Cell Press Acta Medica Okayama 2162-2531 34 2023 MicroRNA-451a inhibits gemcitabine-refractory biliary tract cancer progression by suppressing the MIF-mediated PI3K/AKT pathway 102054 EN Taisuke Obata Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Koichiro Tsutsumi Department of Gastroenterology and Hepatology, Okayama University Hospital Eijiro Ueta Department of Gastroenterology and Hepatology, Okayama University Hospital Takashi Oda Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Tatsuya Kikuchi Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Soichiro Ako Department of Gastroenterology and Hepatology, Okayama University Hospital Yuki Fujii Department of Gastroenterology and Hepatology, Okayama University Hospital Tatsuhiro Yamazaki Department of Gastroenterology and Hepatology, Okayama University Hospital Daisuke Uchida Department of Gastroenterology and Hepatology, Okayama University Hospital Kazuyuki Matsumoto Department of Gastroenterology and Hepatology, Okayama University Hospital Shigeru Horiguchi Department of Gastroenterology and Hepatology, Okayama University Hospital Hironari Kato Department of Gastroenterology and Hepatology, Okayama University Hospital Hiroyuki Okada Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Motoyuki Otsuka Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Gemcitabine is an effective chemotherapeutic agent for biliary tract cancers (BTCs), including gallbladder cancer (GBC) and cholangiocarcinoma (CCA). However, few other effective agents are currently available, particularly for GEM-refractory BTCs. We previously identified microRNA-451a (miR-451a) as a potential therapeutic target in GBC. To elucidate the antineoplastic effects of miR-451a and its underlying mechanisms, we transfected miR-451a into GBC, gemcitabine-resistant GBC (GR-GBC), and gemcitabine-resistant CCA (GR-CCA) cell lines. Furthermore, mimicking in vivo conditions, tumorigenic GBC organoids and three-dimensional (3D) cell culture systems were employed to investigate the anti-proliferative effects of miR-451a on BTCs, and its effect on stem cell properties. We found that miR-451a significantly inhibited cell proliferation, induced apoptosis, and reduced chemoresistant phenotypes, such as epithelial-mesenchymal transition, in both GBC and GR-GBC. The principal mechanism is probably the negative regulation of the phosphatidylinositol 3-kinase/AKT pathway, partially accomplished by directly downregulating macrophage migration inhibitory factor. The Gene Expression Omnibus database revealed that miR-451a was the most significantly downregulated microRNA in CCA tissues. The introduction of miR-451a resulted in similar antineoplastic effects in GR-CCA. Furthermore, miR-451a reduced cell viability in 3D spheroid models and tumorigenic GBC organoids. These findings suggest that the supplementation of miR-451a is a potential treatment strategy for GEM-refractory BTCs. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2211-1247 42 12 2023 Mechanistic dissection of premature translation termination induced by acidic residues-enriched nascent peptide 113569 EN Yuhei Chadani Faculty of Environmental, Life, Natural Science and Technology, Okayama University Takashi Kanamori GeneFrontier Corporation Tatsuya Niwa Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology Kazuya Ichihara Division of Biological Science, Graduate School of Science, Nagoya University Keiichi I. Nakayama Anticancer Strategies Laboratory, TMDU Advanced Research Institute, Tokyo Medical and Dental University Akinobu Matsumoto Division of Biological Science, Graduate School of Science, Nagoya University Hideki Taguchi Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology Ribosomes polymerize nascent peptides through repeated inter-subunit rearrangements between the classic and hybrid states. The peptidyl-tRNA, the intermediate species during translation elongation, stabi-lizes the translating ribosome to ensure robust continuity of elongation. However, the translation of acidic residue-rich sequences destabilizes the ribosome, leading to a stochastic premature translation cessation termed intrinsic ribosome destabilization (IRD), which is still ill-defined. Here, we dissect the molecular mechanisms underlying IRD in Escherichia coli. Reconstitution of the IRD event reveals that (1) the prolonged ribosome stalling enhances IRD-mediated translation discontinuation, (2) IRD depends on temperature, (3) the destabilized 70S ribosome complex is not necessarily split, and (4) the destabilized ribosome is subjected to peptidyl-tRNA hydrolase-mediated hydrolysis of the peptidyl-tRNA without subunit splitting or recycling factors-mediated subunit splitting. Collectively, our data indicate that the translation of acidic-rich sequences alters the conformation of the 70S ribosome to an aberrant state that allows the noncanonical pre-mature termination. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2666-1667 3 4 2022 A protocol to induce expandable limb-bud mesenchymal cells from human pluripotent stem cells 101786 EN Tomoka Takao Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Daisuke Yamada Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takeshi Takarada Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Here, we present a protocol for the selective differentiation of human pluripotent stem cells mimicking human developmental processes into expandable PRRX1+ limb-bud mesenchymal (ExpLBM) cells. This approach enables expansion through serial passage while maintaining capacity for chondrogenic differentiation. For complete details on the use and execution of this protocol, please refer to Yamada et al. (2021, 2022). No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2405-8440 9 8 2023 Bayesian statistical modeling to predict observer-specific optimal windowing parameters in magnetic resonance imaging e19038 EN Kohei Sugimoto Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masataka Oita Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masahiro Kuroda Department of Radiological Technology, Faculty of Health Sciences, Okayama University Magnetic resonance (MR) images require a process known as windowing for optimizing the display conditions. However, the conventional windowing process often fails to achieve the preferred display conditions for observers due to various factors. This study proposes a novel framework for predicting the preferred windowing parameters for each observer using Bayesian statistical modeling. MR images obtained from 1000 patients were divided into training and test sets at a 7:3 ratio. The image intensity and windowing parameters were standardized using previously reported methods. Bayesian statistical modeling was utilized to predict the windowing parameters preferred by three MR imaging (MRI) operators. The performance of the proposed framework was evaluated by assessing the mean relative error (MRE), mean absolute error (MAE), and Pearson's correlation coefficient (ρ) of the test set. In addition, the naive method, which presumes that the average value of the windowing parameters for each acquisition sequence and body region in the training set is optimal, was also used for comparison. Three MRI operators and three radiologists conducted visual assessments. The mean MRE, MAE, and ρ values for the window level and width (WL/WW) in the proposed framework were 12.6 and 13.9, 42.9 and 85.4, and 0.98 and 0.98, respectively. These results outperformed those obtained using the naive method. The visual assessments revealed no significant differences between the original and predicted display conditions, indicating that the proposed framework accurately predicts individualized windowing parameters with the additional advantages of robustness and ease of use. Thus, the proposed framework can effectively predict the windowing parameters preferred by each observer. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2666-1667 3 1 2022 Expression of microbial rhodopsins in Escherichia coli and their extraction and purification using styrene-maleic acid copolymers 101046 EN Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Microbial rhodopsins are photoreceptive membrane proteins showing various light-dependent biological activities. Styrene-maleic acid (SMA) copolymers spontaneously form nanoscale lipid particles containing membrane proteins and associated lipids without detergent, and can be used to characterize membrane molecules. Here, we provide a protocol to functionally express a thermally stable rhodopsin, Rubrobacter xylanophilus rhodopsin, and an unstable rhodopsin, Halobacterium salinarum sensory rhodopsin I, in Escherichia coli. We then describe the preparation of SMA and the extraction and purification of rhodopsin molecules using SMA. <br> For complete details on the use and execution of this protocol, please refer to Ueta et al. (2020). No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2405-8440 9 7 2023 Expression and clinicopathological characteristics of PDX1, PTF1A, and SALL4 in large and small ducts of ectopic pancreas located in gastro-duodenum and jejunum e18241 EN Mengxi Chen Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takehiro Tanaka Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takuro Igawa Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yanyan Han Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fangli Peng Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Zaishun Jin Department of Pathology, Mudanjiang Medical University Tadashi Yoshino Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences An ectopic pancreas is defined as pancreatic tissue outside its normal location, anatomically separated from the pancreas.<br> 　The transcription factor pancreas/duodenum homeobox protein 1 (PDX1) is involved in maintaining the pancreas and functions in early pancreatic development, beta cell differentiation, and endocrine non beta cells. Pancreatic transcription factor 1 subunit alpha (PTF1A) affects exocrine cell formation and regulation of acinar cell identity, and is expressed in exocrine cells as a transcription factor. The depletion of SALL4 disrupts self-renewal and induces differentiation.<br> 　To clarify which of PDX1, PTF1A, or SALL4 determines the difference in Heinrich's classification, we examined the localization and number of positive cells. We analyzed the differential expression of PDX1, PTF1A, and SALL4 in large and small ducts in ectopic pancreas by immunohistochemistry. Results showed that the number of PTF1A-positive cells in large ducts was more widespread in type I than in type II in the gastro-duodenum, and more SALL4-positive cells were noticed in large ducts than in small ducts in the gastro-duodenum of type II. Our results revealed that PTF1A might promote exocrine differentiation in developing the pancreatic tissues, and that those with widespread expression differentiate into exocrine cells. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2589-0042 26 3 2023 Muscarinic acetylcholine receptor-dependent and NMDA receptor-dependent LTP and LTD share the common AMPAR trafficking pathway 106133 EN Tomonari Sumi Research Institute for Interdisciplinary Science, Okayama University Kouji Harada Department of Computer Science and Engineering, Toyohashi University of Technology The forebrain cholinergic system promotes higher brain function in part by signaling through the M1 muscarinic acetylcholine receptor (mAChR). Long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic transmis-sion in the hippocampus are also induced by mAChR. An AMPA receptor (AMPAR) trafficking model for hippocampal neurons has been proposed to simulate N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity in the early phase. In this study, we demonstrated the validity of the hypothesis that the mAChR-dependent LTP/LTD shares a common AMPAR trafficking pathway associated with NMDAR-dependent LTP/LTD. However, unlike NMDAR, Ca2+ influx into the spine cytosol occurs owing to the Ca2+ stored inside the ER and is induced via the activation of inositol 1,4,5-trisphosphate (IP3) receptors during M1 mAChR activation. Moreover, the AMPAR trafficking model implies that alterations in LTP and LTD observed in Alzheimer's disease could be attributed to age-dependent reductions in AMPAR expression levels. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2372-7705 27 2022 Oncolytic virus-mediated p53 overexpression promotes immunogenic cell death and efficacy of PD-1 blockade in pancreatic cancer 3 13 EN Hiroyuki Araki Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Tazawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nobuhiko Kanaya Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshinori Kajiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Motohiko Yamada Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masashi Hashimoto Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Kikuchi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinji Kuroda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryuichi Yoshida Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuzo Umeda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuo Urata Oncolys BioPharma, Inc. Shunsuke Kagawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiyoshi Fujiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Immune checkpoint inhibitors, including anti-programmed cell death 1 (PD-1) antibody, provide improved clinical outcome in certain cancers. However, pancreatic ductal adeno-carcinoma (PDAC) is refractory to PD-1 blockade therapy due to poor immune response. Oncolytic virotherapy is a novel approach for inducing immunogenic cell death (ICD). We demonstrated the therapeutic potential of p53-expressing telo-merase-specific oncolytic adenovirus OBP-702 to induce ICD and anti-tumor immune responses in human PDAC cells with different p53 status (Capan-2, PK-59, PK-45H, Capan-1, MIA PaCa-2, BxPC-3) and murine PDAC cells (PAN02). OBP-702 significantly enhanced ICD with secretion of extracel-lular adenosine triphosphate and high-mobility group box pro-tein B1 by inducing p53-mediated apoptosis and autophagy. OBP-702 significantly promoted the tumor infiltration of CD8+ T cells and the anti-tumor efficacy of PD-1 blockade in a subcutaneous PAN02 syngeneic tumor model. Our results suggest that oncolytic adenovirus-mediated p53 overexpres-sion augments ICD and the efficacy of PD-1 blockade therapy against cold PDAC tumors. Further in vivo experiments would be warranted to evaluate the survival benefit of tumor-bearing mice in combination therapy with OBP-702 and PD-1 blockade. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2372-7705 25 2022 Modulation of p53 expression in cancer-associated fibroblasts prevents peritoneal metastasis of cancer 249 261 EN Toshihiro Ogawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Kikuchi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Motoyasu Tabuchi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ema Mitsui Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuta Une Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Tazawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinji Kuroda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiro Noma Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiaki Ohara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shunsuke Kagawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuo Urata Oncolys BioPharma Toshiyoshi Fujiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Cancer-associated fibroblasts (CAFs) in the tumor microenvironment are associated with the establishment and progression of peritoneal metastasis. This study investigated the efficacy of replicative oncolytic adenovirus-mediated p53 gene therapy (OBP-702) against CAFs and peritoneal metastasis of gastric cancer (GC). Higher CAF expression in the primary tumor was associated with poor prognosis of GC, and higher CAF expression was also observed with peritoneal metastasis in immunohistochemical analysis of clinical samples. And, we found transcriptional alteration of p53 in CAFs relative to normal gastric fibroblasts (NGFs). CAFs increased the secretion of cancer-promoting cytokines, including interleukin-6, and gained resistance to chemotherapy relative to NGFs. OBP-702 showed cytotoxicity to both GC cells and CAFs but not to NGFs. Overexpression of wild-type p53 by OBP-702 infection caused apoptosis and autophagy of CAFs and decreased the secretion of cancer-promoting cytokines by CAFs. Combination therapy using intraperitoneal administration of OBP-702 and paclitaxel synergistically inhibited the tumor growth of peritoneal metastases and decreased CAFs in peritoneal metastases. OBP-702, a replicative oncolytic adenovirus-mediated p53 gene therapy, offers a promising biological therapeutic strategy for peritoneal metastasis, modulating CAFs in addition to achieving tumor lysis. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2589-0042 25 3 2022 Lipid flippase dysfunction as a therapeutic target for endosomal anomalies in Alzheimer's disease 103869 EN Nanaka Kaneshiro Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masato Komai Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ryosuke Imaoka Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Atsuya Ikeda Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuji Kamikubo Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine Takashi Saito Department of Neurocognitive Science, Nagoya City University Graduate School of Medical Sciences Takaomi C. Saido Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science Taisuke Tomita Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo Tadafumi Hashimoto Department of Neuropathology, Graduate School of Medicine, The University of Tokyo Takeshi Iwatsubo Department of Neuropathology, Graduate School of Medicine, The University of Tokyo Takashi Sakurai Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine Takashi Uehara Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Endosomal anomalies because of vesicular traffic impairment have been indicated as an early pathology of Alzheimer'vertical bar disease (AD). However, the mechanisms and therapeutic targets remain unclear. We previously reported thatbCTF, one of the pathogenic metabolites of APP, interacts with TMEM30A. TMEM30A constitutes a lipid flippase with P4-ATPase and regulates vesicular trafficking through the asymmetric distribution of phospholipids. Therefore, the alteration of lipid flippase activity in AD pathology has got attention. Herein, we showed that the interaction between beta CTF and TMEM30A suppresses the physiological formation and activity of lipid flippase in AD model cells, A7, and App(NLG-F/NLG-F) model mice. Furthermore, the T-RAP peptide derived from the beta CTF binding site of TMEM30A improved endosomal anomalies, which could be a result of the restored lipid flippase activity. Our results provide insights into the mechanisms of vesicular traffic impairment and suggest a therapeutic target for AD. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2211-1247 38 5 2022 PD-1 blockade therapy promotes infiltration of tumor-attacking exhausted T cell clonotypes 110331 EN Joji Nagasaki Chiba Cancer Center, Research Institute Takashi Inozume Chiba Cancer Center, Research Institute Nicolas Sax KOTAI Biotechnologies, Inc. Ryo Ariyasu Division of Cancer Immunology, National Cancer Center, Research Institute, Exploratory Oncology Research and Clinical Trial Center (EPOC) Masakazu Ishikawa KOTAI Biotechnologies, Inc. Kazuo Yamashita KOTAI Biotechnologies, Inc. Masahito Kawazu Division of Cellular Signaling, National Cancer Center, Research Institute Toshihide Ueno Division of Cellular Signaling, National Cancer Center, Research Institute Takuma Irie Division of Cancer Immunology, National Cancer Center, Research Institute, Exploratory Oncology Research and Clinical Trial Center (EPOC) Etsuko Tanji Chiba Cancer Center, Research Institute Takao Morinaga Chiba Cancer Center, Research Institute Akiko Honobe Department of Dermatology, University of Yamanashi Takehiro Ohnuma Department of Dermatology, University of Yamanashi Mitsuru Yoshino Department of Thoracic Surgery, Chiba Cancer Center Takekazu Iwata Department of Thoracic Surgery, Chiba Cancer Center Katsushige Kawase Chiba Cancer Center, Research Institute Keita Sasaki Department of Head and Neck Surgery, Chiba Cancer Center Toyoyuki Hanazawa Department of Otolaryngology, Head and Neck Surgery, Chiba University Graduate School of Medicine Vitaly Kochin Department of Immunology, Nagoya University Graduate School of Medicine Tatsuyoshi Kawamura Department of Dermatology, University of Yamanashi Hiroyuki Matsue Department of Dermatology, Chiba University Graduate School of Medicine Masayuki Hino Department of Hematology, Graduate School of Medicine, Osaka City University Hiroyuki Mano Division of Cellular Signaling, National Cancer Center, Research Institute Yutaka Suzuki Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo Hiroyoshi Nishikawa Division of Cancer Immunology, National Cancer Center, Research Institute, Exploratory Oncology Research and Clinical Trial Center (EPOC) Yosuke Togashi Department of Tumor Microenvironment, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences PD-1 blockade exerts clinical efficacy against various types of cancer by reinvigorating T cells that directly attack tumor cells (tumor-specific T cells) in the tumor microenvironment (TME), and tumor-infiltrating lymphocytes (TILs) also comprise nonspecific bystander T cells. Here, using single-cell sequencing, we show that TILs include skewed T cell clonotypes, which are characterized by exhaustion (T-ex) or nonexhaustion signatures (Tnon-ex). Among skewed clonotypes, those in the T-ex, but not those in the Tnon-ex, cluster respond to autologous tumor cell lines. After PD-1 blockade, non-preexisting tumor-specific clonotypes in the T-ex cluster appear in the TME. Tumor-draining lymph nodes (TDLNs) without metastasis harbor a considerable number of such clonotypes, whereas these clonotypes are rarely detected in peripheral blood. We propose that tumor-infiltrating skewed T cell clonotypes with an exhausted phenotype directly attack tumor cells and that PD-1 blockade can promote infiltration of such T-ex clonotypes, mainly from TDLNs. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2589-0042 23 10 2020 Degradation of Mutant Protein Aggregates within the Endoplasmic Reticulum of Vasopressin Neurons 101648 EN Takashi Miyata Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Daisuke Hagiwara Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Yuichi Hodai Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Tsutomu Miwata Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Yohei Kawaguchi Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Junki Kurimoto Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Hajime Ozaki Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Kazuki Mitsumoto Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Hiroshi Takagi Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Hidetaka Suga Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Tomoko Kobayashi Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Mariko Sugiyama Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Takeshi Onoue Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Yoshihiro Ito Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Shintaro Iwama Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Ryoichi Banno Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Mami Matsumoto Section of Electron Microscopy, Supportive Center for Brain Research, National Institute for Physiological Sciences Natsuko Kawakami Section of Electron Microscopy, Supportive Center for Brain Research, National Institute for Physiological Sciences Nobuhiko Ohno Department of Anatomy, Division of Histology and Cell Biology, Jichi Medical University, School of Medicine Hirotaka Sakamoto Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University Hiroshi Arima Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Misfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2372-7705 18 2020 Elimination of MYCN-Amplified Neuroblastoma Cells by Telomerase-Targeted Oncolytic Virus via MYCN Suppression 14 23 EN Terutaka Tanimoto Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Tazawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takeshi Ieda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Nouso Department of Pediatric Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Morimichi Tani Department of Pediatric Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takanori Oyama Department of Pediatric Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuo Urata Oncolys BioPharma, Inc. Shunsuke Kagawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takuo Noda Department of Pediatric Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiyoshi Fujiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. High-risk NB is characterized by MYCN amplification and human telomerase reverse transcriptase (hTERT) rearrangement, contributing to hTERT activation and a poor outcome. For targeting hTERT-activated tumors, we developed two oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702, in which the hTERT promoter drives expression of the viral E1 gene for tumor-specific virus replication. In this study, we demonstrate the therapeutic potential of the hTERT-driven oncolytic adenoviruses OBP-301 and OBP-702 using four human MYCN-amplified NB cell lines (IMR-32, CHP-134, NB-1, LA-N-5) exhibiting high hTERT expression. OBP-301 and OBP-702 exhibited a strong antitumor effect in association with autophagy in NB cells. Virus-mediated activation of E2F1 protein suppressed MYCN expression. OBP-301 and OBP-702 significantly suppressed the growth of subcutaneous CHP-134 tumors. Thus, these hTERT-driven oncolytic adenoviruses are promising antitumor agents for eliminating MYCN-amplified NB cells via E2F1-mediated suppression of MYCN protein. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2372-7705 18 2020 Boosting Replication and Penetration of Oncolytic Adenovirus by Paclitaxel Eradicate Peritoneal Metastasis of Gastric Cancer 262 271 EN Wataru Ishikawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Kikuchi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshihiro Ogawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Motoyasu Tabuchi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Tazawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinji Kuroda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiro Noma Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiko Nishizaki Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shunsuke Kagawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuo Urata Oncolys BioPharma, Inc. Toshiyoshi Fujiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Peritoneal metastasis is the most frequent form of distant metastasis and recurrence in gastric cancer, and the prognosis is extremely poor due to the resistance of systemic chemotherapy. Here, we demonstrate that intraperitoneal (i.p.) administration of a green fluorescence protein (GFP)-expressing attenuated adenovirus with oncolytic potency (OBP-401) synergistically suppressed the peritoneal metastasis of gastric cancer in combination with paclitaxel (PTX). OBP-401 synergistically suppressed the viability of human gastric cancer cells in combination with PTX. PTX enhanced the antitumor effect of OBP-401 due to enhanced viral replication in cancer cells. The combination therapy increased induction of mitotic catastrophe, resulting in accelerated autophagy and apoptosis. Peritoneally disseminated nodules were selectively visualized as GFP-positive spots by i.p. administration of OBP-401 in an orthotopic human gastric cancer peritoneal dissemination model. PTX enhanced the deep penetration of OBP-401 into the disseminated nodules. Moreover, a non-invasive in vivo imaging system demonstrated that the combination therapy of i.p. OBP-401 administration with PTX significantly inhibited growth of peritoneal metastatic tumors and the amount of malignant ascites. i.p. virotherapy with PTX may be a promising treatment strategy for the peritoneal metastasis of gastric cancer. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2666-1667 1 2 2020 Protocol for Genome Editing to Produce Multiple Mutants in Wheat 100053 EN Fumitaka Abe Division of Basic Research, Institute of Crop Science, NARO Yuji Ishida Plant Innovation Center, Japan Tobacco Inc. Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Masaki Endo Division of Applied Genetics, Institute of Agrobiological Sciences, NARO Toshihiko Komari Plant Innovation Center, Japan Tobacco Inc. Seiichi Toki Division of Applied Genetics, Institute of Agrobiological Sciences, NARO Kazuhiro Sato Institute of Plant Science and Resources, Okayama University Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat (Triticum aestivum) cv. Fielder. Using Agrobacterium-delivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated. The technique for acceleration of generation advancement with embryo culture reduces time for mutant production. The mutants produced by this protocol can be used for the analysis of gene function and crop improvement. For complete details on the use and execution of this protocol, please refer to Abe et al. (2019). No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2666-1667 1 1 2020 Highly Efficient CRISPR-Associated Protein 9 Ribonucleoprotein-Based Genome Editing in Euglena gracilis 100023 EN Toshihisa Nomura RIKEN Center for Sustainable Resource Science Mizuki Yoshikawa RIKEN Baton Zone Program Kengo Suzuki RIKEN Baton Zone Program Keiichi Mochida Institute of Plant Science and Resources, Okayama University Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, and biofuels. However, targeted mutagenesis in this species has been a long-standing challenge. We recently developed a transgene-free, highly efficient, genome editing method for E. gracilis using CRISPR/Cas9 ribonucleoproteins (RNPs). Our method achieved mutagenesis rates of approximately 80% or more through an electroporation-based direct delivery of Cas9 RNPs. Therefore, this method is suitable for basic research and industrial applications, such as the breeding of Euglena. For complete details on the use and execution of this protocol, please refer to Nomura et al. (2019). No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 25890042 23 6 2020 BATTLE: Genetically Engineered Strategies for Split-Tunable Allocation of Multiple Transgenes in the Nervous System 101248 EN Keigo Kohara Department of Cellular and Functional Biology, Institute of Biomedical Science, Kansai Medical University Akitoshi Inoue Department of Medical Chemistry, Kansai Medical University, Graduate School of Medicine Yousuke Nakano Department of Anatomy, Kansai Medical University, Graduate School of Medicine Hirokazu Hirai Department of Neurophysiology and Neural Repair, Gunma University Graduate School of Medicine Takuya Kobayashi Department of Medical Chemistry, Kansai Medical University, Graduate School of Medicine, Hirakata Masato Maruyama Faculty of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ryosuke Baba Department of Cellular and Functional Biology, Institute of Biomedical Science, Kansai Medical University Chiho Kawashima Department of Cellular and Functional Biology, Institute of Biomedical Science, Kansai Medical University Elucidating fine architectures and functions of cellular and synaptic connections requires development of new flexible methods. Here, we created a concept called the “battle of transgenes,” based on which we generated strategies using genetically engineered battles of multiple recombinases. The strategies enabled split-tunable allocation of multiple transgenes. We demonstrated the versatility of these strategies and technologies in inducing strong and multi-sparse allocations of multiple transgenes. Furthermore, the combination of our transgenic strategy and expansion microscopy enabled three-dimensional high-resolution imaging of whole synaptic structures in the hippocampus with simultaneous visualizations of endogenous synaptic proteins. These strategies and technologies based on the battle of genes may accelerate the analysis of whole synaptic and cellular connections in diverse life science fields. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2589-0042 23 6 2020 Genetic Factors Associated with Heading Responses Revealed by Field Evaluation of 274 Barley Accessions for 20 Seasons 101146 EN Kazuhiro Sato Institute of Plant Science and Resources, Okayama University Makoto Ishii Institute of Plant Science and Resources, Okayama University Kotaro Takahagi RIKEN Center for Sustainable Resource Science Komaki Inoue RIKEN Center for Sustainable Resource Science Minami Shimizu RIKEN Center for Sustainable Resource Science Yukiko Uehara-Yamaguchi RIKEN Center for Sustainable Resource Science Ryuei Nishii School of Information and Data Sciences, Nagasaki University Keiichi Mochida Institute of Plant Science and Resources, Okayama University Heading time is a key trait in cereals affecting the maturation period for optimal grain filling before harvest. Here, we aimed to understand the factors controlling heading time in barley (Hordeum vulgare). We characterized a set of 274 barley accessions collected worldwide by planting them for 20 seasons under different environmental conditions at the same location in Kurashiki, Japan. We examined interactions among accessions, known genetic factors, and an environmental factor to determine the factors controlling heading response. Locally adapted accessions have been selected for genetic factors that stabilize heading responses appropriate for barley cultivation, and these accessions show stable heading responses even under varying environmental conditions. We identified vernalization requirement and PPD-H1 haplotype as major stabilizing mechanisms of the heading response for regional adaptation in Kurashiki. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2589-0042 23 6 2020 Histidine-Rich Glycoprotein Inhibits High-Mobility Group Box-1-Mediated Pathways in Vascular Endothelial Cells through CLEC-1A 101180 EN Shangze Gao Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hidenori Wake Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology,Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Dengli Wang Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youhei Takahashi Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kiyoshi Teshigawara Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hui Zhong Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shuji Mori Department of Pharmacology, School of Pharmacy, Shujitsu University Keyue Liu Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideo Takahashi Department of Pharmacology, Faculty of Medicine, Kindai University Masahiro Nishibori Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences High-mobility group box-1 (HMGB1) protein has been postulated to play a pathogenic role in severe sepsis. Histidine-rich glycoprotein (HRG), a 75 kDa plasma protein, was demonstrated to improve the survival rate of septic mice through the regulation of neutrophils and endothelium barrier function. As the relalionship of HRG and HMGB1 remains poorly understood, we investigated the effects of HRG on HMGB1-mediated pathway in endothelial cells, focusing on the involvement of specific receptors for HRG. HRC potently inhibited the HMGB1 mobilization and effectively suppressed rHMGB1-induced inflammatory responses and expression of all three HMGB1 receptors in endothelial cells. Moreover, we first clarified that these protective effects of HRG on endothelial cells were mediated through C-type lectin domain family 1 member A (CLEC-1A) receptor. Thus, current study elueiates protective effects of HRG on vascular endothelial cells through inhintion of HMGB1-mediated pathways may contribute to the therapeutic effects of HRG on severe sepsis. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2372-7705 17 2020 Oncolytic Virus-Mediated Targeting of the ERK Signaling Pathway Inhibits Invasive Propensity in Human Pancreatic Cancer 107 117 EN Takeshi Koujima Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Tazawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takeshi Ieda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroyuki Araki Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takuro Fushimi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryohei Shoji Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinji Kuroda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Kikuchi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryuichi Yoshida Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuzo Umeda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fuminori Teraishi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuo Urata Oncolys BioPharma Hiroyuki Mizuguchi Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences Toshiyoshi Fujiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Pancreatic ductal adenocarcinoma (PDAC) cells have an exceptional ability to invade nerves through pronounced crosstalk between nerves and cancer cells; however, the mechanism of PDAC cell invasion remains to be elucidated. Here, we demonstrate the therapeutic potential of telomerase-specific oncolytic adenoviruses, OBP -301 and tumor suppressor p53-armed OBP-702, against human PDAC cells. Highly invasive PDAC cells exhibited higher levels of phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) expression independent of KRAS expression; ERK1/2 inhibitor or small interfering RNA (siRNA) treatment significantly reduced the migration and invasion of PDAC cells, suggesting that the ERK signaling pathway is associated with the invasiveness of PDAC cells. OBP-702 infection suppressed ERK signaling and inhibited PDAC cell migration and invasion more efficiently than OBP-301. OBP-702 also effectively inhibited PDAC cell invasion even when invasiveness was enhanced by administration of motility stimulators, such as nerve and neurosecretory factors. Moreover, noninvasive whole-body imaging analyses showed that OBP-702 significantly suppressed tumor growth in an orthotopic PDAC xenograft model, although both viruses were equally effective against subcutaneous tumors, suggesting that OBP-702 can influence the orthotopic tumor microenvironment. Our data suggest that oncolytic virus-mediated disruption of ERK signaling is a promising antitumor strategy for attenuating the invasiveness of PDAC cells. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 25890042 23 4 2020 Cytotoxic T Lymphocytes Regenerated from iPS Cells Have Therapeutic Efficacy in a Patient-Derived Xenograft Solid Tumor Model 100998 EN Soki Kashima Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University Takuya Maeda Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University Kyoko Masuda Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University Seiji Nagano Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University Takamitsu Inoue Department of Urology, Akita University Graduate School of Medicine Masashi Takeda Department of Urology, Kyoto University Graduate School of Medicine Yuka Kono Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University Takashi Kobayashi Department of Urology, Kyoto University Graduate School of Medicine Shigeyoshi Saito Department of Medical Physics and Engineering, Division of Health Sciences, Osaka University Takahiro Higuchi Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hiroshi Ichise Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University Yuka Kobayashi Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University Keiko Iwaisako Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University Koji Terada Department of Biochemistry and Molecular Biology, Shiga University of Medical School Yasutoshi Agata Department of Biochemistry and Molecular Biology, Shiga University of Medical School Kazuyuki Numakura Department of Urology, Akita University Graduate School of Medicine Mitsuru Saito Department of Urology, Akita University Graduate School of Medicine Shintaro Narita Department of Urology, Akita University Graduate School of Medicine Masaki Yasukawa Department of Hematology, Clinical Immunology and Infectious Diseases, Graduate School of Medicine, Ehime University Osamu Ogawa Department of Urology, Kyoto University Graduate School of Medicine Tomonori Habuchi Department of Urology, Akita University Graduate School of Medicine Hiroshi Kawamoto Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University Current adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which cytotoxic T lymphocytes (CTLs) are regenerated from iPSCs that were originally derived from T cells and succeeded in regenerating CTLs specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. In this study, we extended our strategy to solid tumors. The regenerated WT1-specific CTLs had a strong therapeutic effect in orthotopic xenograft model using a renal cell carcinoma (RCC) cell line. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/β genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of RCC, demonstrating the feasibility of our strategy against solid tumors. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 23290501 17 2020 Hair cell transduction efficiency of single- and dual-AAV serotypes in adult murine cochleae 1167 1177 EN Ryotaro Omichi Department of Otolaryngology–Head and Neck Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hidekane Yoshimura Molecular Otolaryngology and Renal Research Laboratories, Carver College of Medicine, University of Iowa Seiji B. Shibata Molecular Otolaryngology and Renal Research Laboratories, Carver College of Medicine, University of Iowa Luk H. Vandenberghe Grousbeck Gene Therapy Center, Ocular Genomics Institute, Schepens Eye Research Institute and Mass Eye and Ear Richard J.H. Smith Molecular Otolaryngology and Renal Research Laboratories, Carver College of Medicine, University of Iowa Gene delivery is a key component for the treatment of genetic hearing loss. To date, a myriad of adeno-associated virus (AAV) serotypes and surgical approaches have been employed to deliver transgenes to cochlear hair cells, but the efficacy of dual transduction remains unclear. Herein, we investigated cellular tropism of single injections of AAV serotype 1 (AAV1), AAV2, AAV8, AAV9, and Anc80L65, and quantitated dual-vector co-transduction rates following co-injection of AAV2 and AAV9 vectors in adult murine cochlea. We used the combined round window membrane and canal fenestration (RWM+CF) injection technique for vector delivery. Single AAV2 injections were most robust and transduced 96.7% ± 1.1% of inner hair cells (IHCs) and 83.9% ± 2.0% of outer hair cells (OHCs) throughout the cochlea without causing hearing impairment or hair cell loss. Dual AAV2 injection co-transduced 96.9% ± 1.7% of IHCs and 65.6% ± 8.95% of OHCs. Together, RWM+CF-injected single or dual AAV2 provides the highest auditory hair cell transduction efficiency of the AAV serotypes we studied. These findings broaden the application of cochlear gene therapy targeting hair cells. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2589-0042 21 2019 2-Methylthio Conversion of N6-Isopentenyladenosine in Mitochondrial tRNAs by CDK5RAP1 Promotes the Maintenance of Glioma-Initiating Cells 42 56 EN Takahiro Yamamoto Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University Atsushi Fujimura Neutron Therapy Research Center, Okayama University Fan-Yan Wei Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University Naoki Shinojima Department of Neurosurgery, Faculty of Life Sciences, Kumamoto University Jun-Ichiro Kuroda Department of Neurosurgery, Faculty of Life Sciences, Kumamoto University Akitake Mukasa Department of Neurosurgery, Faculty of Life Sciences, Kumamoto University Kazuhito Tomizawa Neutron Therapy Research Center, Okayama University 2-Methylthio-N-6-isopentenyl modification of adenosine (ms(2)i(6)A) is an evolutionally conserved modification found in mitochondrial (mt)-tRNAs. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) specifically converts N6-isopentenyladenosine (i(6)A) to ms(2)i(6)A at position A37 of four mt-DNA-encoded tRNAs, and the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Here, we report that the ms 2 conversion mediated by CDK5RAP1 in mt-tRNAs is required to sustain glioma-initiating cell (GIC)-related traits. CDK5RAP1 maintained the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs. This regulation was not related to the translational control of mt-proteins. CDK5RAP1 abrogated the antitumor effect of i(6)A by converting i(6)A to ms (2)i(6) A and protected GICs from excessive autophagy triggered by i(6)A. The elevated activity of CDK5RAP1 contributed to the amelioration of the tumor-suppressive effect of i(6)A and promoted GIC maintenance. This work demonstrates that CDK5RAP1 is crucial for the detoxification of endogenous i(6)A and that GICs readily utilize this mechanism for survival. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 15250016 28 3 2020 Immune Modulation by Telomerase-Specific Oncolytic Adenovirus Synergistically Enhances Antitumor Efficacy with Anti-PD1 Antibody 794 804 EN Nobuhiko Kanaya Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinji Kuroda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshihiko Kakiuchi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kento Kumon Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomoko Tsumura Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masashi Hashimoto Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiaki Morihiro Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tetsushi Kubota Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Katsuyuki Aoyama Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Kikuchi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiko Nishizaki Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shunsuke Kagawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Tazawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroyuki Mizuguchi Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University Yasuo Urata Oncolys BioPharma Toshiyoshi Fujiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The clinical benefit of monotherapy involving immune checkpoint inhibitors (ICIs) such as anti-programmed death-1 antibody (PD-1 Ab) is limited to small populations. We previously developed a telomerase-specific oncolytic adenovirus, Telomelysin (OBP-301), the safety of which was confirmed in a phase I clinical study. Here, we examined the potential of OBP-502, an OBP-301 variant, as an agent for inducing immunogenic cell death (ICD) and synergistically enhancing the efficacy of OBP-502 with PD-1 Ab using CT26 murine colon cancer and PAN02 murine pancreatic cancer cell lines. OBP-502 induced the release of ICD molecules such as adenosine triphosphate (ATP) and high-mobility group box protein 1 (HMGB1) from CT26 and PAN02 cells, leading to recruitment of CD8-positive lymphocytes and inhibition of Foxp3-positive lymphocyte infiltration into tumors. Combination therapy involving OBP-502 intratumoral administration and PD-1 Ab systemic administration significantly suppressed the growth of not only OBP-502-treated tumors but also tumors not treated with OBP-502 (so-called abscopal effect) in CT26 and PAN02 bilateral subcutaneous tumor models, in which active recruitment of CD8-positve lymphocytes was observed even in tumors not treated with OBP-502. This combined efficacy was similar to that observed in a CT26 rectal orthotopic tumor model involving liver metastases. In conclusion, telomerase-specific oncolytic adenoviruses are promising candidates for combined therapies with ICIs. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 25890042 23 2 2020 PLOD2 is essential to functional activation of integrin β1 for invasion/metastasis in head and neck squamous cell carcinomas. 100850 EN Yushi Ueki Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Ken Saito Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Hidekazu Iioka Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Izumi Sakamoto ACROSCALE Inc. Yasuhiro Kanda Department of Immunology, Niigata University Graduate School of Medical and Dental Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Arata Horii Department of Otolaryngology, Head and Neck Surgery, Niigata University Graduate School of Medical and Dental Sciences Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin β1, enabling integrin β1 to initiate tumor invasion/metastasis. Integrin β1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis in vivo, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin β1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin β1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 22111247 28 5 2019 Genome-Edited Triple-Recessive Mutation AltersSeed Dormancy in Wheat 1362 1369 EN Fumitaka Abe Division of Wheat and Barley Research, Institute of Crop Science, NARO Emdadul Haque Division of Wheat and Barley Research, Institute of Crop Science, NARO Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Tsuyoshi Tanaka Division of Basic Research, Institute of Crop Science, NARO Yoko Kamiya Kihara Institute for Biological Research, Yokohama City University Masafumi Mikami Graduate School of Nanobioscience, Yokohama City University Kanako Kawaura Kihara Institute for Biological Research, Yokohama City University Masaki Endo Division of Applied Genetics, Institute of Agrobiological Sciences, NARO Kazumitsu Onishi Department of Agro-Environmental Science, Obihiro University of Agriculture and Veterinary Medicine Takeshi Hayashi Division of Basic Research, Institute of Crop Science, NARO Kazuhiro Sato Institute of Plant Science and Resources, Okayama University 1Common wheat has three sets of sub-genomes, making mutations difficult to observe, especially for traits controlled by recessive genes. Here, we produced hexaploid wheat lines with loss of function of homeoalleles of Qsd1, which controls seed dormancy in barley, by Agrobacterium-mediated CRISPR/Cas9. Of the eight transformed wheat events produced, three independent events carrying multiple mutations in wheat Qsd1 homeoalleles were obtained. Notably, one line had mutations in every homeoallele. We crossed this plant with wild-type cultivar Fielder to generate a transgene-free triple-recessive mutant, as revealed by Mendelian segregation. The mutant showed a significantly longer seed dormancy period than wild-type, which may result in reduced pre-harvest sprouting of grains on spikes. PCR, southern blotting, and whole-genome shotgun sequencing revealed that this segregant lacked transgenes in its genomic sequence. This technique serves as a model for trait improvement in wheat, particularly for genetically recessive traits, based on locus information from diploid barley. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2329-0501 13 2019 Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors 243 252 EN Hirofumi Noguchi Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus Chika Miyagi-Shiohira Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus Yoshiki Nakashima Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus Takao Kinjo Department of Basic Laboratory Sciences, School of Health Sciences in Faculty of Medicine, University of the Ryukyus Naoya Kobayashi Okayama Saidaiji Hospital Issei Saitoh Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University Masami Watanabe Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences A. M. James Shapiro Clinical Islet Transplant Program and Department of Surgery, University of Alberta Tatsuya Kin Clinical Islet Transplant Program and Department of Surgery, University of Alberta We recently demonstrated the generation of mouse induced tissue-specific stem (iTS) cells through transient overexpression of reprogramming factors combined with tissue-specific selection. Here we induced expandable tissue-specific progenitor (iTP) cells from human pancreatic tissue through transient expression of genes encoding the reprogramming factors OCT4 (octamer-binding transcription factor 4), p53 small hairpin RNA (shRNA), SOX2 (sex-determining region Y-box 2), KLF4 (Kruppel-like factor 4), L-MYC, and LIN28. Transfection of episomal plasmid vectors into human pancreatic tissue efficiently generated iTP cells expressing genetic markers of endoderm and pancreatic progenitors. The iTP cells differentiated into insulin-producing cells more efficiently than human induced pluripotent stem cells (iPSCs). iTP cells continued to proliferate faster than pancreatic tissue cells until days 100–120 (passages 15–20). iTP cells subcutaneously inoculated into immunodeficient mice did not form teratomas. Genomic bisulfite nucleotide sequence analysis demonstrated that the OCT4 and NANOG promoters remained partially methylated in iTP cells. We compared the global gene expression profiles of iPSCs, iTP cells, and pancreatic cells (islets >80%). Microarray analyses revealed that the gene expression profiles of iTP cells were similar, but not identical, to those of iPSCs but different from those of pancreatic cells. The generation of human iTP cells may have important implications for the clinical application of stem/progenitor cells. No potential conflict of interest relevant to this article was reported.