87.5%) about the usefulness of this screening on the grounds of clarity, simplicity, ease, and time-saving effects. There was no significant increase in infections after implementation of this method. Conclusions Systematic screening with a predetermined simple questionnaire is effective as an infection screening method, with a high negative predictive value. This approach contributes to high satisfaction of nurses and a time-efficient practice by focusing on screen-positive cases without increasing infections.

SummaryChondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitablein vitrosystem has been reported to analyze the pathology in humans. Here, utilizing human chondrodysplasia-specific iPSCs, we examined the UPR caused by mutations inMATN3orCOL10A1. In growth plate-like structures formed from iPSC-derived sclerotomein vivo, the hypertrophic zone was disrupted, and induced hypertrophic chondrocytesin vitroshowed varying levels of ER stress depending on the mutation. Autophagy inducers and chemical chaperones succeeded in reducing ER stress only in some mutants, while transcriptome analysis revealed many mutation-specific changes in genes involved in apoptosis, metabolism, and protein trafficking. In this way, our system has allowed the precise evaluation of the UPR caused by each mutation, opening up new avenues for treatment of individual chondrodysplasia patients.

application/pdf, 研究報告

Osteoarthritis is a leading cause of pain and joint immobility, the incidence of which is increasing worldwide. Currently, total joint replacement is the only treatment for end-stage disease. Scaffold-based tissue engineering is a promising alternative approach for joint repair but is subject to limitations such as poor cytocompatibility and degradation-associated toxicity. To overcome these limitations, a completely scaffold-free Kenzan method for bio-3D printing was used to fabricate cartilage constructs feasible for repairing large chondral defects. Human induced pluripotent stem cell (iPSC)-derived neural crest cells with high potential to undergo chondrogenesis through mesenchymal stem cell differentiation were used to fabricate the cartilage. Unified, self-sufficient, and functional cartilaginous constructs up to 6 cm2 in size were assembled by optimizing fabrication time during chondrogenic induction. Maturation for 3 weeks facilitated the self-organisation of the cells, which improved the construct’s mechanical strength (compressive and tensile properties) and induced changes in glycosaminoglycan and type II collagen expression, resulting in improved tissue function. The compressive modulus of the construct reached the native cartilage range of 0.88 MPa in the 5th week of maturation. This paper reports the fabrication of anatomically sized and shaped cartilage constructs, achieved by combining novel iPSCs and bio-3D printers using a Kenzan needle array technology, which may facilitate chondral resurfacing of articular cartilage defects.

application/pdf, 研究報告

application/pdf, 研究報告

Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed moremore » than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly different in response to the induction of SYT–SSX, and more than half of SYT–SSX target genes in hPSCs were not induced in hMSCs. These results suggest the importance of cellular context for correct understanding of SYT–SSX function, and demonstrated how our new system will help to overcome this issue.« less

application/pdf, 我々は2011年3月の東日本大震災によって引き起こされた経済活動における事象の記録，震災という未曾有の経済的混乱の状況下での被害状況および復興のようすを，若い世代に伝えたいと考える。その一環として，本稿では意外な株価上昇の原因をwebマイニングにより探るケーススタディについて述べる。震災後，ほとんどの企業が株価下落となったが，大幅に株価が上昇した企業もあった。また，震災の影響でマイナス要因が多いのも関わらず，株価が上昇した企業もあった。我々は，そのような事例として，仙台市の酒のやまや，オリエンタルランドを例にとり，その株価上昇の原因をwebテキストマイニングによって分析した。

The prevalence and specificity of unique fusion oncogenes are high in a number of soft tissue sarcomas (STSs). The close relationship between fusion genes and clinicopathological features suggests that a correlation may exist between the function of fusion proteins and cellular context of the cell-of-origin of each tumor. However, most STSs are origin-unknown tumors and this issue has not yet been investigated in detail. In the present study, we examined the effects of the cellular context on the function of the synovial sarcoma (SS)-specific fusion protein, SS18-SSX, using human pluripotent stem cells (hPSCs) containing the drug-inducible SS18-SSX gene. We selected the neural crest cell (NCC) lineage for the first trial of this system, induced SS18-SSX at various differentiation stages from PSCs to NCC-derived mesenchymal stromal cells (MSCs), and compared its biological effects on each cell type. We found that the expression of FZD10, identified as an SS-specific gene, was induced by SS18-SSX at the PSC and NCC stages, but not at the MSC stage. This stage-specific induction of FZD10 correlated with stage-specific changes in histone marks associated with the FZD10 locus and also with the loss of the BAF47 protein, a member of the SWI/SNF chromatin-remodeling complex. Furthermore, the global gene expression profile of hPSC-derived NCCs was the closest to that of SS cell lines after the induction of SS18- SSX. These results clearly demonstrated that the cellular context is an important factor in the function of SS18-SSX as an epigenetic modifier.

Osteosarcoma is a malignant bone tumor in children and adolescents characterized by intrinsic therapeutic resistance. The IGF2 is expressed at elevated levels in osteosarcoma after treatment with chemotherapy, prompting an examination of its functional contributions to resistance. We found that continuous exposure to IGF2 or insulin in the absence of serum created a dormant growth state in osteosarcoma cells that conferred resistance to various chemotherapeutic drugs in vitro. Mechanistic investigations revealed that this dormant state correlated with downregulation of downstream signaling by the IGF1 receptor, heightened cell survival, enhanced autophagy, and the presence of extracellular glutamine. Notably, inhibiting autophagy or depleting glutamine was sufficient to increase chemotherapeutic sensitivity in osteosarcoma xenografts in mice. Clinically, we confirmed that IGF expression levels were elevated in human osteosarcoma specimens from patients who received chemotherapy. Together, our results suggest that activation of IGF or insulin signaling preserves the survival of osteosarcoma cells under chemotherapeutic stress, providing a drug-resistant population that may engender minimal residual disease. Attenuating this survival mechanism may help overcome therapeutic resistance in osteosarcoma. Cancer Res; 74(22); 6531–41. ©2014 AACR.

Somatic mutations in the isocitrate dehydrogenase (IDH)1/2 genes endow encoding proteins with neomorphic activity to produce the potential oncometabolite, 2-hydroxyglutarate (2-HG), which induces the hypermethylation of histones and DNA. The incidence of IDH1/2 mutations in cartilaginous tumors was previously shown to be the highest among various types of tumors, except for those in the central nervous system. Mutations have been detected in both benign (enchondromas) and malignant (chondrosarcomas) types of cartilaginous tumors, whereas they have rarely been found in other mesenchymal tumors such as osteosarcomas. To address this unique tumor specificity, we herein examined the effects of IDH1 R132C, which is the most prevalent mutant in cartilaginous tumors, on the differentiation properties of human mesenchymal stem cells (hMSCs). The induction of the IDH1 R132C gene into MSCs markedly increased the amount of 2-HG and up-regulated global histone methylation. The induction of IDH1 R132C promoted the chondrogenic differentiation of hMSCs by enhancing the expression of SOX9 and COL2A1 genes in association with an increase in the active mark (H3K4me3), but disrupted cartilage matrix formation. On the other hand, IDH1 R132C inhibited expression of the ALPL gene in association with an increase in the repressive mark (H3K9me3), and subsequently inhibited the osteogenic properties of hMSCs and human osteosarcoma cells. Since osteogenic properties are an indispensable feature for the diagnosis of osteosarcoma, the inhibitory effects of IDH1 R132C on osteogenic properties may contribute to the lack of osteosarcomas with the IDH1 R132C mutation. These results suggested that IDH1 R132C contributed to the formation of cartilaginous tumors by dysregulating the chondrogenic and osteogenic differentiation of hMSCs via gene-specific histone modulation.