担当区分：筆頭著者,　責任著者   記述言語：英語  

The cell polarity regulator Crumbs3 (Crb3) promotes colon cancer cell migration and metastasis. However, the underlying mechanism of cancer cell migration regulated by Crb3 has not been fully elucidated. Here, we demonstrated that Crb3 is associated with cell migration by regulating glycosphingolipid (GSL) expression in human colon cancer cells. Crb3-knockout (KO) cells showed a remarkable increase in ganglioside GM3 (GM3) on the cell surface. Reduced migration by Crb3-KO cells was restored by forced expression of both Crb3 and Neuraminidase3 (Neu3). Immunofluorescent staining revealed that most Crb3 is colocalized with the recycling endosome marker Rab11. These findings show that Crb3 may promote colon cancer cell migration by regulating the expression of GSLs on the cell surface.

DOI： 10.1016/j.bbrc.2019.08.161

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

S100A11, a member of the S100 family of proteins, is actively secreted from pancreatic ductal adenocarcinoma (PDAC) cells. However, the role of the extracellular S100A11 in PDAC progression remains unclear. In the present study, we investigated the extracellular role of S100A11 in crosstalking between PDAC cells and surrounding fibroblasts in PDAC progression. An abundant S100A11 secreted from pancreatic cancer cells stimulated neighboring fibroblasts through receptor for advanced glycation end products (RAGE) upon S100A11 binding and was followed by not only an enhanced cancer cell motility in vitro but also an increased number of the PDAC-derived circulating tumor cells (CTCs) in vivo. Mechanistic investigation of RAGE downstream in fibroblasts revealed a novel contribution of a mitogen-activated protein kinase kinase kinase (MAPKKK), tumor progression locus 2 (TPL2), which is required for positive regulation of PDAC cell motility through induction of cyclooxygenase 2 (COX2) and its catalyzed production of prostaglandin E2 (PGE2), a strong chemoattractive fatty acid. The extracellularly released PGE2 from fibroblasts was required for the rise in cellular migration as well as infiltration of their adjacent PDAC cells in a coculture setting. Taken together, our data reveal a novel role of the secretory S100A11 in PDAC disseminative progression through activation of surrounding fibroblasts triggered by the S100A11-RAGE-TPL2-COX2 pathway. The findings of this study will contribute to the establishment of a novel therapeutic antidote to PDACs that are difficult to treat by regulating cancer-associated fibroblasts (CAFs) through targeting the identified pathway.

DOI： 10.3727/096504019X15555408784978

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes.

DOI： 10.1016/j.neo.2019.04.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The fertile stroma in pancreatic ductal adenocarcinomas (PDACs) has been suspected to greatly contribute to PDAC progression. Since the main cell constituents of the stroma are fibroblasts, there is crosstalking(s) between PDAC cells and surrounding fibroblasts in the stroma, which induces a fibroblast proliferation burst. We have reported that several malignant cancer cells including PDAC cells secrete a pronounced level of S100A11, which in turn stimulates proliferation of cancer cells via the receptor for advanced glycation end products (RAGE) in an autocrine manner. Owing to the RAGE+ expression in fibroblasts, the extracellular abundant S100A11 will affect adjacent fibroblasts. In this study, we investigated the significance of the paracrine axis of S100A11-RAGE in fibroblasts for their proliferation activity. In in vitro settings, extracellular S100A11 induced upregulation of fibroblast proliferation. Our mechanistic studies revealed that the induction is through RAGE-MyD88-mTOR-p70 S6 kinase upon S100A11 stimulation. The paracrine effect on fibroblasts is linked mainly to triggering growth but not cellular motility. Thus, the identified pathway might become a potential therapeutic target to suppress PDAC progression through preventing PDAC-associated fibroblast proliferation.

DOI： 10.3727/096504018X15433161908259

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Despite receiving rituximab-combined chemotherapy, follicular lymphoma (FL) patients often suffer tumor recurrence and understand that the cause of relapse in FL would thus significantly ameliorate the tumor therapeutics. In the present study, we show that TRA-1-60-expressing cells are a unique population in FL, converge to the conventional stem cell marker Oct3/4 and ALDH1-positive population, and resist current B-lymphoma agents. TRA-1-60 expression was observed in scattered lymphoma cells in FL tissues only as well as in resting B-lymphocytes inside germinal centers. Retrospective comparison between recurrent and cognate primary tissues showed that the number of TRA-1-60-positive cells from rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP)-treated FL had increased relative to primary tissue, a finding corroborated by assays on different rituximab-treated FL cell lines, FL-18 and DOHH2, wherein TRA-positive cell numbers increased over 10-fold compared to the untreated sample. Concordantly, scanty TRA-1-60-positive FL-18 cells implanted s.c. into mice evinced potent tumor-initiating capacity in vivo, where tumors were 12-fold larger in volume (P = 0.0021 < 0.005) and 13-fold heavier in weight (P = 0.0015 < 0.005) compared to those xenografted from TRA-negative cells. To explain these results, gene expression profiling and qPCR analysis indicated that TRA-1-60-positive cells defined a distinct population from that of TRA-negative cells, with upregulation of multiple drug transporters and therapeutic resistance genes. Hence, TRA-1-60-expressing cells in FL are considered to be vigorously intractable against conventional therapeutic agents, which may explain its refractory recurrence.

DOI： 10.1111/cas.13870

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.4049/jimmunol.1700219

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記述言語：英語  

Pancreatic ductal adenocarcinoma (PDAC) is currently one of the most intractable malignancies with a typical scirrhous pattern in histology. Due to its abundant tumor stroma and scant vascularization, chemotherapeutic agents are considered inefficiently permeable to cancer nests, making it highly difficult to cure the patients with PDAC. However, PDAC is also considered to owe its intractability to other critical factors such as cellular interaction between tumor cells and tumor microenvironment as well as architectural barriers, which increases in therapeutic resistance. Here, we report a specific cellular interaction between PDAC cells and mesenchymal stem cells (MSCs) intermingled in PDAC stroma, which facilitates cancer invasion. Secretory phenotype profiling revealed that production of Amphiregulin (AREG) and MMP-3 were specifically upregulated under the coexistence of BxPC3 cells with human MSCs (approximately four to ten folds in AREG, and twenty to sixty-folds in MMP-3 compared to that of BxPC3 cells alone), whereas MMP-9 expression was decreased (less than one-tenth comparing with that of BxPC3 cells alone). Blockage of AREG production by its specific siRNA removed MSC-mediated driving force of BxPC3 invasiveness. Immunohistochemical analysis of tissue samples obtained both from PDAC patients and PDAC imitating mouse xenografted models revealed that significant coexpression of AREG and its receptor EGFR were detected on the cancer cells at invasive front. These results strongly suggested that cellular interaction between cancer cells and MSCs in the PDAC stroma might be critical to cancer progression, especially in the process of local invasion and the early stage development of metastasis.

DOI： 10.7150/jca.24415

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

S100A11, a small Ca2+ binding protein, acts extracellularly as a mediator of cancer progression. That raises the question of how a protein that lacks the classical secretory signal is able to be secreted outside cells without being damaged. Some insights into this question have been obtained, and there has been accumulating evidence indicating a pivotal role of a non-classical vesicle-mediated pathway using lysosomes or peroxisomes for the protein secretion. To obtain a more precise insight into the secretory mechanism of S100A11, we first screened representative cancer cells exhibiting significantly active secretion of S100A11. From the results of profiling, we turned our attention to aggressive cancer mesothelioma cells. In mesothelioma cells, we found that abundant dimeric S100A11 was produced selectively in the peroxisome after transportation of monomeric S100A11 through an interaction with PEX14, a peroxisome membrane protein, resulting in peroxisomal secretion of dimerized S100A11. In an extracellular environment in vitro, dimerized S100A11 promoted mesothelial cell invasion indirectly with the help of fibroblast cells. Overall, the results indicate that the peroxisome functions as an essential vesicle for the production of dimerized S100A11 and the subsequent secretion of the protein from mesothelioma cells and that peroxisome-mediated secretion of dimerized S100A11 might play a critical role in mesothelioma progression in a tumor microenvironment.

DOI： 10.1007/s12307-016-0185-2

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

p14(ARF) is one of the major tumor suppressors conventionally identified both as the mdm2-binding molecule restoring p53 function in the nucleus, and as a nucleophosmin-binding partner inside the nucleolous to stabilize ribosomal RNA. However, its recently reported mitochondrial localization has pointed to novel properties as a tumor suppressor. At the same time, functional peptides are gaining much attention in nanomedicine for their in vivo utility as non-invasive biologics. We previously reported the p14(ARF)-specific peptide that restored the sensitivity to gefitinib on the gefitinib-resistant lung cancer cells. Based on the information of this prototype peptide, here we generated the more powerful anti-tumor peptide "r9-CatB-p14 MIS," which comprises the minimal inhibitory sequence of the mitochondrial targeting p14(ARF) protein in combination with the proteolytic cleavage site for cathepsin B, which is activated in various tumor cells, fused with the nine-polyarginine-domain for cell penetration, and demonstrated its novel action of regulating mitochondrial function in accordance with localization of endogenous p14(ARF). The p14 MIS peptide showed a potent tumor inhibiton in vitro and in vivo against not only lung cancer cells but also tumor cells of diverse lineages, via modulating mitochondrial membrane potential, with minimal cytotoxicity to non-neoplastic cells and tissues. Hence, this mitochondrially targeted p14 peptide agent provides a novel basis for non-invasive peptide-based antitumor therapeutics.

DOI： 10.1111/cas.12991

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担当区分：筆頭著者  

DOI： 10.1007/978-1-4939-2730-2_6

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Coxsackie and adenovirus receptor (CAR) is essential for adenovirus infection to target cells, and its constitutive expression in various cancerous and normal tissues has been reported. Recently, the biological role of CAR in human cancers of several different origins has been investigated with respect to tumor progression, metastasis and tumorigenesis. However, its biological function in tumor cells remains controversial. Here we report the critical role of CAR in growth regulation of oral squamous cell carcinomas (SCCs) in vitro and in vivo via the specific interaction with Rho-associated protein kinase (ROCK). Loss of endogenous CAR expression by knockdown using specific small interfering RNA (siRNA) against CAR facilitates growth suppression of SCC cells due to cell dissociation, followed by apoptosis. The consequent morphological reaction was reminiscent of anoikis, rather than epithelial-mesenchymal transition, and the dissociation of oral SCC cells was triggered not by lack of contact with extracellular matrix, but by loss of cell-to-cell contact caused by abnormal translocation of E-cadherin from surface membrane to cytoplasm. Immunoprecipitation assays of the CAR-transfected oral SCC cell line, HSC-2, with or without ROCK inhibitor (Y-27632) revealed that CAR directly associates with ROCK! and ROCKII, which results in inhibition of ROCK activity and contributes to maintenance of cell-to-cell adhesion for their growth and survival. Based on these findings, in vivo behavior of CAR-downregulated HSC-2 cells from siRNA knockdown was compared with that of normally CAR-expressing cells in intraperitoneally xenografted mouse models. The mice engrafted with CAR siRNA-pretreated HSC-2 cells showed poor formation of metastatic foci in contrast to those implanted with the control siRNA-pretreated cells. Thus, CAR substantially has an impact on growth and survival of oral SCC cells as a negative regulator of ROCK in vitro and in vivo.

DOI： 10.1038/onc.2013.66

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：8  

Activation of the epidermal growth factor receptor (EGFR) has been observed in many malignant tumors and its constitutive signal transduction facilitates the proliferation of tumors. EGFR-tyrosine kinase inhibitors, such as gefitinib, are widely used as a molecular-targeting agent for the inactivation of EGFR signaling and show considerable therapeutic effect in non-small cell lung cancers harboring activating EGFR mutations. However, prolonged treatment inevitably produces tumors with additional gefitinib-resistant mutations in EGFR, which is a critical issue for current therapeutics. We aimed to characterize the distinct molecular response to gefitinib between the drug-resistant and drug-sensitive lung adenocarcinoma cells in order to learn about therapeutics based on the molecular information. From the quantitative PCR analysis, we found a specific increase in p14(ARF) expression in gefitinib-sensitive lung adenocarcinoma clones, which was absent in gefitinib-resistant clones. Moreover, mitochondria-targeted p14(ARF) triggered the most augmented apoptosis in both clones. We identified the amino acid residues spanning from 38 to 65 as a functional core of mitochondrial p14(ARF) (p14 38-65 a.a.), which reduced the mitochondrial membrane potential and caused caspase-9 activation. The synthesized peptide covering the p14 38-65 a.a. induced growth suppression of the gefitinib-resistant clones without affecting nonneoplastic cells. Notably, transduction of the minimized dose of the p14 38-65 peptide restored the response to gefitinib like that in the sensitive clones. These findings suggest that the region of p14(ARF) 38-65 a.a. is critical in the pharmacologic action of gefitinib against EGFR-mutated lung adenocarcinoma cells and has potential utility in the therapeutics of gefitinib-resistant cancers.

DOI： 10.1158/1535-7163.MCT-12-1239

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

The diverse localization of transcripts in cells suggests that there are many specific RNA-protein interactions that have yet to be identified. Progress has been limited, however, by the lack of a robust method to detect and isolate the RNA-binding proteins. Here we describe the use of an RNA aptamer, scaffolded to a tRNA, to create an affinity matrix that efficiently pulls down transcript-specific RNA-binding proteins from cell lysates. The addition of the tRNA scaffold to a Streptavidin aptamer (tRSA) increased binding efficiency by similar to 10-fold. The tRSA system with an attached G-quartet sequence also could efficiently and specifically capture endogenous Fragile X Mental Retardation Protein (FMRP), which recognizes this RNA sequence. An alternative method, using biotinylated RNA, captured FMRP less efficiently than did our tRSA method. Finally we demonstrate the identification of novel RNA-binding proteins that interact with intron2 or 3&apos;-UTR of the polarity protein Crumbs3 transcript. Proteins captured by these RNA sequences attached to the tRNA scaffold were identified by mass spectrometry. GFP-tagged versions of these proteins also showed specific interaction with either the Crb3 intron2 or 3&apos;-UTR. Our tRSA technique should find wide application in mapping the RNA-protein interactome.

DOI： 10.1093/nar/gkq1316

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記述言語：英語   出版者・発行元：NATURE PUBLISHING GROUP  

Translation of localized mRnas is an important mechanism for controlling spatially discrete cellular processes. The polarity protein Par-3 is locally translated in axons in response to factors such as NGF and netrin-1, and this increased expression is necessary for factor-stimulated axonal outgrowth.

DOI： 10.1038/ncb0809-919

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The Wnt family of secreted ligands plays critical roles during embryonic development and tumorigenesis. Here we show that Kaiso, a dual specific DNA-binding protein, functions as a bimodal regulator of canonical Wnt signaling. Loss-of-function analysis of Kaiso abrogated Wnt-mediated reporter activity and axis duplication, whereas gain-of-function analysis of Kaiso dose-dependently resulted in synergistic and suppressive effects. Our analyses further suggest Kaiso can regulate TCF/LEF1-activity for these effects via modulating HDAC1 and beta-catenin-complex formation. Our studies together provide insights into why Kaiso null mice display resistance to intestinal tumors when crossed onto an Apc(Min/+) background. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2009.01.012

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Rab GTPases are key regulators of intracellular membrane trafficking. We sought to elucidate the roles of Rab GTPases in Xenopus gastrulation, and found that a Xenopus homolog of Rab40 (XRab40) is required for normal gastrulation. XRab40 is localized at the Golgi apparatus and interacts with ElonginB/C and Cullin5 to form a ubiquitin ligase. XRab40/XCullin5 functions cooperatively and regulates the ubiquitination and localization of Rap2 GTPase. Furthermore, XRab40/XCullin5 regulates the membrane localization of Dishevelled (Dsh), a key signaling molecule in the Wnt pathway, through Rap2 and its effector Misshapen/Nck-interacting kinase (XMINK). XMINK interacts with Dsh, and is translocated to the plasma membrane by Wnt activation. We propose a novel signaling cascade consisting of XRab40/XCullin5, Rap2 and XMINK, which plays a crucial role in the regulation of the noncanonical Wnt pathway.

DOI： 10.1038/sj.emboj.7601781

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Gastrulation movements are critical for establishing the three germ layers and the architecture of vertebrate embryos. During Xenopus laevis gastrulation, mesodermal tissue migrates on the blastocoel roof and elongates along the antero-posterior axis(1),(2). During this process, cells in the dorsal mesoderm are polarized and intercalate with each other, which is defined as convergent extension and is known to be regulated by the non-canonical Wnt pathway(3-5). Here, we show that paxillin plays an essential role in this process. Paxillin is a focal-adhesion associated protein implicated in the regulation of actin cytoskeletal organization and cell motility(6),7, but its role in Xenopus embryogenesis has not yet been clarified. We demonstrate that the Wnt pathway controls the ubiquitination and stability of paxillin, and that this regulatory mechanism is essential for convergent extension movements. We identified a RING finger protein XRNF185, which physically binds to paxillin and the proteasome. XRNF185 destabilizes paxillin at focal adhesions and promotes mesodermal cell migration during convergent extension. We propose a mechanism to regulate gastrulation movements that involves paxillin ubiquitination and stability controlled by Wnt signalling.

DOI： 10.1038/ncb1607

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROCKEFELLER UNIV PRESS  

Myristoylated alanine-rich C kinase substrate (MARCKS) is an actin-binding, membrane-associated protein expressed during Xenopus embryogenesis. We analyzed its function in cytoskeletal regulation during gastrulation. Here, we show that blockade of its function impaired morphogenetic movements, including convergent extension. MARCKS was required for control of cell morphology, motility, adhesion, protrusive activity, and cortical actin formation in embryonic cells. We also demonstrate that the noncanonical Wnt pathway promotes the formation of lamellipodia- and filopodia-like protrusions and that MARCKS is necessary for this activity. These findings show that MARCKS regulates the cortical actin formation that is requisite for dynamic morphogenetic movements.

DOI： 10.1083/jcb.200310027

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

Protein kinase C (PKC) has been implicated in the Wnt signaling pathway; however, its molecular role is poorly understood. We identified novel genes encoding delta-type PKC in the Xenopus EST databases. Loss of PKCdelta function revealed that it was essential for convergent extension during gastrulation. We then examined the relationship between PKCdelta and the Wnt pathway. PKCdelta was translocated to the plasma membrane in response to Frizzled signaling. In addition, loss of PKCdelta function inhibited the translocation of Dishevelled and the activation of c-Jun N-terminal kinase (INK) by Frizzled. Furthermore, PKCdelta formed a complex with Dishevelled, and the activation of PKCdelta by phorbol ester was sufficient for Dishevelled translocation and INK activation. Thus, PKCdelta plays an essential role in the Wnt/JNK pathway by regulating the localization and activity of Dishevelled.

DOI： 10.1101/gad.1101303

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記述言語：英語   会議種別：口頭発表（一般）  

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記述言語：日本語   会議種別：口頭発表（一般）  

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記述言語：日本語   会議種別：口頭発表（一般）  

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記述言語：英語   会議種別：口頭発表（一般）  

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記述言語：英語   会議種別：口頭発表（一般）  

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記述言語：日本語   会議種別：口頭発表（一般）  

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記述言語：日本語   会議種別：口頭発表（一般）  

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記述言語：日本語   会議種別：口頭発表（一般）  

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記述言語：日本語   会議種別：口頭発表（一般）  

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