Elsevier BV Acta Medica Okayama 0732-8893 115 3 2026 Investigation of the cefazolin inoculum effect in blood culture-isolated methicillin-susceptible Staphylococcus aureus strains: A Japanese multicenter study 117345 EN Shinnosuke Fukushima Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shuma Tsuji Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Kazuyoshi Gotoh Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Sakura Ogawa Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Norihito Koyanagi Department of Clinical Laboratory, Chutoen General Medical Center Yuji Ito Department of General Internal Medicine, Chutoen General Medical Center Hiroshi Koganemaru Department of Infectious Diseases, Tokyo Metropolitan Institute for Geriatrics and Gerontology Atsushi Yoshida Department of Infectious Diseases, Tokyo Metropolitan Institute for Geriatrics and Gerontology Hideharu Hagiya Department of Infectious Diseases, Okayama University Hospital Background: Cefazolin inoculum effect (CInE) is a microbiological phenomenon where the MIC of cefazolin against methicillin-susceptible Staphylococcus aureus (MSSA) strains increases with higher bacterial volumes.<br> Method: We retrospectively investigated the prevalence and characteristics of the CInE among MSSA strains isolated from blood cultures at three Japanese hospitals. The collected isolates were screened for blaZ using PCR, and the cefazolin minimum inhibitory concentration (MIC) for the blaZ-positive MSSA isolates was measured at standard and high inoculum volumes. CInE-positive MSSA strains were defined as those with a cefazolin MIC ≥16 μg/mL at 107 CFU/mL and ≤8 μg/mL at 105 CFU/mL. In these blaZ-positive strains, we performed blaZ typing and tested a modified nitrocefin-based rapid examination to detect the CInE.<br> Results: We collected 329 MSSA strains isolated from blood cultures. Of these, 96 (29.2%) were positive for the blaZ gene, with the following genotypes: type A (15, 15.6%), type B (3, 3.1%), type C (77, 80.2%), type D (0, 0.0%), and non-type (1, 1.0%). Among 96 blaZ-positive MSSA isolates, 11 exhibited the CInE, all of which harbored blaZ type A. The rapid nitrocefin test detected CInE positivity with high sensitivity (100%), specificity (94.1%), and diagnostic accuracy (94.8%).<br> Conclusion: This study highlighted the low prevalence of CInE-presenting MSSA isolates in Japan. When the cefazolin MIC is ≥1 μg/mL or the penicillin G MIC is ≥0.25 μg/mL, the rapid nitrocefin test may be useful for considering the CInE in patients with high bacterial volume MSSA infections. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1746-6148 22 1 2026 Genetic and phenotypic identities of Staphylococcus coagulans isolated from pustules of dogs with superficial bacterial folliculitis 98 EN Takafumi Osumi Animal Medical Center, Faculty of Agriculture, Tokyo University of Agriculture and Technology Yuuki Shinomiya Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology Thamonwan Wanganuttara Department of Bacteriology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ichiro Imanishi Kimberly and Eric J. Waldman Department of Dermatology, Icahn School of Medicine at Mount Sinai Yotaro Shimazaki Animal Medical Center, Faculty of Agriculture, Tokyo University of Agriculture and Technology Keita Iyori 1sec Co. Ltd. Yoichi Toyoda 1sec Co. Ltd. Kaori Ide Animal Medical Center, Faculty of Agriculture, Tokyo University of Agriculture and Technology Jumpei Uchiyama Department of Bacteriology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Koji Nishifuji Animal Medical Center, Faculty of Agriculture, Tokyo University of Agriculture and Technology Background Staphylococcus coagulans, formerly called Staphylococcus schleiferi subsp. coagulans is the second most common isolate from skin lesions of dogs with superficial bacterial folliculitis (SBF). However, the clinical significance of S. coagulans in pustules of canine SBF remains uncertain. This study aimed to investigate the prevalence and genotypic and phenotypic diversity of S. coagulans isolated from pustules in two dogs with SBF.<br> Results Two dogs with SBF were included in this study. S. schleiferi/coagulans was isolated as the sole organism from three pustules in case #1, whereas it coexisted with S. pseudintermedius in two of seven pustules in case #2. S. pseudintermedius was the sole organism in the remaining five pustules in case #2. Whole genome sequences revealed that all isolates tested were annotated as S. coagulans. The isolates from the same pustules exhibited identical genotypic and phenotypic profiles, indicating clonal multiplication. S. coagulans isolated from different pustules exhibited similar yet distinct genotypic and phenotypic profiles.<br> Conclusions S. coagulans with identical genetic and phenotypic profiles can be identified as the sole pathogen or coexist with S. pseudintermedius in the pustules of the same dogs with SBF. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1347-9032 2025 Genotype–Phenotype Correlations of Li–Fraumeni Syndrome in Japan Children's Cancer Group LFS20 Study Cohort EN Fumito Yamazaki Department of Pediatrics, Keio University School of Medicine Yoshiko Nakano Department of Genetic Medicine and Services, National Cancer Center Hospital Masashi Sanada Department of Advanced Diagnosis, Clinical Research Center, NHO Nagoya Medical Center Hiroki Kurahashi Division of Molecular Genetics, Center for Medical Science, Fujita Health University Shunsuke Miyai Division of Molecular Genetics, Center for Medical Science, Fujita Health University Arisa Ueki Department of Clinical Genetic Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer Research Yuko Watanabe Department of Pediatric Oncology, National Cancer Center Hospital Daisuke Hasegawa Department of Pediatrics, St. Luke's International Hospital Shuhei Karakawa Department of Pediatrics, Hiroshima University Hospital Toshifumi Ozaki Department of Orthopaedic Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Akira Hirasawa Department of Clinical Genomic Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akiko M. Saito Clinical Research Center, NHO Nagoya Medical Center Eisuke Inoue Showa Medical University Research Administration Center, Showa Medical University Motohiro Kato Department of Pediatrics, The University of Tokyo Hiroyoshi Hattori Department of Clinical Genetics, NHO Nagoya Medical Center Li–Fraumeni syndrome (LFS) is a cancer predisposition syndrome caused by germline pathogenic variants in the TP53 gene. With the increasing use of multi-gene panel testing, TP53 variants have been identified in individuals who do not meet established TP53 testing criteria, such as the Chompret criteria. The term “attenuated LFS” has been proposed for some of these cases, particularly those with adult-onset cancer. We analyzed participants of the Japanese nationwide prospective clinical trial of the cancer surveillance program (Japan Children's Cancer Group LFS-20), along with clinical information including their family histories, to better understand their genotypic and phenotypic characteristics. We identified 32 distinct TP53 variants from 41 families (45 participants), including four missense variants with conflicting classifications of pathogenicity in ClinVar. Among these families, 36 (88%) met the LFS criteria (hereafter referred to as “LFS” in contrast to attenuated LFS), while 5 (12%) were classified as attenuated LFS. Including 30 additional family members carrying the same variant, we analyzed 75 individuals with TP53 variants. Of these, 40 with LFS and 6 with attenuated LFS had cancer. Multiple primary cancers occurred in 22 individuals (21 LFS, 1 attenuated LFS). LFS-core tumors accounted for 66% (58/88) of cancers in the LFS group and 63% (5/8) in the attenuated LFS group; of note, all core tumors in the attenuated group were limited to breast cancer. Hotspot missense variants were detected in 11 of 36 LFS families and in none of 5 attenuated LFS families, and non-hotspot null variants were found in 14 and 1, respectively. Our study revealed genotype–phenotype correlations in several respects. UMIN-CTR: UMIN000045855. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1472-6831 25 1 2025 Evaluation of Streptococcus mutans strains possessing genes encoding collagen-binding proteins in the Japanese population 1908 EN Makoto Okuda Department of Pediatric Dentistry, Graduate School of Dentistry, The University of Osaka Yuto Suehiro Department of Pediatric Dentistry, Graduate School of Dentistry, The University of Osaka Jinthana Lapirattanakul Department of Oral Microbiology, Faculty of Dentistry, Mahidol University Shuhei Naka Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Michiyo Matsumoto-Nakano Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryota Nomura Department of Pediatric Dentistry, Graduate School of Biomedical and Health Sciences, Hiroshima University Rena Okawa Department of Pediatric Dentistry, Graduate School of Dentistry, The University of Osaka Kazuhiko Nakano Department of Pediatric Dentistry, Graduate School of Dentistry, The University of Osaka Background Streptococcus mutans harbors collagen-binding protein genes, namely cnm and cbm, which are implicated in its virulence and pathogenicity in both oral and extraoral infections. Although both genes were initially identified in S. mutans isolated from Japanese populations, their geographical prevalence, distribution, and genetic relatedness within Japan remain largely unexplored. This study investigates the prevalence of S. mutans strains carrying cnm and cbm genes across Japan, correlates these findings with clinical data, and analyzes the genetic relatedness of cnm-positive and cnm-negative strains using multilocus sequence typing (MLST).<br> Methods Dental plaque specimens were collected from 1248 individuals from eight Japanese cities (Hiroshima, Fukuoka, Nagasaki, Niigata, Okayama, Osaka, Tokushima, and Tokyo) and plated on selective medium for S. mutans isolation. S. mutans was confirmed in 523 subjects by colony morphology and PCR using species-specific primers, and the presence of the cnm and cbm genes was determined by PCR with gene-specific primers. Demographic (age, sex) and oral examination (caries prevalence, caries experience, number of teeth) data were recorded. MLST was employed to genotype selected cnm-positive and cnm-negative S. mutans strains to assess their clonal relationships.<br> Results Among 523 subjects possessing S. mutans (aged 3–90 years), we detected cnm-positive strains in all cities; specifically, the prevalence ranged from 5.5% in Okayama to 25.0% in Tokushima. In contrast, cbm-positive strains were less common and undetectable in some regions. Furthermore, subjects harboring cnm-positive S. mutans were significantly older (p = 0.002) and had higher caries prevalence and experience (p < 0.001). MLST revealed evolutionary relationships among cnm-positive strains across the cities but no discernible region-specific clustering. Clonal relationships partially reflected cnm gene distribution, particularly for exclusively cnm-positive or cnm-negative clonal complexes, but inconsistencies involving serotypes and cnm presence within some clonal complexes and sequence types were also noted.<br> Conclusions The cnm-positive S. mutans strains are widely distributed throughout Japan and are associated with increased age and caries burden. Although core genome analysis revealed some clonal patterns, the non-uniform distribution of the non-core cnm gene is likely influenced by horizontal gene transfer, providing S. mutans with adaptive advantages irrespective of its core genetic background or serotype. No potential conflict of interest relevant to this article was reported.

Informa UK Limited Acta Medica Okayama 2167-8421 2025 Novel in-frame duplication variant of SOD1 in a Japanese family with familial amyotrophic lateral sclerosis EN Masanori Nakajima Department of Neurology, Kyorin University School of Medicine Hiroya Naruse Department of Neurology, Graduate School of Medicine, The University of Tokyo Yuichi Riku Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University Kunihiro Ueda Department of Neurology, Graduate School of Medicine, The University of Tokyo Takashi Matsukawa Department of Neurology, Graduate School of Medicine, The University of Tokyo Jun Mitsui Department of Neurology, Graduate School of Medicine, The University of Tokyo Yoshitsugu Nakamura Division of Neurology, Department of Internal Medicine IV, Osaka Medical and Pharmaceutical University Shimon Ishida Division of Neurology, Department of Internal Medicine IV, Osaka Medical and Pharmaceutical University Takashi Yamada Department of Pathology, Osaka Medical and Pharmaceutical University Naoki Moro Department of Neurology, Kyorin University School of Medicine Naoki Kotsuki Department of Neurology, Kyorin University School of Medicine Kentaro Nagai Department of Neurology, Kyorin University School of Medicine Shin-ichi Tokushige Department of Neurology, Kyorin University School of Medicine Ayumi Uchibori Department of Neurology, Kyorin University School of Medicine Chizuko Oishi Department of Neurology, Kyorin University School of Medicine Hiroyuki Yabata Department of Neurology, Shiga University of Medical Science Makoto Urushitani Department of Neurology, Shiga University of Medical Science Yasushi Iwasaki Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University Hiroyuki Ishiura Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tatsushi Toda Department of Neurology, Graduate School of Medicine, The University of Tokyo Shoji Tsuji Department of Neurology, Graduate School of Medicine, The University of Tokyo Yaeko Ichikawa Department of Neurology, Kyorin University School of Medicine Objectives: To analyze the cases of a family with a novel in-frame duplication variant (NM_000454.5:c.357_357 + 2dup, p.Val120dup) of SOD1 and a structural model of the mutated SOD1 protein. Methods: The clinical profiles of three patients in the family were analyzed, including the neuropathological findings of the proband’s mother. Genetic analyses were conducted for three patients. cDNA and in silico structural analyses were performed to evaluate the effects of duplication variants on the structure of SOD1. Results: The clinical features of the patients included predominant involvement of the lower motor neurons, asymmetric onset of motor symptoms in the lower limbs, and a relatively rapid progression of muscular weakness and respiratory insufficiency. Neuropathological findings revealed severe loss of spinal cord motor neurons, and immunohistochemistry using an anti-misfolded SOD1 antibody revealed aggregates in the spinal cord. Genetic analyses revealed a c.357_357 + 2dup at the exon 4–intron 4 boundary of SOD1 in three patients. cDNA analysis of the proband suggested the presence of a valine (p.Val120dup) duplication in the heterozygous state, and the SOD1 transcript level showed no significant differences from those of healthy controls. In silico structural analyses predicted that p.Val120dup could affect the structure of the β-barrels and copper ion binding site of SOD1, suggesting an abnormal conformation of SOD1 that is predicted to interfere with the binding of copper ions. Conclusion: We identified a novel in-frame duplication variant in the C-terminus of β7 of SOD1. This genotype–structure–phenotype study of SOD1 provides valuable insights into disease-causing mechanisms. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 79 6 2025 COVID-19 and the Risks of Migraine and Headache: A Mendelian Randomization Study 413 419 EN Zhiyun Jiang Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University Ying Xi Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University Original Article 10.18926/AMO/69843 Several observational studies suggested that migraine headache attacks were associated with coronavirus disease 2019 (COVID-19). We investigated genetic causal links between COVID-19 phenotypes and the development of headache and migraine, including migraine with aura (MA) and migraine without aura (MO). We conducted a two-sample Mendelian randomization (MR) analysis to estimate the genetic association in European populations. The inverse-variance weighted (IVW) method was used as the main approach in the MR analyses, together with weighted median and MR-Egger methods. We also performed a series of sensitivity tests to assess the robustness of the MR results. The MR results demonstrated that COVID-19 severity, hospitalization, and susceptibility had no causal effect on the risks of headache, migraine, MA, or MO. No horizontal pleiotropy was detected, and the results were robust as supported by the sensitivity analysis findings. Our analyses identified no casual effect of COVID-19 severity, hospitalization, or susceptibility on the risks of headache or migraine in European populations. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1347-9032 2025 Comparative Analysis of a Dual DNA–RNA Panel and a DNA-Only Panel for Sarcoma: Real-World Data From a Nationwide Genomic Database EN Eiji Nakata Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kiichiro Ninomiya Center for Comprehensive Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Tatsunori Osone Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Daisuke Ennishi Center for Comprehensive Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Shuta Tomida Center for Comprehensive Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Tomohiro Fujiwara Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Toshiyuki Kunisada Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Mashu Futagawa Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Akira Hirasawa Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Shinichi Toyooka Center for Comprehensive Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Toshifumi Ozaki Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Next-generation sequencing-based comprehensive cancer genomic profiling is promising in cancer management; however, most studies rely on tumor-only DNA panels from single institutions. In 2023, Japan introduced an insurance-covered cancer genomic profiling test—the GenMine TOP Cancer Genome Profiling System—a dual DNA–RNA panel with matched tumor–normal testing. This study evaluated its utility compared to a conventional DNA-only test (FoundationOne CDx) in managing sarcoma patients using a nationwide genetic profiling database provided by the Center for Cancer Genomics and Advanced Therapeutics. This study included 1046 patients registered between August 2023 and October 2024. The dual DNA–RNA test identified significantly more fusion genes (20.3% vs. 7.4%, p < 0.001) and therapeutically targetable kinase fusions (3.5% vs. 1.2%, p = 0.019) than the DNA-only test. Among patients with translocation-related sarcomas, histology-specific fusion genes were identified in 77.5% using the dual panel, compared to 40.0% with the DNA-only panel (p < 0.001). In non-gastrointestinal stromal tumor sarcomas, the dual test showed a trend toward higher rates of genotype-matched therapy (4.3% vs. 2.6%, p = 0.25) and a significantly higher rate of molecular targeted therapy (4.3% vs. 1.5%, p = 0.03). Additionally, 5.7% of patients had pathogenic germline variants identified through tumor–normal matched analysis. These findings suggest that a dual DNA–RNA panel with matched tumor–normal testing may improve diagnostic accuracy and inform treatment decisions in the routine clinical management of sarcoma. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1936-5209 19 3 2025 Oregon Wolfe barley genetic stocks – Research and teaching tools for next generation scientists e70004 EN Margaret R. Krause Department of Crop and Soil Science, Oregon State University Juan David Arbelaez Department of Crop Sciences, University of Illinois at Urbana-Champaign Åsmund Asdal Nordic Genetic Resource Centre Ramzi Belkodja CIHEAM-Zaragoza Nancy Boury Department of Plant Pathology, Entomology, and Microbiology, Iowa State University Victoria C. Blake Department of Plant Sciences and Plant Pathology, Montana State University Patrick J. Brown Department of Plant Sciences, University of California-Davis Ana Casas Departamento de Genética y Producción Vegetal, Estación Experimental Aula Dei–CSIC Luis Cistué Departamento de Genética y Producción Vegetal, Estación Experimental Aula Dei–CSIC Alba Farré‐Martínez AGROTECNIO-CERCA Center, Universidad de Lleida Scott Fisk Department of Crop and Soil Science, Oregon State University Gregory S. Fuerst U.S. Department of Agriculture-Agricultural Research Service, Corn Insects and Crop Genetics Research Unit, Iowa State University Estela Giménez Department of Biotechnology-Plant Biology, School of Agricultural, Food and Biosystems Engineering, Universidad Politécnica de Madrid Carla Guijarro‐Real Department of Biotechnology-Plant Biology, School of Agricultural, Food and Biosystems Engineering, Universidad Politécnica de Madrid Katy Guthrie Department of Agronomy and Plant Genetics, University of Minnesota Margaret Halstead Aardevo North America Laura Helgerson Department of Crop and Soil Science, Oregon State University Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Ernesto Igartua Departamento de Genética y Producción Vegetal, Estación Experimental Aula Dei–CSIC Morten Lillemo Department of Plant Sciences, Norwegian University of Life Sciences Marina Martínez‐García Department of Biotechnology-Plant Biology, School of Agricultural, Food and Biosystems Engineering, Universidad Politécnica de Madrid Mariona Martínez‐Subirà AGROTECNIO-CERCA Center, Universidad de Lleida Susan McCouch Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University Laurie McGhee Colfax-Mingo Community High School Travis Nickols Department of Crop and Soil Science, Oregon State University Nick Peters Department of Plant Pathology, Entomology, and Microbiology, Iowa State University Raymond Porter Haupert Institute for Agricultural Studies, Huntington University Ignacio Romagosa AGROTECNIO-CERCA Center, Universidad de Lleida Anja Karine Ruud Department of Plant Sciences, Norwegian University of Life Sciences Kazuhiro Sato Institute of Plant Science and Resources, Okayama University Silvio Salvi Department of Agricultural and Food Sciences, University of Bologna Giuseppe Sangiorgi Department of Agricultural and Food Sciences, University of Bologna Rebekka Schüller Department of Crop Sciences, University of Illinois at Urbana-Champaign Taner Z. Sen Crop Improvement and Genetics Research Unit, U.S. Department of Agriculture-Agricultural Research Service José Miguel Soriano AGROTECNIO-CERCA Center, Universidad de Lleida Robert M. Stupar Department of Agronomy and Plant Genetics, University of Minnesota To‐Chia Ting Agronomy Department, Purdue University Kelly Vining Department of Crop and Soil Science, Oregon State University Maria von Korff Institute of Plant Genetics, Heinrich-Heine-Universität Düsseldorf Agatha Walla Institute of Plant Genetics, Heinrich-Heine-Universität Düsseldorf Diane R. Wang Agronomy Department, Purdue University Robbie Waugh Division of Plant Sciences, School of Life Sciences, University of Dundee Roger P. Wise Department of Plant Pathology, Entomology, and Microbiology, Iowa State University Robert Wolfe Agriculture and Agri-Food Canada Eric Yao Crop Improvement and Genetics Research Unit, U.S. Department of Agriculture-Agricultural Research Service Patrick M. Hayes Department of Crop and Soil Science, Oregon State University The Oregon Wolfe Barley (OWB) mapping population (Reg. no. MP-4, NSL 554937 MAP) is a resource for genetics research and instruction. The OWBs are a set of doubled haploid barley (Hordeum vulgare L.) lines developed at Oregon State University from the F1 of a cross between Dr. Robert Wolfe's dominant and recessive marker stocks. Exhibiting a high level of genetic and phenotypic diversity, the OWBs are used throughout the world as a research tool for barley genetics. To date, these endeavors have led to 56 peer-reviewed publications, as well as three reports in the Barley Genetics Newsletter. At the same time, the OWBs are widely used as an instructor resource at the K–12, undergraduate, graduate, and professional levels. They are currently used at universities and/or institutes in German, Italy, Norway, Spain, and the United States and are currently being developed further for educational use in other countries. Genotype and phenotype data, lesson plans, and seed availability information are available herein and online. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 2045-7634 14 15 2025 Real‐World Data of Comprehensive Cancer Genomic Profiling Tests Performed in the Routine Clinical Setting in Sarcoma e71098 EN Eiji Nakata Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Daisuke Ennishi Center for Comprehensive Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Tatsunori Osone Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kiichiro Ninomiya Center for Comprehensive Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Shuta Tomida Center for Comprehensive Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Takuto Itano Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Tomohiro Fujiwara Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Toshiyuki Kunisada Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Naoyuki Ida Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hideki Yamamoto Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Mashu Futagawa Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Tatsunori Shimoi Department of Medical Oncology, National Cancer Center Hospital Hiroyuki Yanai Department of Pathology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Akira Hirasawa Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Shinichi Toyooka Center for Comprehensive Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Masahiro Tabata Center for Clinical Oncology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Toshifumi Ozaki Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Introduction: Next-generation sequencing-based comprehensive cancer genomic profiling (CGP) tests are beneficial for refining diagnosis and personalized treatment of various cancers. However, the clinical impact of CGP, as covered by public health insurance in the management of sarcomas, remains unknown. Especially, the data on the utility of the newly emerging dual DNA–RNA panel compared to the conventional DNA-only panel in clinical settings is lacking. Therefore, we evaluated the utility of CGP in routine clinical practice for sarcoma treatment.<br> Patients and Methods: In this study, three types of DNA panel and one DNA–RNA panel, reimbursed by Japanese public health insurance, were utilized. We detected oncogenic and druggable gene mutations and genotype-matched therapies.<br> Results: One hundred and thirty-six patients were included in this study. Based on the detection of highly histology-specific translocations in the sequencing results, 2.2% of patients were re-classified. In patients with translocation-related sarcomas, a DNA–RNA panel identified more histology-specific fusion genes than DNA panels (p = 0.0035). Specifically, 86.8% and 39.0% of patients had oncogenic and druggable genomic alterations, respectively. Of these, 9.6% underwent genotype-matched therapy, with a 36.3% response rate and an 81.8% disease control rate. Patients who were administered genomically matched therapy had better overall survival (OS) than those who did not in patients with metastatic or advanced sarcoma with no prior chemotherapy (3-year OS: 83.3% vs. 48.0%, p = 0.42). Patients with TP53 and RB1 mutations had worse OS than those without. Germline findings were detected in 11.0% of the patients, one of whom had a truly germline origin.<br> Conclusions: This study suggests that publicly reimbursed CGP tests, particularly the dual DNA–RNA panel, could be beneficial for refining diagnostic precision in selected sarcoma subtypes, treatment decisions, detecting the germline findings, and prognosis prediction in routine clinical settings for sarcoma. The implementation of genotype-matched therapies showed favorable clinical outcomes and improved the prognosis. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 2052-7276 11 11 2024 A low-cost dpMIG-seq method for elucidating complex inheritance in polysomic crops: a case study in tetraploid blueberry uhae248 EN Kyoka Nagasaka Graduate School of Agriculture, Kyoto University Kazusa Nishimura Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Ko Motoki Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Keigo Yamagata Graduate School of Agriculture, Kyoto University Soichiro Nishiyama Graduate School of Agriculture, Kyoto University Hisayo Yamane Graduate School of Agriculture, Kyoto University Ryutaro Tao Graduate School of Agriculture, Kyoto University Ryohei Nakano Graduate School of Agriculture, Kyoto University Tetsuya Nakazaki Graduate School of Agriculture, Kyoto University Next-generation sequencing (NGS) library construction often requires high-quality DNA extraction, precise adjustment of DNA concentration, and restriction enzyme digestion to reduce genome complexity, which results in increased time and cost in sample preparation and processing. To address these challenges, a PCR-based method for rapid NGS library preparation, named dpMIG-seq, has been developed and proven effective for high-throughput genotyping. However, the application of dpMIG-seq has been limited to diploid and polyploid species with disomic inheritance. In this study, we obtained genome-wide single nucleotide polymorphism (SNP) markers for tetraploid blueberry to evaluate genotyping and downstream analysis outcomes. Comparison of genotyping qualities inferred across samples with different DNA concentrations and multiple bioinformatics approaches revealed high accuracy and reproducibility of dpMIG-seq-based genotyping, with Pearson's correlation coefficients between replicates in the range of 0.91 to 0.98. Furthermore, we demonstrated that dpMIG-seq enables accurate genotyping of samples with low DNA concentrations. Subsequently, we applied dpMIG-seq to a tetraploid F1 population to examine the inheritance probability of parental alleles. Pairing configuration analysis supported the random meiotic pairing of homologous chromosomes on a genome-wide level. On the other hand, preferential pairing was observed on chr-11, suggesting that there may be an exception to the random pairing. Genotypic data suggested quadrivalent formation within the population, although the frequency of quadrivalent formation varied by chromosome and cultivar. Collectively, the results confirmed applicability of dpMIG-seq for allele dosage genotyping and are expected to catalyze the adoption of this cost-effective and rapid genotyping technology in polyploid studies. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2025 Genotypes and phenotypes of neurofibromatosis type 1 patients in Japan: A Hereditary Tumor Cohort Study EN Mashu FUTAGAWA Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2054-345X 11 1 2024 Genotypes and phenotypes of neurofibromatosis type 1 patients in Japan: A Hereditary Tumor Cohort Study 42 EN Mashu Futagawa Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tetsuya Okazaki Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eiji Nakata Department of Orthopedic Surgery, Okayama University Hospital Chika Fukano Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Risa Osumi Department of Clinical Genetics and Genomic Medicine, Okayama University Hospital Fumino Kato Department of Clinical Genetics and Genomic Medicine, Okayama University Hospital Yusaku Urakawa Department of Genetic Medicine, School of Medicine, Fujita Health University Hideki Yamamoto Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshifumi Ozaki Department of Orthopedic Surgery, Okayama University Hospital Akira Hirasawa Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Neurofibromatosis type 1 (NF1) presents with a broad spectrum of clinical manifestations, including an increased risk of tumor development and hypertension. Comprehensive data on genotype‒phenotype correlations in patients with NF1 are limited. Therefore, in this study, we aimed to elucidate the detailed genetic and clinical characteristics of NF1 in a hereditary tumor cohort. We performed sequencing and copy number assays in a clinical laboratory and analyzed the clinical data of 44 patients with suspected NF1. Germline pathogenic variants were detected in 36 patients (81.8%), and 20.7% of the variants were novel. Notably, 40.0% of adult patients presented with malignancies; female breast cancer occurred in 20.0% of patients, which was a higher rate than that previously reported. Hypertension was observed in 30.6% of the adult patients, with one patient experiencing sudden death and another developing pheochromocytoma. Three patients with large deletions in NF1 exhibited prominent cutaneous, skeletal, and neurological manifestations. These results highlight the importance of regular surveillance, particularly for patients with malignancies and hypertension. Our findings provide valuable insights for genetic counseling and clinical management, highlighting the multiple health risks associated with NF1 and the need for comprehensive and multidisciplinary care. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 25 13 2024 Genome-Wide Association Study with Three Control Cohorts of Japanese Patients with Esotropia and Exotropia of Comitant Strabismus and Idiopathic Superior Oblique Muscle Palsy 6986 EN Toshihiko Matsuo Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ichiro Hamasaki Department of Ophthalmology, Okayama University Hospital Yoichiro Kamatani Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo Takahisa Kawaguchi Center for Genomic Medicine, Graduate School of Medicine, Kyoto University Izumi Yamaguchi Center for Genomic Medicine, Graduate School of Medicine, Kyoto University Fumihiko Matsuda Center for Genomic Medicine, Graduate School of Medicine, Kyoto University Akira Saito StaGen Co., Ltd. Kazuyuki Nakazono StaGen Co., Ltd. Shigeo Kamitsuji StaGen Co., Ltd. Esotropia and exotropia in the entity of comitant strabismus are multifactorial diseases with both genetic and environmental backgrounds. Idiopathic superior oblique muscle palsy, as the predominant entity of non-comitant (paralytic) strabismus, also has a genetic background, as evidenced by varying degrees of muscle hypoplasia. A genome-wide association study (GWAS) was conducted of 711 Japanese patients with esotropia (n= 253), exotropia (n = 356), and idiopathic superior oblique muscle palsy (n = 102). The genotypes of single nucleotide polymorphisms (SNPs) were determined by Infinium Asian Screening Array. Three control cohorts from the Japanese population were used: two cohorts from BioBank Japan (BBJ) and the Nagahama Cohort. BBJ (180K) was genotyped by a different array, Illumina Infinium OmniExpressExome or HumanOmniExpress, while BBJ (ASA) and the Nagahama Cohort were genotyped by the same Asian array. After quality control of SNPs and individuals, common SNPs between the case cohort and the control cohort were chosen in the condition of genotyping by different arrays, while all SNPs genotyped by the same array were used for SNP imputation. The SNPs imputed with R-square values ≥ 0.3 were used to compare the case cohort of each entity or the combined entity with the control cohort. In comparison with BBJ (180K), the esotropia group and the exotropia group showed CDCA7 and HLA-F, respectively, as candidate genes at a significant level of p < 5 × 10−8, while the idiopathic superior oblique muscle palsy group showed DAB1 as a candidate gene which is involved in neuronal migration. DAB1 was also detected as a candidate in comparison with BBJ (ASA) and the Nagahama Cohort at a weak level of significance of p < 1 × 10−6. In comparison with BBJ (180K), RARB (retinoic acid receptor-β) was detected as a candidate at a significant level of p < 5 × 10−8 in the combined group of esotropia, exotropia, and idiopathic superior oblique muscle palsy. In conclusion, a series of GWASs with three different control cohorts would be an effective method with which to search for candidate genes for multifactorial diseases such as strabismus. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2076-2607 12 5 2024 Isolation of Vibrio cholerae and Vibrio vulnificus from Estuarine Waters, and Genotyping of V. vulnificus Isolates Using Loop-Mediated Isothermal Amplification 877 EN Shin-Ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Megumi Kurata Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Riho Hirose Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masaya Yoshikawa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yong Liang Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yosuke Yamagishi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tamaki Mizuno Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Bacteria in the genus Vibrio are ubiquitous in estuarine and coastal waters. Some species (including Vibrio cholerae and Vibrio vulnificus) are known human pathogens causing ailments like cholera, diarrhea, or septicemia. Notably, V. vulnificus can also cause a severe systemic infection (known as vibriosis) in eels raised in aquaculture facilities. Water samples were periodically collected from the estuary of the Asahi River, located in the southern part of Okayama City, Japan. These samples were directly plated onto CHROMagar Vibrio plates, and colonies displaying turquoise-blue coloration were selected. Thereafter, polymerase chain reaction was used to identify V. cholerae and V. vulnificus. A total of 30 V. cholerae strains and 194 V. vulnificus strains were isolated during the warm season when the water temperature (WT) was higher than 20 degrees C. Concurrently, an increase in coliforms was observed during this period. Notably, V. vulnificus has two genotypes, designated as genotype 1 and genotype 2. Genotype 1 is pathogenic to humans, while genotype 2 is pathogenic to both humans and eels. The loop-mediated isothermal amplification method was developed to rapidly determine genotypes at a low cost. Of the 194 strains isolated, 80 (41.2%) were identified as genotype 1 strains. Among the 41 strains isolated when the WTs were higher than 28 degrees C, 25 strains (61.0%) belonged to genotype 1. In contrast, of the 32 strains isolated when the WTs were lower than 24 degrees C, 27 strains (84.4%) belonged to genotype 2. These results suggest that the distribution of the two genotypes was influenced by WT. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2024 The Genotypic and Phenotypic Characteristics Contributing to Flomoxef Sensitivity in Clinical Isolates of ESBL-Producing E.coli Strains from Urinary Tract Infections EN Kazuma SAKAEDA Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0340-5443 78 3 2024 Experimental quantification of genetic and ontogenetic effects on fighting behavior in the broad-horned flour beetle 34 EN Toshiki Nishitani Graduate School of Environmental, Life, Natural and Technology, Okayama University Kentarou Matsumura Graduate School of Environmental, Life, Natural and Technology, Okayama University Erik Postma Centre for Ecology & Conservation, University of Exeter, Penryn Campus Manmohan Dev Sharma Centre for Ecology & Conservation, University of Exeter, Penryn Campus David J Hosken Centre for Ecology & Conservation, University of Exeter, Penryn Campus Takahisa Miyatake Graduate School of Environmental, Life, Natural and Technology, Okayama University Most animal behaviors show large within- and among-individual variation, and this includes competitive male behaviors. With male fighting for example, aggressiveness often correlates with dominance, and contest duration varies with age. However, few studies have directly quantified how mean aggressiveness and contest duration, the variation among individuals in both traits, and the relationship among them, vary with age. Here we address these gaps and examine the effect of male age and genotype on two key aspects of male fighting behavior - aggressiveness (here measured as latency to fight) and contest duration - and the relationship between them. We do this using isogenic lines of the broad-horned flour beetle Gnatocerus cornutus. We observed fighting behavior of paired males of similar body size and age. Using uni- and multivariate mixed models, we show that although there was a significant difference between younger and older males in contest duration, mean aggressiveness was not affected by male age. However, the variation in aggression and fight duration varied with age, being greater in younger and older males respectively. Additionally, although there was a positive correlation between aggressiveness and contest duration in younger males, this relationship was not found in older males. Finally, the only significant genetic effect was for aggression in younger males. Our study shows that age differentially shapes key components of male fighting behavior as well as the relationship among them, highlighting the dynamic nature and context-dependence of fighting. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 1340-2838 31 1 2023 MCPtaggR: R package for accurate genotype calling in reduced representation sequencing data by eliminating error-prone markers based on genome comparison dsad027 EN Tomoyuki Furuta Institute of Plant Science and Resources, Okayama University Toshio Yamamoto Institute of Plant Science and Resources, Okayama University Reduced representation sequencing (RRS) offers cost-effective, high-throughput genotyping platforms such as genotyping-by-sequencing (GBS). RRS reads are typically mapped onto a reference genome. However, mapping reads harbouring mismatches against the reference can potentially result in mismapping and biased mapping, leading to the detection of error-prone markers that provide incorrect genotype information. We established a genotype-calling pipeline named mappable collinear polymorphic tag genotyping (MCPtagg) to achieve accurate genotyping by eliminating error-prone markers. MCPtagg was designed for the RRS-based genotyping of a population derived from a biparental cross. The MCPtagg pipeline filters out error-prone markers prior to genotype calling based on marker collinearity information obtained by comparing the genome sequences of the parents of a population to be genotyped. A performance evaluation on real GBS data from a rice F2 population confirmed its effectiveness. Furthermore, our performance test using a genome assembly that was obtained by genome sequence polishing on an available genome assembly suggests that our pipeline performs well with converted genomes, rather than necessitating de novo assembly. This demonstrates its flexibility and scalability. The R package, MCPtaggR, was developed to provide functions for the pipeline and is available at https://github.com/tomoyukif/MCPtaggR. No potential conflict of interest relevant to this article was reported.

American Association for the Advancement of Science (AAAS) Acta Medica Okayama 2643-6515 5 2023 Deep Learning Enables Instant and Versatile Estimation of Rice Yield Using Ground-Based RGB Images 0073 EN Yu Tanaka Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Tomoya Watanabe Graduate School of Mathematics, Kyushu University Keisuke Katsura Graduate School of Agriculture, Tokyo University of Agriculture and Technology Yasuhiro Tsujimoto Japan International Research Center for Agricultural Sciences Toshiyuki Takai Japan International Research Center for Agricultural Sciences Takashi Sonam Tashi Tanaka Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kensuke Kawamura Japan International Research Center for Agricultural Sciences Hiroki Saito Tropical Agriculture Research Front, Japan International Research Center for Agricultural Sciences Koki Homma Graduate School of Agricultural Science, Tohoku University Salifou Goube Mairoua Africa Rice Center (AfricaRice) Kokou Ahouanton Africa Rice Center (AfricaRice) Ali Ibrahim Africa Rice Center (AfricaRice), Regional Station for the Sahel Kalimuthu Senthilkumar Africa Rice Center (AfricaRice) Vimal Kumar Semwal Africa Rice Center (AfricaRice), Nigeria Station Eduardo Jose Graterol Matute Latin American Fund for Irrigated Rice - The Alliance of Bioversity International and CIAT Edgar Corredor Latin American Fund for Irrigated Rice - The Alliance of Bioversity International and CIAT Raafat El-Namaky Rice Research and Training Center, Field Crops Research Institute, ARC Norvie Manigbas Philippine Rice Research Institute (PhilRice) Eduardo Jimmy P. Quilang Philippine Rice Research Institute (PhilRice) Yu Iwahashi Graduate School of Agriculture, Kyoto University Kota Nakajima Graduate School of Agriculture, Kyoto University Eisuke Takeuchi Graduate School of Agriculture, Kyoto University Kazuki Saito Graduate School of Agriculture, Kyoto University Rice (Oryza sativa L.) is one of the most important cereals, which provides 20% of the world’s food energy. However, its productivity is poorly assessed especially in the global South. Here, we provide a first study to perform a deep-learning-based approach for instantaneously estimating rice yield using red-green-blue images. During ripening stage and at harvest, over 22,000 digital images were captured vertically downward over the rice canopy from a distance of 0.8 to 0.9 m at 4,820 harvesting plots having the yield of 0.1 to 16.1 t·ha−1 across 6 countries in Africa and Japan. A convolutional neural network applied to these data at harvest predicted 68% variation in yield with a relative root mean square error of 0.22. The developed model successfully detected genotypic difference and impact of agronomic interventions on yield in the independent dataset. The model also demonstrated robustness against the images acquired at different shooting angles up to 30° from right angle, diverse light environments, and shooting date during late ripening stage. Even when the resolution of images was reduced (from 0.2 to 3.2 cm·pixel−1 of ground sampling distance), the model could predict 57% variation in yield, implying that this approach can be scaled by the use of unmanned aerial vehicles. Our work offers low-cost, hands-on, and rapid approach for high-throughput phenotyping and can lead to impact assessment of productivity-enhancing interventions, detection of fields where these are needed to sustainably increase crop production, and yield forecast at several weeks before harvesting. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 1340-2838 30 5 2023 Genetic basis of lineage-specific evolution of fruit traits in hexaploid persimmon dsad015 EN Ayano Horiuchi Graduate School of Environmental and Life Science, Okayama University Kanae Masuda Graduate School of Environmental and Life Science, Okayama University Kenta Shirasawa Kazusa DNA Research Institute Noriyuki Onoue Institute of Fruit Tree and Tea Science, NARO Ryusuke Matsuzaki Institute of Fruit Tree and Tea Science, NARO Ryutaro Tao Graduate School of Agriculture, Kyoto University Yasutaka Kubo Graduate School of Environmental and Life Science, Okayama University Koichiro Ushijima Graduate School of Environmental and Life Science, Okayama University Takashi Akagi Graduate School of Environmental and Life Science, Okayama University Frequent polyploidization events in plants have led to the establishment of many lineage-specific traits representing each species. Little is known about the genetic bases for these specific traits in polyploids, presumably due to plant genomic complexity and their difficulties in applying genetic approaches. Hexaploid Oriental persimmon (Diospyros kaki) has evolved specific fruit characteristics, including wide variations in fruit shapes and astringency. In this study, using whole-genome diploidized/quantitative genotypes from ddRAD-Seq data of 173 persimmon cultivars, we examined their population structures and potential correlations between their structural transitions and variations in nine fruit traits. The population structures of persimmon cultivars were highly randomized and not substantially correlated with the representative fruit traits focused on in this study, except for fruit astringency. With genome-wide association analytic tools considering polyploid alleles, we identified the loci associated with the nine fruit traits; we mainly focused on fruit-shape variations, which have been numerically characterized by principal component analysis of elliptic Fourier descriptors. The genomic regions that putatively underwent selective sweep exhibited no overlap with the loci associated with these persimmon-specific fruit traits. These insights will contribute to understanding the genetic mechanisms by which fruit traits are independently established, possibly due to polyploidization events. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 1943-2631 224 2 2023 GBScleanR: robust genotyping error correction using a hidden Markov model with error pattern recognition iyad055 EN Tomoyuki Furuta Institute of Plant Science and Resources, Okayama University Toshio Yamamoto Institute of Plant Science and Resources, Okayama University Motoyuki Ashikari Bioscience and Biotechnology Center, Nagoya University Reduced-representation sequencing (RRS) provides cost-effective and time-saving genotyping platforms. Despite the outstanding advantage of RRS in throughput, the obtained genotype data usually contain a large number of errors. Several error correction methods employing the hidden Markov model (HMM) have been developed to overcome these issues. These methods assume that markers have a uniform error rate with no bias in the allele read ratio. However, bias does occur because of uneven amplification of genomic fragments and read mismapping. In this paper, we introduce an error correction tool, GBScleanR, which enables robust and precise error correction for noisy RRS-based genotype data by incorporating marker-specific error rates into the HMM. The results indicate that GBScleanR improves the accuracy by more than 25 percentage points at maximum compared to the existing tools in simulation data sets and achieves the most reliable genotype estimation in real data even with error-prone markers. No potential conflict of interest relevant to this article was reported.

Japanese Society of Breeding Acta Medica Okayama 1344-7610 73 3 2023 Elucidation of genetic variation and population structure of melon genetic resources in the NARO Genebank, and construction of the World Melon Core Collection 269 277 EN Gentaro Shigita Graduate School of Environmental and Life Science, Okayama University Tran Phuong Dung Graduate School of Environmental and Life Science, Okayama University Mst. Naznin Pervin Graduate School of Environmental and Life Science, Okayama University Thanh-Thuy Duong Graduate School of Environmental and Life Science, Okayama University Odirich Nnennaya Imoh Graduate School of Environmental and Life Science, Okayama University Yuki Monden Graduate School of Environmental and Life Science, Okayama University Hidetaka Nishida Graduate School of Environmental and Life Science, Okayama University Katsunori Tanaka Faculty of Agriculture and Life Science, Hirosaki University Mitsuhiro Sugiyama Institute of Vegetable and Floriculture Science, National Agriculture and Food Research Organization (NARO) Yoichi Kawazu Institute of Vegetable and Floriculture Science, National Agriculture and Food Research Organization (NARO) Norihiko Tomooka Research Center of Genetic Resources, National Agriculture and Food Research Organization (NARO) Kenji Kato Graduate School of Environmental and Life Science, Okayama University Numerous genetic resources of major crops have been introduced from around the world and deposited in Japanese National Agriculture and Food Research Organization (NARO) Genebank. Understanding their genetic variation and selecting a representative subset (“core collection”) are essential for optimal management and efficient use of genetic resources. In this study, we conducted genotyping-by-sequencing (GBS) to characterize the genetic relationships and population structure in 755 accessions of melon genetic resources. The GBS identified 39,324 single-nucleotide polymorphisms (SNPs) that are distributed throughout the melon genome with high density (one SNP/10.6 kb). The phylogenetic relationships and population structure inferred using this SNP dataset are highly associated with the cytoplasm type and geographical origin. Our results strongly support the recent hypothesis that cultivated melon was established in Africa and India through multiple independent domestication events. Finally, we constructed a World Melon Core Collection that covers at least 82% of the genetic diversity and has a wide range of geographical origins and fruit morphology. The genome-wide SNP dataset, phylogenetic relationships, population structure, and the core collection provided in this study should largely contribute to genetic research, breeding, and genetic resource preservation in melon. No potential conflict of interest relevant to this article was reported.

nature portfolio Acta Medica Okayama 2045-2322 13 1 2023 Identification of genetic loci associated with renal dysfunction after lung transplantation using an ethnic-specific single-nucleotide polymorphism array 8912 EN Yasuaki Tomioka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichiro Sugimoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Haruchika Yamamoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shuta Tomida Center for Comprehensive Genomic Medicine, Okayama University Hospital Toshio Shiotani Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shin Tanaka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Shien Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken Suzawa Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaroh Miyoshi Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinji Otani Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mikio Okazaki Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masaomi Yamane Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Renal dysfunction is a long-term complication associated with an increased mortality after lung transplantation (LT). We investigated the association of single-nucleotide polymorphisms (SNPs) with the development of renal dysfunction after LT using a Japanese-specific SNP array. First, eligible samples of 34 LT recipients were genotyped using the SNP array and divided into two groups, according to the presence of homozygous and heterozygous combinations of mutant alleles of the 162 renal-related SNPs. To identify candidate SNPs, the renal function tests were compared between the two groups for each SNP. Next, we investigated the association between the candidate SNPs and the time course of changes of the estimated glomerular filtration rate (eGFR) in the 99 recipients until 10 years after the LT. Delta eGFR was defined as the difference between the postoperative and preoperative eGFR values. Eight SNPs were identified as the candidate SNPs in the 34 recipients. Validation analysis of these 8 candidate SNPs in all the 99 recipients showed that three SNPs, namely, rs10277115, rs4690095, and rs792064, were associated with significant changes of the Delta eGFR. Pre-transplant identification of high-risk patients for the development of renal dysfunction after LT based on the presence of these SNPs might contribute to providing personalized medicine. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2079-6382 12 3 2023 The Genotypic and Phenotypic Characteristics Contributing to Flomoxef Sensitivity in Clinical Isolates of ESBL-Producing E. coli Strains from Urinary Tract Infections 522 EN Kazuma Sakaeda Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takuya Sadahira Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Maruyama Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takehiro Iwata Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masami Watanabe Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Koichiro Wada Koichiro Wada Department of Urology, School of Medicine, Shimane University Motoo Araki Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University We carried out a molecular biological analysis of extended-spectrum beta-lactamase (ESBL)-producing E. coli strains and their sensitivity to flomoxef (FMOX). Sequence type (ST) analysis by multilocus sequence typing (MLST) and classification of ESBL genotypes by multiplex PCR were performed on ESBL-producing E. coli strains isolated from urine samples collected from patients treated at our institution between 2008 and 2018. These sequences were compared with results for antimicrobial drug susceptibility determined using a micro-liquid dilution method. We also analyzed cases treated with FMOX at our institution to examine its clinical efficacy. Of the 911 E. coli strains identified, 158 (17.3%) were ESBL-producing. Of these, 67.7% (107/158) were strain ST-131 in ST analysis. Nearly all (154/158; 97.5%) were CTX-M genotypes, with M-14 and M-27 predominating. The isolated strains were sensitive to FMOX in drug susceptibility tests. Among the patient samples, 33 cases received FMOX, and of these, 5 had ESBL-producing E. coli. Among these five cases, three received FMOX for surgical prophylaxis as urinary carriers of ESBL-producing E. coli, and postoperative infections were prevented in all three patients. The other two patients received FMOX treatment for urinary tract infections. FMOX treatment was successful for one, and the other was switched to carbapenem. Our results suggest that FMOX has efficacy for perioperative prophylactic administration in urologic surgery involving carriers of ESBL-producing bacteria and for therapeutic administration for urinary tract infections. Use of FMOX avoids over-reliance on carbapenems or beta-lactamase inhibitors and thus is an effective antimicrobial countermeasure. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 77 1 2023 Prevalence of Inducible Macrolide, Lincosamide, and Streptogramin B (inducible MLSB) Resistance in Clindamycin-Susceptible Staphylococcus aureus at Okayama University Hospital 1 9 EN Lutfun Nahar Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideharu Hagiya Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takahiro Nada Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koji Iio Microbiology Division, Clinical Laboratory, Okayama University Hospital Kazuyoshi Gotoh Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fumio Otsuka Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/64355 Inducible resistance to the macrolide, lincosamide, and streptogramin B (iMLSB) antibiotic family is a latent mechanism for antimicrobial resistance in Staphylococcus aureus. We here investigated the frequency and genotypic profiles of iMLSB resistance in clindamycin (CLDM)-susceptible S. aureus isolated in Okayama University Hospital from June 2020 to June 2021. We phenotypically screened the iMLSB resistance via D-zone test and performed PCR testing for the erythromycin ribosomal methylase (erm) genes: ermA and ermC. Among 432 CLDM-susceptible S. aureus isolates, 138 (31.9%) exhibited an iMLSB-resistance phenotype, with methicillinresistant S. aureus isolates (MRSA; 61 isolates: 58.6%) exhibiting higher positivity than methicillin-sensitive S. aureus isolates (MSSA; 77 isolates: 23.5%) (p<0.001). Male patients had a higher frequency of iMLSB resistance than females (OR [95%CI]: 1.8 [1.2-2.8]; p=0.007). Genotypically, ermA predominated in both MSSA (70.1%) and MRSA (86.9%) compared to ermC (14.3% in MSSA and 11.5% in MRSA). A single strain of MRSA possessed both ermA and ermC, while 12 (15.6%) MSSA isolates were negative for both ermA and ermC, suggesting the presence of other genetic mechanisms. Collectively, these results show that approximately 33% of CLDM-susceptible S. aureus isolates at our university hospital exhibited iMLSB resistance, predominantly caused by ermA in both MSSA and MRSA. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0300-8177 478 8 2022 Significance of UGT1A6, UGT1A9, and UGT2B7 genetic variants and their mRNA expression in the clinical outcome of renal cell carcinoma 1779 1790 EN Jun Matsumoto Department of Personalized Medicine and Preventive Healthcare Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Anzu Nishimoto Department of Personalized Medicine and Preventive Healthcare Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shogo Watari Department of Urology, National Hospital Organization Okayama Medical Center Hideo Ueki Department of Urology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Shoya Shiromizu Department of Pharmacy, Okayama University Hospital Naohiro Iwata Department of Pharmacy, Okayama University Hospital Tatsuaki Takeda Department of Pharmacy, Okayama University Hospital Soichiro Ushio Department of Pharmacy, Okayama University Hospital Makoto Kajizono Department of Pharmacy, Okayama University Hospital Masachika Fujiyoshi Department of Pharmacy, Tottori University Hospital Toshihiro Koyama Department of Pharmaceuticals Biomedicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Motoo Araki Department of Urology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Koichiro Wada Department of Urology, Faculty of Medicine, Shimane University Yoshito Zamami Department of Pharmacy, Okayama University Hospital Yasutomo Nasu Department of Urology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Noritaka Ariyoshi Department of Personalized Medicine and Preventive Healthcare Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University UDP-glucuronosyltransferase (UGT) metabolizes a number of endogenous and exogenous substrates. Renal cells express high amounts of UGT; however, the significance of UGT in patients with renal cell carcinoma (RCC) remains unknown. In this study, we profile the mRNA expression of UGT subtypes (UGT1A6, UGT1A9, and UGT2B7) and their genetic variants in the kidney tissue of 125 Japanese patients with RCC (Okayama University Hospital, Japan). In addition, we elucidate the association between the UGT variants and UGT mRNA expression levels and clinical outcomes in these patients. The three representative genetic variants, namely, UGT1A6 541A > G, UGT1A9 i399C > T, and UGT2B7-161C > T, were genotyped, and their mRNA expression levels in each tissue were determined. We found that the mRNA expression of the three UGTs (UGT1A6, UGT1A9, and UGT2B7) are significantly downregulated in RCC tissues. Moreover, in patients with RCC, the UGT2B7-161C > T variant and high UGT2B7 mRNA expression are significantly correlated with preferable cancer-specific survival (CSS) and overall survival (OS), respectively. As such, the UGT2B7-161C > T variant and UGT2B7 mRNA expression level were identified as significant independent prognostic factors of CSS and CSS/OS, respectively. Taken together, these findings indicate that UGT2B7 has a role in RCC progression and may, therefore, represent a potential prognostic biomarker for patients with RCC. No potential conflict of interest relevant to this article was reported.

Taylor & Francis Ltd Acta Medica Okayama 1742-9145 18 1 2023 Identification of quantitative trait loci associated with sorghum susceptibility to Asian stem borer damage 2153182 EN Cyprian Osinde Institute of Plant Science and Resources, Okayama University Wataru Sakamoto Institute of Plant Science and Resources, Okayama University Hiromi Kajiya-Kanegae Graduate School of Agricultural and Life Sciences, The University of Tokyo Islam S. Sobhy Institute of Plant Science and Resources, Okayama University Arthur K. Tugume Department of Plant Science, Microbiology and Biotechnology Makerere University Anthony M. Nsubuga Department of Plant Science, Microbiology and Biotechnology Makerere University Ivan Galis Institute of Plant Science and Resources, Okayama University Sorghum (Sorghum bicolor (L.) Moench) is an important crop originated in Africa that shows susceptibility to herbivores. In this study, we identified two sorghum genotypes with highly contrasting levels of stem damage caused by the caterpillars of Asian stem borer (Ostrinia furnacalis Guenee). Recombinant inbred lines (RILs) from genetic cross between resistant (BTx623) and susceptible (NOG) sorghum were used to perform a quantitative trait locus (QTL) analysis in the field. Two major QTLs responsible for higher NOG infestation by stem borer in three independent field seasons were detected on chromosomes 7 and 9, interestingly in positions that overlapped with two major QTLs for plant height. As plant height and stem borer damage were highly correlated, we propose that sorghum height-associated morphological or physiological traits could be important for stem borer establishment and/or damage in sorghum. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 76 6 2022 Association of Genetic Polymorphism with Taxane-induced Peripheral Neuropathy: Sub-analysis of a Randomized Phase II Study to Determine the Optimal Dose of 3-week Cycle Nab-Paclitaxel in Metastatic Breast Cancer Patients 661 671 EN Yuko Abe Department of Thoracic, Breast, and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Naruto Taira Department of Breast and Endocrine surgery, Kawasaki Medical School Hospital Kosuke Kashiwabara Clinical Research Promotion Center, University of Tokyo Hospital Junji Tsurutani Advanced Cancer Translational Research Institute, Showa University Masahiro Kitada Breast Disease Center, Asahikawa Medical University Hospital Masato Takahashi Department of Breast Surgery, National Hospital Organization Hokkaido Cancer Center Hiroaki Kato Department of Breast Surgery, Teine Keijinkai Hospital Yuichiro Kikawa Department of Breast Surgery, Kansai Medical University Hospital Eiko Sakata Department of Breast Surgery, Niigata City General Hospital Yoichi Naito Department of Medical Oncology, National Cancer Center Hospital East Yoshie Hasegawa Department of Breast Surgery, Hachinohe City Hospital Tsuyoshi Saito Department of Breast Surgery, Japanese Red Cross Saitama Hospital Tsutomu Iwasa Department of Medical Oncology, Kindai University Faculty of Medicine Tsutomu Takashima Department of Breast and Endocrine Surgery, Osaka City University Graduate School of Medicine Tomohiko Aihara Breast Center, Aihara Hospital Hirofumi Mukai Department of Medical Oncology, National Cancer Center Hospital East Fumikata Hara Breast Oncology Center, Cancer Institute Hospital of Japanese Foundation for Cancer Research Tadahiko Shien Department of Breast and Endocrine surgery, Okayama University Hospital Hiroyoshi Doihara Department of Breast surgery, Kawasaki Medical School General Medical Center Shinichi Toyooka Department of Thoracic, Breast, and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/64116 Chemotherapy-induced peripheral neuropathy (CIPN) is an important clinical challenge that threatens patients’ quality of life. This sub-study of the ABROAD trial investigated the influence of single nucleotide polymorphisms (SNPs) on CIPN, using genotype data from a randomized study to determine the optimal dose of a 3-week-cycle regimen of nab-paclitaxel (q3w nab-PTX) in patients with metastatic breast cancer (MBC). Patients with HER2-negative MBC were randomly assigned to three doses of q3w nab-PTX (SD: 260 mg/m2 vs. MD: 220 mg/m2 vs. LD: 180 mg/m2). Five SNPs (EPHA4-rs17348202, EPHA5-rs7349683, EPHA6-rs301927, LIMK2-rs5749248, and XKR4-rs4737264) were analyzed based on the results of a previous genome-wide association study. Per-allele SNP associations were assessed by a Cox regression to model the cumulative dose of nab-PTX up to the onset of severe or worsening sensory neuropathy. A total of 141 patients were enrolled in the parent study; 91(65%) were included in this sub-study. Worsening of CIPN was significantly greater in the cases with XKR4 AC compared to those with a homozygote AA (HR 1.86, 95%CI: 1.00001−3.46, p=0.049). There was no significant correlation of CIPN with any other SNP. A multivariate analysis showed that the cumulative dose of nab-PTX was most strongly correlated with CIPN (p<0.01). No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 76 6 2022 Knockdown of LncRNA SBF2-AS1 Inhibited Gastric Cancer Tumorigenesis via the Wnt/LRP5 Signaling Pathway 625 633 EN Department of General surgery, Affiliated Qingdao Hiser Hospital of Qingdao University (Qingdao Hospital of Traditional Chinese Medicine) Qingmei Li Department of General surgery, Affiliated Qingdao Hiser Hospital of Qingdao University (Qingdao Hospital of Traditional Chinese Medicine) Ye Wang Department of General surgery, Affiliated Qingdao Hiser Hospital of Qingdao University (Qingdao Hospital of Traditional Chinese Medicine) Yunjie Ge Department of Healthcare Internal Medicine, Affiliated Qingdao Municipal Hospital of Qingdao University Original Article 10.18926/AMO/64112 This investigation aimed to uncover the impact of a long noncoding RNA, SET-binding factor 2 antisense RNA1 (SBF2-AS1) on the malignant progression of gastric cancer (GC) and to further explore its underlying mechanism. SBF2-AS1 expression was quantified by qRT-PCR in GC cell lines and GC tissues. In vitro loss-of-function studies of SBF2-AS1, accompanied by flow cytometry, CCK-8, and cell invasion tests, were applied to elucidate the impact of SBF2-AS1 on the tumor progression of GC cells. Finally, Western blotting and a luciferase assay were used to detect WNT/LRP5 signaling pathway activation. SBF2-AS1 was aberrantly expressed in GC cell lines (p<0.05) and GC tissues (p<0.05). Cell invasive and proliferative capabilities were inhibited via SBF2-AS1 knockdown, resulting in apoptosis of NCI-N87 and MKN74 cells. Additionally, online database analysis uncovered a positive correlation between SBF2-AS1 and the Wnt/LRP5 signaling pathway (p<0.05). SBF2-AS1 knockdown blocked the Wnt/LRP5 signaling pathway, whereas the effects of SBF2-AS1 knockdown on the malignant genotype of MKN74 as well as NCI-N87 cells were partially restored by triggering the Wnt/ LRP5 signaling pathway. High expression of SBF2-AS1 was found in GC, the malignant progression of which was repressed via SBF2-AS1 knockdown by inhibiting the Wnt/LRP5 signaling pathway. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0140-7791 45 11 2022 FE UPTAKE‐INDUCING PEPTIDE1 maintains Fe translocation by controlling Fe deficiency response genes in the vascular tissue of Arabidopsis 3322 3337 EN Satoshi Okada Group of Environmental Stress Response Systems, Institute of Plant Science and Resources, Okayama University Gui J. Lei Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Naoki Yamaji Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Sheng Huang Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Jian F. Ma Group of Plant Stress Physiology, Institute of Plant Science and Resources, Okayama University Keiichi Mochida Crop Design Research Team, Institute of Plant Science and Resources, Okayama University Takashi Hirayama Group of Environmental Stress Response Systems, Institute of Plant Science and Resources, Okayama University FE UPTAKE-INDUCING PEPTIDE1 (FEP1), also named IRON MAN3 (IMA3) is a short peptide involved in the iron deficiency response in Arabidopsis thaliana. Recent studies uncovered its molecular function, but its physiological function in the systemic Fe response is not fully understood. To explore the physiological function of FEP1 in iron homoeostasis, we performed a transcriptome analysis using the FEP1 loss-of-function mutant fep1-1 and a transgenic line with oestrogen-inducible expression of FEP1. We determined that FEP1 specifically regulates several iron deficiency-responsive genes, indicating that FEP1 participates in iron translocation rather than iron uptake in roots. The iron concentration in xylem sap under iron-deficient conditions was lower in the fep1-1 mutant and higher in FEP1-induced transgenic plants compared with the wild type (WT). Perls staining revealed a greater accumulation of iron in the cortex of fep1-1 roots than in the WT root cortex, although total iron levels in roots were comparable in the two genotypes. Moreover, the fep1-1 mutation partially suppressed the iron overaccumulation phenotype in the leaves of the oligopeptide transporter3-2 (opt3-2) mutant. These data suggest that FEP1 plays a pivotal role in iron movement and in maintaining the iron quota in vascular tissues in Arabidopsis. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 76 2 2022 Changes in Plasma Clozapine Levels after Smoking Cessation in Japanese Inpatients with Schizophrenia: A Retrospective Cohort Study 137 143 EN Masaru Tsukahara Department of Psychiatry, Okayama Psychiatric Medical Center Ryuhei So Department of Psychiatry, Okayama Psychiatric Medical Center Yuji Yada Department of Psychiatry, Okayama Psychiatric Medical Center Masafumi Kodama Department of Psychiatry, Okayama Psychiatric Medical Center Yoshiki Kishi Department of Psychiatry, Okayama Psychiatric Medical Center Norihito Yamada Department of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/63407 Although reported for Caucasians, changes in plasma clozapine levels after smoking cessation in East Asians remain unclear. We here investigated plasma clozapine levels before and after smoking cessation in Japanese inpatients with schizophrenia. We conducted a retrospective chart review of 14 inpatients with schizophrenia who were being treated with clozapine between June 1, 2019, and July 31, 2019 and who were smokers as of July 1, 2019, the day on which a smoking ban was instituted in the tertiary public psychiatric hospital. The primary outcome was individual differences in plasma clozapine levels between before and after the smoking ban, which were compared using paired t-tests. The mean plasma clozapine level was significantly increased, by 213.4 ng/mL (95% CI 119.9-306.8; p<0.01) or 53.2%. Four of the 14 inpatients experienced clinically significant side effects, such as myoclonus, drooling, and amnesia, due to the development of high plasma clozapine levels. Our findings indicated that close monitoring of plasma clozapine levels before and after smoking cessation and prior dose adjustment of clozapine may be necessary, to prevent a significant risk of developing high plasma clozapine levels, even in Japanese patients. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 1340-2838 29 1 2022 Chromosome-scale assembly of barley cv. 'Haruna Nijo' as a resource for barley genetics dsac001 EN Areej Sakkour Institute of Plant Science and Resources, Okayama University Martin Mascher Department Genebank, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Axel Himmelbach Department Genebank, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Georg Haberer Plant Genome and Systems Biology (PGSB), Helmholtz Center Munich, German Research Center for Environmental Health Thomas Lux Plant Genome and Systems Biology (PGSB), Helmholtz Center Munich, German Research Center for Environmental Health Manuel Spannagl Plant Genome and Systems Biology (PGSB), Helmholtz Center Munich, German Research Center for Environmental Health Nils Stein Department Genebank, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Shoko Kawamoto Department of Informatics, National Institute of Genetics Kazuhiro Sato Institute of Plant Science and Resources, Okayama University Cultivated barley (Hordeum vulgare ssp. vulgare) is used for food, animal feed, and alcoholic beverages and is widely grown in temperate regions. Both barley and its wild progenitor (H. vulgare ssp. spontaneum) have large 5.1-Gb genomes. High-quality chromosome-scale assemblies for several representative barley genotypes, both wild and domesticated, have been constructed recently to populate the nascent barley pan-genome infrastructure. Here, we release a chromosome-scale assembly of the Japanese elite malting barley cultivar 'Haruna Nijo' using a similar methodology as in the barley pan-genome project. The 4.28-Gb assembly had a scaffold N50 size of 18.9 Mb. The assembly showed high collinearity with the barley reference genome 'Morex' cultivar, with some inversions. The pseudomolecule assembly was characterized using transcript evidence of gene projection derived from the reference genome and de novo gene annotation achieved using published full-length cDNA sequences and RNA-Seq data for 'Haruna Nijo'. We found good concordance between our whole-genome assembly and the publicly available BAC clone sequence of 'Haruna Nijo'. Interesting phenotypes have since been identified in Haruna Nijo; its genome sequence assembly will facilitate the identification of the underlying genes. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2075-1729 12 1 2021 Whole Exome-Sequencing of Pooled Genomic DNA Samples to Detect Quantitative Trait Loci in Esotropia and Exotropia of Strabismus in Japanese 41 EN Jingjing Zhang Regenerative and Reconstructive Medicine (Ophthalmology), Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toshihiko Matsuo Regenerative and Reconstructive Medicine (Ophthalmology), Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ichiro Hamasaki Department of Ophthalmology, Okayama University Hospital Kazuhiro Sato Barley and Wild Plant Resource Center, Institute of Plant Science and Resources (IPSR), Okayama University Background: Esotropia and exotropia are two major phenotypes of comitant strabismus. It remains controversial whether esotropia and exotropia would share common genetic backgrounds. In this study, we used a quantitative trait locus (QTL)-sequencing pipeline for diploid plants to screen for susceptibility loci of strabismus in whole exome sequencing of pooled genomic DNAs of individuals. Methods: Pooled genomic DNA (2.5 ng each) of 20 individuals in three groups, Japanese patients with esotropia and exotropia, and normal members in the families, was sequenced twice after exome capture, and the first and second sets of data in each group were combined to increase the read depth. The SNP index, as the ratio of variant genotype reads to all reads, and Δ(SNP index) values, as the difference of SNP index between two groups, were calculated by sliding window analysis with a 4 Mb window size and 10 kb slide size. The rows of 200 “N”s were inserted as a putative 200-b spacer between every adjoining locus to depict Δ(SNP index) plots on each chromosome. SNP positions with depth &lt; 20 as well as SNP positions with SNP index of &lt;0.3 were excluded. Results: After the exclusion of SNPs, 12,242 SNPs in esotropia/normal group and 12,108 SNPs in exotropia/normal group remained. The patterns of the Δ(SNP index) plots on each chromosome appeared different between esotropia/normal group and exotropia/normal group. When the consecutive groups of SNPs on each chromosome were set at three patterns: SNPs in each cytogenetic band, 50 consecutive sliding SNPs, and SNPs in 4 Mb window size with 10 kb slide size, p values (Wilcoxon signed rank test) and Q values (false discovery rate) in a few loci as Manhattan plots showed significant differences in comparison between the Δ(SNP index) in the esotropia/normal group and exotropia/normal group. Conclusions: The pooled DNA sequencing and QTL mapping approach for plants could provide overview of genetic background on each chromosome and would suggest different genetic backgrounds for two major phenotypes of comitant strabismus, esotropia and exotropia. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2021 Genetic analysis in Japanese patients with osteogenesis imperfecta: genotype and phenotype spectra in 96 probands EN Yosuke Higuchi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 1664-302X 12 2021 Comprehensive Comparative Genomics and Phenotyping of Methylobacterium Species 740610 EN Ola Alessa Institute of Plant Science and Resources, Okayama University Yoshitoshi Ogura Division of Microbiology, Department of Infectious Medicine, Kurume University School of Medicine Yoshiko Fujitani Institute of Plant Science and Resources, Okayama University Hideto Takami Atmosphere and Ocean Research Institute, The University of Tokyo Tetsuya Hayashi Department of Bacteriology, Graduate School of Medical Sciences, Kyushu University Nurettin Sahin Egitim Fakultesi, Mugla Sitki Kocman University Akio Tani Institute of Plant Science and Resources, Okayama University The pink-pigmented facultative methylotrophs (PPFMs), a major bacterial group found in the plant phyllosphere, comprise two genera: Methylobacterium and Methylorubrum. They have been separated into three major clades: A, B (Methylorubrum), and C. Within these genera, however, some species lack either pigmentation or methylotrophy, which raises the question of what actually defines the PPFMs. The present study employed a comprehensive comparative genomics approach to reveal the phylogenetic relationship among the PPFMs and to explain the genotypic differences that confer their different phenotypes. We newly sequenced the genomes of 29 relevant-type strains to complete a dataset for almost all validly published species in the genera. Through comparative analysis, we revealed that methylotrophy, nitrate utilization, and anoxygenic photosynthesis are hallmarks differentiating the PPFMs from the other Methylobacteriaceae. The Methylobacterium species in clade A, including the type species Methylobacterium organophilum, were phylogenetically classified into six subclades, each possessing relatively high genomic homology and shared phenotypic characteristics. One of these subclades is phylogenetically close to Methylorubrum species; this finding led us to reunite the two genera into a single genus Methylobacterium. Clade C, meanwhile, is composed of phylogenetically distinct species that share relatively higher percent G+C content and larger genome sizes, including larger numbers of secondary metabolite clusters. Most species of clade C and some of clade A have the glutathione-dependent pathway for formaldehyde oxidation in addition to the H4MPT pathway. Some species cannot utilize methanol due to their lack of MxaF-type methanol dehydrogenase (MDH), but most harbor an XoxF-type MDH that enables growth on methanol in the presence of lanthanum. The genomes of PPFMs encode between two and seven (average 3.7) genes for pyrroloquinoline quinone-dependent alcohol dehydrogenases, and their phylogeny is distinctly correlated with their genomic phylogeny. All PPFMs were capable of synthesizing auxin and did not induce any immune response in rice cells. Other phenotypes including sugar utilization, antibiotic resistance, and antifungal activity correlated with their phylogenetic relationship. This study provides the first inclusive genotypic insight into the phylogeny and phenotypes of PPFMs. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 75 6 2021 Pulmonary Enteric Adenocarcinoma Harboring a BRAF G469V Mutation 759 762 EN Dai Shimizu Department of Thoracic Surgery, Okayama University Hospital Hiromasa Yamamoto Department of Thoracic Surgery, Okayama University Hospital Kazuhiko Shien Department of Thoracic Surgery, Okayama University Hospital Kohei Taniguchi Department of Diagnostic Pathology, Okayama University Hospital Kentaroh Miyoshi Department of Thoracic Surgery, Okayama University Hospital Kei Namba Department of Thoracic Surgery, Okayama University Hospital Kumi Mesaki Department of Thoracic Surgery, Okayama University Hospital Seiichiro Sugimoto Department of Thoracic Surgery, Okayama University Hospital Junichi Soh Department of Thoracic Surgery, Okayama University Hospital Masaomi Yamane Department of Thoracic Surgery, Okayama University Hospital Shinichi Toyooka Department of Thoracic Surgery, Okayama University Hospital Case Report 10.18926/AMO/62819 Pulmonary enteric adenocarcinoma (PEAC) is a rare subtype of lung cancer that should be differentiated from colorectal cancer metastasis. Little is known about its genetic background. An 84-year-old male with adenocarcinoma of the lung underwent left upper lobectomy. The histology of the surgical specimen was suggestive of PEAC. Gastrointestinal and colorectal fiberscopy revealed no evidence of colorectal cancer. Next-generation sequencing of the tumor identified a G469V substitution in serine/threonine-protein kinase B-raf (BRAF). Based on the higher prevalence of the G469 substitution in BRAF-mutant lung adenocarcinoma than in BRAFmutant colorectal cancer, the tumor likely originated from the lung. Identification of mutational genotype may be of some help in distinguishing PEAC from the lung metastasis of colorectal cancer. No potential conflict of interest relevant to this article was reported.

Elsevier Ltd. Acta Medica Okayama 07335210 101 2021 Germplasm evaluation for crop improvement: Analysis of grain quality and cadmium accumulation in barley 103297 EN Kazuhiro Sato Institute of Plant Science and Resources, Okayama University Kazuyoshi Takeda Institute of Plant Science and Resources, Okayama University Jian Feng Ma Institute of Plant Science and Resources, Okayama University Evaluating genetic variation in barley (Hordeum vulgare) germplasm, combined with genome-wide genotyping, is vital for identifying genes controlling important grain-quality traits. For example, in addition to traditional grain quality properties such as starch and protein contents, grain safety parameters such as heavy metal content, are important in the use of barley for human food and animal feed. A number of genes affecting grain quality have been identified by map-based cloning strategies and functionally analyzed by genetic transformation experiments. Moreover, germplasm evaluation yielded information that enabled the introgression of a key gene controlling grain cadmium accumulation into an elite barley cultivar, reducing the content of this heavy metal in grain. Genotyping of molecular markers and resequencing of germplasm accessions may provide information about how grain quality–related loci evolved and how the current allelic diversity was established. In this review, we describe germplasm resources for barley grain quality–related traits and the methods used to analyze the functions of genes controlling these traits, illustrating cadmium accumulation as an example. We also discuss future directions for the efficient identification of grain quality–related genes. No potential conflict of interest relevant to this article was reported.

Elsevier Ltd. Acta Medica Okayama 13474367 40 2021 Blood concentrations of tacrolimus upon conversion from rabeprazole to vonoprazan in renal transplant recipients: Correlation with cytochrome P450 gene polymorphisms 100407 EN Shogo Watari Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Motoo Araki Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Jun Matsumoto Department of Personalized Medicine and Preventive Healthcare Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kasumi Yoshinaga Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Sekito Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Maruyama Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yosuke Mitsui Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takuya Sadahira Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Risa Kubota Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shingo Nishimura Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Koichiro Wada Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yasuyuki Kobayashi Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hidemi Takeuchi Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Katsuyuki Tanabe Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Masashi Kitagawa Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Hiroshi Morinaga Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Shinji Kitamura Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Hitoshi Sugiyama Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Noritaka Ariyoshi Department of Personalized Medicine and Preventive Healthcare Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Masami Watanabe Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Toyohiko Watanabe Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yasutomo Nasu Department of Urology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University We evaluated the impact of vonoprazan on blood concentrations of tacrolimus via a retrospective analysis of 52 renal transplant recipients who took tacrolimus and converted from rabeprazole to vonoprazan between August 2018 and September 2019. We compared tacrolimus trough levels upon conversion among groups that were classified based on cytochrome P450 (CYP) gene polymorphisms. CYP3A5 groups were heterozygous or homozygous for CYP3A5∗1 and CYP3A5∗3 alleles. CYP2C19 genotypes were classified as extensive (∗1/∗1), intermediate (∗1/∗2 and ∗1/∗3) or poor metabolizers (∗2/∗2, ∗2/∗3 and ∗3/∗3). Tacrolimus trough levels increased only 0.3 ng/mL upon conversion in the CYP3A5∗3/∗3 group: 5.8 [3.4-7.2] vs 6.1 [3.8-7.9]; p = 0.06. No statistically significance changes in tacrolimus levels also occurred in the CYP3A5∗1/∗1 or CYP3A5∗1/∗3 groups. Subgroup analyses of CYP3A5∗3/∗3 demonstrated low changes for all three CYP2C19 subgroups: 5.2 [4.3-6.5] vs 6.2 [4.3-7.9]; p = 0.07, 6.1 [3.4-7.2] vs 6.7 [4.6-7.9]; p = 0.12 and 5.4 [3.6-6.5] vs 4.7 [3.8-6.3]; p = 1.00, respectively. Conversion to vonoprazan thus resulted in little increase of tacrolimus trough levels, even in the group predicted to be most susceptible (CYP3A5∗3/∗3 and 2C19∗1/∗1), thus supporting the safety of concomitant use of vonoprazan with tacrolimus. No potential conflict of interest relevant to this article was reported.

Frontiers Media SA Acta Medica Okayama 1664-302X 12 2021 Virulence of Cholera Toxin Gene-Positive Vibrio cholerae Non-O1/non-O139 Strains Isolated From Environmental Water in Kolkata, India 726273 EN Eizo Takahashi Collaborative Research Center of Okayama University for Infectious Diseases in India Sadayuki Ochi Department of Health Pharmacy, Yokohama University of Pharmacy Tamaki Mizuno Graduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama University Daichi Morita Collaborative Research Center of Okayama University for Infectious Diseases in India Masatomo Morita Department of Bacteriology I, National Institute of Infectious Diseases Makoto Ohnishi Department of Bacteriology I, National Institute of Infectious Diseases Hemanta Koley National Institute of Cholera and Enteric Diseases Moumita Dutta National Institute of Cholera and Enteric Diseases Goutam Chowdhury National Institute of Cholera and Enteric Diseases Asish K. Mukhopadhyay National Institute of Cholera and Enteric Diseases Shanta Dutta National Institute of Cholera and Enteric Diseases Shin-Ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences of Okayama University Keinosuke Okamoto Collaborative Research Center of Okayama University for Infectious Diseases in India Cholera toxin (CT)-producing Vibrio cholerae O1 and O139 cause acute diarrheal disease and are proven etiological agents of cholera epidemics and pandemics. On the other hand, V. cholerae non-O1/non-O139 are designated as non-agglutinable (NAG) vibrios and are not associated with epidemic cholera. The majority of NAG vibrios do not possess the gene for CT (ctx). In this study, we isolated three NAG strains (strains No. 1, 2, and 3) with ctx from pond water in Kolkata, India, and examined their pathogenic properties. The enterotoxicity of the three NAG strains in vivo was examined using the rabbit ileal intestinal loop test. Strain No. 1 induced the accumulation of fluid in the loop, and the volume of fluid was reduced by simultaneous administration of anti-CT antiserum into the loop. The volume of fluid in the loop caused by strains No. 2 and 3 was small and undetectable, respectively. Then, we cultured these three strains in liquid medium in vitro at two temperatures, 25 degrees C and 37 degrees C, and examined the amount of CT accumulated in the culture supernatant. CT was accumulated in the culture supernatant of strain No.1 when the strain was cultured at 25 degrees C, but that was low when cultured at 37 degrees C. The CT amount accumulated in the culture supernatants of the No. 2 and No. 3 strains was extremely low at both temperature under culture conditions examined. In order to clarify the virulence properties of these strains, genome sequences of the three strains were analyzed. The analysis showed that there was no noticeable difference among three isolates both in the genes for virulence factors and regulatory genes of ctx. However, vibrio seventh pandemic island-II (VSP-II) was retained in strain No. 1, but not in strains No. 2 or 3. Furthermore, it was revealed that the genotype of the B subunit of CT in strain No. 1 was type 1 and those of strains No. 2 and 3 were type 8. Histopathological examination showed the disappearance of villi in intestinal tissue exposed to strain No. 1. In addition, fluid accumulated in the loop due to the action of strain No. 1 had hemolytic activity. This indicated that strain No. 1 may possesses virulence factors to induce severe syndrome when the strain infects humans, and that some strains of NAG vibrio inhabiting pond water in Kolkata have already acquired virulence, which can cause illness in humans. There is a possibility that these virulent NAG vibrios, which have acquired genes encoding factors involved in virulence of V. cholerae O1, may emerge in various parts of the world and cause epidemics in the future. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 75 4 2021 Two Types of Polyp Shape Observed in the Stomach of Patients with Peutz-Jeghers Syndrome 471 477 EN Masaya Iwamuro Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tatsuya Toyokawa Department of Gastroenterology, Fukuyama Medical Center Kazuhiro Matsueda Department of Gastroenterology and Hepatology, Kurashiki Central Hospital Shinichiro Hori Department of Endoscopy, Shikoku Cancer Center Masao Yoshioka Department of Internal Medicine, Okayama Saiseikai General Hospital Yuki Moritou Department of Internal Medicine, Hiroshima City Hiroshima Citizens Hospital Takehiro Tanaka Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Motowo Mizuno Department of Gastroenterology and Hepatology, Kurashiki Central Hospital Hiroyuki Okada Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/62399 The characteristics of gastric polyps in patients with Peutz-Jeghers (PJ) syndrome (PJS) have not been fully investigated. The objective of this study was to reveal the endoscopic and pathologic findings of gastric polyps in patients with PJS. We reviewed 11 patients with PJS treated at 6 institutions, and summarized the endo-scopic and pathologic features of their gastric polyps. The polyps were mainly classified into 2 types: (i) soli-tary or sporadic polyps > 5 mm, reddish in color with a sessile or semi-pedunculated morphology (n = 9); and (ii) multiple sessile polyps ≤ 5 mm with the same color tone as the peripheral mucosa (n = 9). Patients who underwent endoscopic mucosal resection for polyps > 5 mm were diagnosed with PJ polyps (n = 2), whereas those who underwent biopsy were diagnosed with hyperplastic polyps. Polyps ≤ 5 mm were pathologically diagnosed as fundic gland polyps or hyperplastic polyps. This study revealed that patients with PJS present with 2 types of polyps in the stomach. Endoscopic mucosal resection of polyps > 5 mm seems necessary for the pathologic diagnosis of PJ polyps. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 75 4 2021 The Cell Cycle Checkpoint Gene, RAD17 rs1045051, Is Associated with Prostate Cancer Risk 415 421 EN Jingkai Sun Department of Urology, Zhujiang Hospital, Southern Medical University Wenfeng Lin Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Qixu Wang Department of Urology, Zhujiang Hospital, Southern Medical University Akiko Sakai Department of Molecular Genetics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ruizhi Xue Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masami Watanabe Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Chunxiao Liu Department of Urology, Zhujiang Hospital, Southern Medical University Takuya Sadahira Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasutomo Nasu Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Abai Xu Department of Urology, Zhujiang Hospital, Southern Medical University Peng Huang Department of Urology, Zhujiang Hospital, Southern Medical University Original Article 10.18926/AMO/62379 Human RAD17, as an agonist of checkpoint signaling, plays an essential role in mediating DNA damage. This hospital-based case-control study aimed to explore the association between RAD17 rs1045051, a missense sin-gle nucleotide polymorphism (SNP), and prostate cancer risk. Subjects were 358 prostate cancer patients and 314 cancer-free urology patients undergoing treatment at the Zhujiang Hospital of Southern Medical University in China. RAD17 gene polymorphism rs1045051 was evaluated by the SNaPshot method. Compared with the RAD17 gene polymorphism rs1045051 AA genotype, there was a higher risk of prostate cancer for the CC gen-otype (adjusted odds ratio [AOR] = 1.731, 95% confidence interval [95%CI] = 1.031−2.908, p = 0.038). Compared with the A allele, the C allele was significantly associated with the disease status (AOR = 1.302, 95%CI = 1.037−1.634, p = 0.023). All these findings indicate that in the SNP rs1045051, both the CC genotype and C allele may have a substantial influence on the prostate cancer risk. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 2160-1836 2021 Chromosome-scale assembly of wild barley accession “OUH602” jkab244 EN Kazuhiro Sato Institute of Plant Science and Resources, Okayama University Martin Mascher Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben Axel Himmelbach Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben Georg Haberer Plant Genome and Systems Biology (PGSB), Helmholtz Center Munich, German Research Center for Environmental Health Manuel Spannagl Plant Genome and Systems Biology (PGSB), Helmholtz Center Munich, German Research Center for Environmental Health Nils Stein Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben Barley (Hordeum vulgare) was domesticated from its wild ancestral form ca. 10,000 years ago in the Fertile Crescent and is widely cultivated throughout the world, except for in tropical areas. The genome size of both cultivated barley and its conspecific wild ancestor is approximately 5 Gb. High-quality chromosome-level assemblies of 19 cultivated and one wild barley genotype were recently established by pan-genome analysis. Here, we release another equivalent short-read assembly of the wild barley accession “OUH602.” A series of genetic and genomic resources were developed for this genotype in prior studies. Our assembly contains more than 4.4 Gb of sequence, with a scaffold N50 value of over 10 Mb. The haplotype shows high collinearity with the most recently updated barley reference genome, “Morex” V3, with some inversions. Gene projections based on “Morex” gene models revealed 46,807 protein-coding sequences and 43,375 protein-coding genes. Alignments to publicly available sequences of bacterial artificial chromosome (BAC) clones of “OUH602” confirm the high accuracy of the assembly. Since more loci of interest have been identified in “OUH602,” the release of this assembly, with detailed genomic information, should accelerate gene identification and the utilization of this key wild barley accession. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1660-4601 18 12 2021 Comprehensive Analysis of Risk Factors for Periodontitis Focusing on the Saliva Microbiome and Polymorphism 6430 EN Naoki Toyama Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Daisuke Ekuni Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Daisuke Matsui Department of Epidemiology for Community Health and Medicine, Kyoto Prefectural University of Medicine Teruhide Koyama Department of Epidemiology for Community Health and Medicine, Kyoto Prefectural University of Medicine Masahiro Nakatochi Public Health Informatics Unit, Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine Yukihide Momozawa Laboratory for Genotyping Development, RIKEN Center for Integrative Medical Sciences Michiaki Kubo Laboratory for Genotyping Development, RIKEN Center for Integrative Medical Sciences Manabu Morita Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Few studies have exhaustively assessed relationships among polymorphisms, the microbiome, and periodontitis. The objective of the present study was to assess associations simultaneously among polymorphisms, the microbiome, and periodontitis. We used propensity score matching with a 1:1 ratio to select subjects, and then 22 individuals (mean age +/- standard deviation, 60.7 +/- 9.9 years) were analyzed. After saliva collection, V3-4 regions of the 16S rRNA gene were sequenced to investigate microbiome composition, alpha diversity (Shannon index, Simpson index, Chao1, and abundance-based coverage estimator) and beta diversity using principal coordinate analysis (PCoA) based on weighted and unweighted UniFrac distances. A total of 51 single-nucleotide polymorphisms (SNPs) related to periodontitis were identified. The frequencies of SNPs were collected from Genome-Wide Association Study data. The PCoA of unweighted UniFrac distance showed a significant difference between periodontitis and control groups (p < 0.05). There were no significant differences in alpha diversity and PCoA of weighted UniFrac distance (p > 0.05). Two families (Lactobacillaceae and Desulfobulbaceae) and one species (Porphyromonas gingivalis) were observed only in the periodontitis group. No SNPs showed significant expression. These results suggest that periodontitis was related to the presence of P. gingivalis and the families Lactobacillaceae and Desulfobulbaceae but not SNPs. No potential conflict of interest relevant to this article was reported.

Taylor ＆ Francis Ltd. Acta Medica Okayama 2000-2297 13 1 2021 Efficacy of FimA antibody and clindamycin in silkworm larvae stimulated with Porphyromonas gulae 1914499 EN Sho Yoshida Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroaki Inaba Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryota Nomura Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry Masaru Murakami Departments of Pharmacology, Veterinary Public Health II and Molecular Biology, School of Veterinary Medicine, Azabu University Hidemi Yasuda Yasuda Veterinary Clinic Kazuhiko Nakano Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry Michiyo Matsumoto-Nakano Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Objective: Porphyromonas gulae, a major periodontal pathogen in animals, possesses fimbriae that have been classified into three genotypes (A, B, C) based on the diversity of fimA genes encoding fimbrillin protein (FimA). P. gulae strains with type C fimbriae were previously shown to be more virulent than other types. In this study, we further examined the host toxicity mediated by P. gulae fimbriae by constructing recombinant FimA (rFimA) expression vectors for each genotype and raised antibodies to the purified proteins. Methods and Results: All larvae died within 204 h following infection with P. gulae type C at the low-dose infection, whereas type A and B did not. Among fimA types, the survival rates of the larvae injected with rFimA type C were remarkably decreased, while the survival rates of the larvae injected with rFimA type A and type B were greater than 50%. Clindamycin treatment inhibited the growth of type C strains in a dose-dependent manner, resulting in an increased rate of silkworm survival. Finally, type C rFimA-speci?c antiserum prolonged the survival of silkworm larvae stimulated by infection with P. gulae type C strain or injection of rFimA type C protein. Conclusion: These results suggested that type C fimbriae have high potential for enhancement of bacterial pathogenesis, and that both clindamycin and anti-type C rFimA-specific antibodies are potent inhibitors of type C fimbriae-induced toxicity. This is the first report to establish a silkworm infection model using P. gulae for toxicity assessment. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 2324-9269 2021 Genetic analysis in Japanese patients with osteogenesis imperfecta: Genotype and phenotype spectra in 96 probands e1675 EN Yousuke Higuchi Department of Pediatrics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kosei Hasegawa Department of Pediatrics, Okayama University Hospital Natsuko Futagawa Department of Pediatrics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Miho Yamashita Faculty of Human Life Sciences, Notre Dame Seishin University Hiroyuki Tanaka Department of Pediatrics, Okayama Saiseikai General Hospital Hirokazu Tsukahara Department of Pediatrics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background Osteogenesis imperfecta (OI) is a rare connective-tissue disorder characterized by bone fragility. Approximately 90% of all OI cases are caused by variants in COL1A1 or COL1A2. Additionally, IFITM5 variants are responsible for the unique OI type 5. We previously analyzed COL1A1/2 variants in 22 Japanese families with OI through denaturing high-performance liquid chromatography screening, but our detection rate was low (41%). Methods To expand the genotype-phenotype correlations, we performed a genetic analysis of COL1A1/2 and IFITM5 in 96 non-consanguineous Japanese OI probands by Sanger sequencing. Results Of these individuals, 54, 41, and 1 had type 1 (mild), type 2-4 (moderate-to-severe), and type 5 phenotypes, respectively. In the mild group, COL1A1 nonsense and splice-site variants were prevalent (n = 30 and 20, respectively), but there were also COL1A1 and COL1A2 triple-helical glycine substitutions (n = 2 and 1, respectively). In the moderate-to-severe group, although COL1A1 and COL1A2 glycine substitutions were common (n = 14 and 18, respectively), other variants were also detected. The single case of type 5 had the characteristic c.-14C>T variant in IFITM5. Conclusion These results increase our previous detection rate for COL1A1/2 variants to 99% and provide insight into the genotype-phenotype correlations in OI. No potential conflict of interest relevant to this article was reported.

Spandidos Publications Acta Medica Okayama 2049-9434 13 5 2020 Human NINEIN polymorphism at codon 1111 is associated with the risk of colorectal cancer 45 EN Yukiko Yasuda Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akiko Sakai Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Sachio Ito Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kaori Sasai Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akisada Ishizaki Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshiya Okano Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seito Kawahara Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshimi Jitsumori Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nagahide Matsubara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kenji Shimizu Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Katayama Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences NINEIN serves an essential role in centrosome function as a microtubule organizing center, and in the reformation of the interphase centrosome architecture following mitosis. In the present study, the association between NINEIN Pro1111Ala (rs2236316), a missense single nucleotide polymorphism, and the risk of colorectal cancer (CRC), related to smoking and alcohol consumption habits in 200 patients with CRC and 1,141 cancer‑free control participants were assessed in a case‑control study performed in Japan. The results showed that the NINEIN Ala/Ala genotype compared with the Pro/Pro genotype was significantly more associated with an increased risk of CRC, and the males with the Ala/Ala genotype exhibited a significantly increased risk of CRC compared with those with Pro/Pro and Pro/Ala genotypes. Stratified analyses of the Ala/Ala genotype with CRC risk further showed an increased association in never/light drinkers (<23 g of ethanol/day), in male never/light drinkers and in male patients with rectal cancer. These findings suggest that the genetic variant of the NINEIN Pro1111Ala polymorphism has a significant effect on CRC susceptibility in the Japanese population. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 1664-462X 11 2020 Postharvest Properties of Ultra-Late Maturing Peach Cultivars and Their Attributions to Melting Flesh (M) Locus: Re-evaluation of M Locus in Association With Flesh Texture 554158 EN Ryohei Nakano Experimental Farm of Graduate School of Agriculture, Kyoto University Takashi Kawai Graduate School of Environmental and Life Science, Okayama University Yosuke Fukamatsu Graduate School of Environmental and Life Science, Okayama University Kagari Akita Graduate School of Environmental and Life Science, Okayama University Sakine Watanabe Graduate School of Environmental and Life Science, Okayama University Takahiro Asano Graduate School of Environmental and Life Science, Okayama University Daisuke Takata Faculty of Food and Agricultural Sciences, Fukushima University Mamoru Sato Faculty of Food and Agricultural Sciences, Fukushima University Fumio Fukuda Graduate School of Environmental and Life Science, Okayama University Koichiro Ushijima Graduate School of Environmental and Life Science, Okayama University The postharvest properties of two ultra-late maturing peach cultivars, "Tobihaku" (TH) and "Daijumitsuto" (DJ), were investigated. Fruit were harvested at commercial maturity and held at 25 degrees C. TH exhibited the characteristics of normal melting flesh (MF) peach, including rapid fruit softening associated with appropriate level of endogenous ethylene production In contrast, DJ did not soften at all during 3 weeks experimental period even though considerable ethylene production was observed. Fruit of TH and DJ were treated with 5,000 ppm of propylene, an ethylene analog, continuously for 7 days. TH softened rapidly whereas DJ maintained high flesh firmness in spite of an increase in endogenous ethylene production, suggesting that DJ but not TH lacked the ability to be softened in response to endogenous and exogenous ethylene/propylene. DNA-seq analysis showed that tandem endo-polygalacturonase (endoPG) genes located at melting flesh (M) locus, Pp-endoPGM (PGM), and Pp-endoPGF (PGF), were deleted in DJ. The endoPG genes at M locus are known to control flesh texture of peach fruit, and it was suggested that the non-softening property of DJ is due to the lack of endoPG genes. On the other hand, TH possessed an unidentified M haplotype that is involved in determination of MF phenotype. Structural identification of the unknown M haplotype, designated as M-0, through comparison with previously reported M haplotypes revealed distinct differences between PGM on M-0 haplotype (PGM-M-0) and PGM on other haplotypes (PGM-M-1). Peach M haplotypes were classified into four main haplotypes: M-0 with PGM-M-0; M-1 with both PGM-M-1 and PGF; M-2 with PGM-M-1; and M-3 lacking both PGM and PGF. Re-evaluation of M locus in association with MF/non-melting flesh (NMF) phenotypes in more than 400 accessions by using whole genome shotgun sequencing data on database and/or by PCR genotyping demonstrated that M-0 haplotype was the common haplotype in MF accessions, and M-0 and M-1 haplotypes were dominant over M-2 and M-3 haplotypes and co-dominantly determined the MF trait. It was also assumed on the basis of structural comparison of M haplotypes among Prunus species that the ancestral haplotype of M-0 diverged from those of the other haplotypes before the speciation of Prunus persica. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 6 2020 A Japanese Patient with Gastric Cancer and Dihydropyrimidine Dehydrogenase Deficiency Presenting with DPYD Variants 557 562 EN Mikako Ishiguro Department of Internal Medicine, Tsuyama Chuo Hospital Ryuta Takenaka Department of Internal Medicine, Tsuyama Chuo Hospital Kenichiro Ogura Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences Akira Hiratsuka Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences Hiromasa Takeda Department of Internal Medicine, Tsuyama Chuo Hospital Daisuke Kawai Department of Internal Medicine, Tsuyama Chuo Hospital Hirofumi Tsugeno Department of Internal Medicine, Tsuyama Chuo Hospital Shigeatsu Fujiki Department of Internal Medicine, Tsuyama Chuo Hospital Hiroyuki Okada Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Case Report 10.18926/AMO/61217 A 63-year-old Japanese male with stomach adenocarcinoma received oral 5-fluorouracil derivative, cisplatin and trastuzumab chemotherapy. On day 8, severe diarrhea and mucositis developed; chemotherapy was stopped. On day 14, the patient developed renal dysfunction and febrile neutropenia. He also suffered from pneumonia due to Candida albicans. Systemic symptoms improved after intensive conservative treatment. Best supportive care was continued until the patient died from gastric cancer. The dihydropyrimidine dehydroge-nase protein level was low at 3.18 U/mg protein. The result of DPYD genotyping revealed three variants at posi-tions 1615 (G > A), 1627 (A > G), and 1896 (T > C) in exons 13, 13, and 14, respectively. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 6 2020 Reality of Gastric Cancer in Young Patients: The Importance and Difficulty of the Early Diagnosis, Prevention and Treatment 461 466 EN Yoshiyasu Kono Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromitsu Kanzaki Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masaya Iwamuro Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiji Kawano Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshiro Kawahara Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroyuki Okada Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Review 10.18926/AMO/61204 Gastric cancer usually arises in middle-aged to older patients, and is rarely found in younger patients. The clin-ical characteristics, etiology, prognosis, preventive methods and treatment of gastric cancer in young patients have not been fully investigated because of its low prevalence. In this review, we discuss the current under-standing and clinical problems associated with gastric cancer in young patients. Helicobacter pylori (H. pylori), which is a major cause of gastric cancer, especially in older populations, is closely associated with gastric cancer in young patients as well as in older patients. Gastric cancer in young patients tends to be diagnosed at an advanced stage with alarm symptoms. However, young patients with advanced gastric cancer tend to have a favorable general condition and organ function, so they can tolerate intensive systematic chemotherapy. Unfortunately, the prognosis of gastric cancer in young patients with an advanced stage is not favorable. We should not take this rare disease lightly, given its poor prognosis if patients are diagnosed at an unresectable stage. The evaluation of the H. pylori infection status and performance of H. pylori eradication therapy to prevent gastric cancer in young patients as well as the development of more intensive chemotherapy regimens for unre-sectable gastric cancer in young patients are warranted. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0197-0186 141 2020 Scn1a and Cacna1a mutations mutually alter their original phenotypes in rats 104859 EN Iori Ohmori Graduate School of Education, Okayama University Kiyoka Kobayashi Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mamoru Ouchida Department of Molecular Oncology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University This study aimed to examine the effects of Cacna1a mutation on the phenotype of Scn1a-associated epilepsy in rats. We used rats with an N1417H missense mutation in the Scn1a gene and others with an M251K mutation in the Cacna1a gene. Scn1a/Cacna1a double mutant rats were generated by mating both Scn1a and Cacna1a mutants. We investigated general health and the epileptic phenotype in all these genotypes. The onset threshold of hyperthermia-induced seizures was examined at 5 weeks and spontaneous seizures were monitored using video-EEG recordings from 6 to 12 weeks of age. Scn1a/Cacna1a double mutants showed significantly reduced threshold for hyperthermia-sensitive seizures onset compared with the Scn1a mutants and had absence seizures having 6–7 c/s spike-wave bursts with changes in the spike-wave pattern, whereas Cacna1a mutants had regular 6–7 c/s spike-wave bursts. In Scn1a/Cacna1a double mutants, 6–7 c/s spike-wave bursts were accompanied with eyelid myoclonia and continuously shifting generalized clonic seizures, which were not observed in either Scn1a or Cacna1a mutants. Although a curvature of the spine was observed in rats of all these genotypes, the degree of curvature was more pronounced in Scn1a/Cacna1a double mutants, followed by Cacna1a and Scn1a mutants. Our results indicate that Cacna1a and Scn1a mutations mutually alter their original phenotypes in rats. The phenotype of absence seizures with eyelid myoclonia, generalized clonic seizures, and of spine curvature in the Scn1a/Cacna1a double mutants were similar to that observed in patients with Dravet syndrome. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 15 10 2020 Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung e0240333 EN Munenori Habuta Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Yasue Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School Ken-Ichi T. Suzuki Department of Mathematical and Life Sciences, Hiroshima University Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keita Sato Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitomi Kono Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ayuko Takayama Center for the Development of New Model Organisms, National Institute for Basic Biology Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Miyaishi Department of Legal Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichi Oyadomari Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University Eiji Tanaka Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known thatFibroblast growth factor 10(Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in theFgf10-CRISPR F0 mice. However, how the lung phenotype in theFgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in theFgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functionalFgf10genotypes. Firstly, according to a previous report,Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of theFgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 +/- 6.2% in type I, 25.3 +/- 2.7% in type II, and 54.3 +/- 9.5% in type III (mean +/- standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in theFgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functionalFgf10genotypes. These data suggest theFgf10gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, theFgf10-CRISPR F0 mouse would present an ideal experimental system to explore it. No potential conflict of interest relevant to this article was reported.

OXFORD UNIV PRESS Acta Medica Okayama 1340-2838 27 3 2020 Genome-wide study on the polysomic genetic factors conferring plasticity of flower sexuality in hexaploid persimmon dsaa012 EN Kanae Masuda Graduate School of Environmental and Life Science, Okayama University Eiji Yamamoto Kazusa DNA Research Institute Kenta Shirasawa Kazusa DNA Research Institute Noriyuki Onoue Institute of Fruit Tree and Tea Science, NARO Atsushi Kono Institute of Fruit Tree and Tea Science, NARO Koichiro Ushijima Graduate School of Environmental and Life Science, Okayama University Yasutaka Kubo Graduate School of Environmental and Life Science, Okayama University Ryutaro Tao Graduate School of Agriculture, Kyoto University Isabelle M. Henry Department of Plant Biology and Genome Center, University of California Takashi Akagi Graduate School of Environmental and Life Science, Okayama University Sexuality is one of the fundamental mechanisms that work towards maintaining genetic diversity within a species. In diploid persimmons (Diospyros spp.), separated sexuality, the presence of separate male and female individuals (dioecy), is controlled by the Y chromosome-encoded small-RNA gene, OGI. On the other hand, sexuality in hexaploid Oriental persimmon (Diospyros kaki) is more plastic, with OGI-bearing genetically male individuals, able to produce both male and female flowers (monoecy). This is thought to be linked to the partial inactivation of OGI by a retrotransposon insertion, resulting in DNA methylation of the OGI promoter region. To identify the genetic factors regulating branch sexual conversion, genome-wide correlation/association analyses were conducted using ddRAD-Seq data from an F-1 segregating population, and using both quantitative and diploidized genotypes, respectively. We found that allelic ratio at the Y-chromosomal region, including OGI, was correlated with male conversion based on quantitative genotypes, suggesting that OGI can be activated in cis in a dosage-dependent manner. Genome-wide association analysis based on diploidized genotypes, normalized for the effect of OGI allele dosage, detected three fundamental loci associated with male conversion. These loci underlie candidate genes, which could potentially act epigenetically for the activation of OGI expression. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 0024-4066 130 1 2020 Genetic variation and phenotypic plasticity in circadian rhythms in an armed beetle, Gnatocerus cornutus (Tenebrionidae) 34 40 EN Kentarou Matsumura Graduate School of Environmental and Life Science, Okayama University Masato S Abe Center for Advanced Intelligence Project, RIKEN Manmohan D Sharma Centre for Ecology and Conservation, School of Biosciences, University of Exeter David J Hosken Centre for Ecology and Conservation, School of Biosciences, University of Exeter Taishi Yoshii Graduate School of Natural Science and Technology, Okayama University Takahisa Miyatake Graduate School of Environmental and Life Science, Okayama University Circadian rhythms, their free-running periods and the power of the rhythms are often used as indicators of biological clocks, and there is evidence that the free-running periods of circadian rhythms are not affected by environmental factors, such as temperature. However, there are few studies of environmental effects on the power of the rhythms, and it is not clear whether temperature compensation is universal. Additionally, genetic variation and phenotypic plasticity in biological clocks are important for understanding the evolution of biological rhythms, but genetic and plastic effects are rarely investigated. Here, we used 18 isofemale lines (genotypes) of Gnatocerus cornutus to assess rhythms of locomotor activity, while also testing for temperature effects. We found that total activity and the power of the circadian rhythm were affected by interactions between sex and genotype or between sex, genotype and temperature. The males tended to be more active and showed greater increases in activity, but this effect varied across both genotypes and temperatures. The period of activity varied only by genotype and was thus independent of temperature. The complicated genotype–sex–environment interactions we recorded stress the importance of investigating circadian activity in more integrated ways. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 3 2020 Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland 199 208 EN Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichi Oyadomari Division of Molecular Biology, Institute for Genome Research, University of Tokushima Kazuhiko Ochiai Department of Basic Science, School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University Masami Watanabe Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/59950 Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland. No potential conflict of interest relevant to this article was reported.

Genetics Society of America Acta Medica Okayama 2160-1836 G3: Genes Genomes Genetics 1 2019 Medaka Population Genome Structure and Demographic History Described via Genotyping-by-Sequencing 217 228 EN Takafumi Katsumura Graduate School of Natural Science and Technology, Okayama University Shoji Oda Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo Hiroshi Mitani Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo Hiroki Oota Department of Anatomy, Kitasato University School of Medicine Medaka is a model organism in medicine, genetics, developmental biology and population genetics. Lab stocks composed of more than 100 local wild populations are available for research in these fields. Thus, medaka represents a potentially excellent bioresource for screening disease-risk- and adaptation-related genes in genome-wide association studies. Although the genetic population structure should be known before performing such an analysis, a comprehensive study on the genome-wide diversity of wild medaka populations has not been performed. Here, we performed genotyping-by-sequencing (GBS) for 81 and 12 medakas captured from a bioresource and the wild, respectively. Based on the GBS data, we evaluated the genetic population structure and estimated the demographic parameters using an approximate Bayesian computation (ABC) framework. The genome-wide data confirmed that there were substantial differences between local populations and supported our previously proposed hypothesis on medaka dispersal based on mitochondrial genome (mtDNA) data. A new finding was that a local group that was thought to be a hybrid between the northern and the southern Japanese groups was actually an origin of the northern Japanese group. Thus, this paper presents the first population-genomic study of medaka and reveals its population structure and history based on chromosomal genetic diversity. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 2 2020 Identification of a Novel ACVRL1 Gene Mutation (c.100T>A, p.Cys34Ser) in a Japanese Patient with Possible Hereditary Hemorrhagic Telangiectasia (Osler-Weber-Rendu Disease) 165 169 EN Hiroshi Umemura Division of Laboratory Medicine, Department of Pathology and Microbiology, Nihon University School of Medicine Katsuhiro Miura Division of Hematology and Rheumatology, Department of Medicine, Nihon University School of Medicine Hiromu Naruse Division of Laboratory Medicine, Department of Pathology and Microbiology, Nihon University School of Medicine Yoshihiro Hatta Division of Hematology and Rheumatology, Department of Medicine, Nihon University School of Medicine Masami Takei Division of Hematology and Rheumatology, Department of Medicine, Nihon University School of Medicine Tomohiro Nakayama Division of Laboratory Medicine, Department of Pathology and Microbiology, Nihon University School of Medicine Case Report 10.18926/AMO/58276 Hereditary hemorrhagic telangiectasia (HHT; also known as Osler-Weber-Rendu disease) is an autosomal dominant genetic disorder that causes frequent epistaxis, mucocutaneous telangiectasia, and visceral arteriovenous malformations. Four genes (ENG, ACVRL1, SMAD4, and GDF2) have been identified as pathogenic in HHT. We describe the case of a 50-year-old Japanese man highly suspected of having HHT due to recurrent epistaxis, mucocutaneous telangiectasia, and a family history. Genomic analysis revealed a novel missense mutation of c.100T>A, p.Cys34Ser in the patient’s ACVRL1 gene. We used 6 freeware programs to perform an in silico analysis of this mutation. The results demonstrated the mutation’s high pathogenicity. No potential conflict of interest relevant to this article was reported.

Japan Society of Human Genetics Acta Medica Okayama 1434-5161 65 2019 Effect of CYP3A5*3 genetic variant on the metabolism of direct-acting antivirals in vitro : a different effect on asunaprevir versus daclatasvir and beclabuvir 143 153 EN Jun Matsumoto Department of Personalized Medicine and Preventive Healthcare Sciences, Graduate Su Nwe San Department of Pharmacokinetics, Pharmaceutical Sciences, International University of Health and Welfare Masachika Fujiyoshi Department of Personalized Medicine and Preventive Healthcare Sciences, Graduate Ayano Kawauchi Department of Pharmacokinetics, Pharmaceutical Sciences, International University of Health and Welfare Natsumi Chiba Department of Pharmacokinetics, Pharmaceutical Sciences, International University of Health and Welfare Ran Tagai Department of Pharmacokinetics, Pharmaceutical Sciences, International University of Health and Welfare Ryoko Sanbe Department of Pharmacokinetics, Pharmaceutical Sciences, International University of Health and Welfare Shiho Yanaka Department of Pharmacokinetics, Pharmaceutical Sciences, International University of Health and Welfare Hiroaki Sakaue Department of Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences Yoshinori Kato Department of Pharmacokinetics, Pharmaceutical Sciences, International University of Health and Welfare Hiroyoshi Nakamura Department of Pharmacokinetics, Pharmaceutical Sciences, International University of Health and Welfare Harumi Yamada Department of Pharmacokinetics, Pharmaceutical Sciences, International University of Health and Welfare Noritaka Ariyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University Direct-acting antivirals, asunaprevir (ASV), daclatasvir (DCV), and beclabuvir (BCV) are known to be mainly metabolized by CYP3A enzymes; however, the differences in the detailed metabolic activities of CYP3A4 and CYP3A5 on these drugs are not well clarified. The aim of the present study was to elucidate the relative contributions of CYP3A4 and CYP3A5 to the metabolism of ASV, DCV, and BCV, as well as the effect of CYP3A5*3 genetic variant in vitro. The amount of each drug and their major metabolites were determined using LC-MS/MS. Recombinant CYP3As and CYP3A5*3-genotyped human liver microsomes (CYP3A5 expressers or non-expressers) were used for the determination of their metabolic activities. The contribution of CYP3A5 to ASV metabolism was considerable compared to that of CYP3A4. Consistently, ASV metabolic activity in CYP3A5 expressers was higher than those in CYP3A5 non-expresser. Moreover, CYP3A5 expression level was significantly correlated with ASV metabolism. In contrast, these observations were not found in DCV and BCV metabolism. To our knowledge, this is the first study to directly demonstrate the effect of CYP3A5*3 genetic variants on the metabolism of ASV. The findings of the present study may provide basic information on ASV, DCV, and BCV metabolisms. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 1340-2838 26 5 2019 Development of molecular markers associated with resistance to Meloidogyne incognita by performing quantitative trait locus analysis and genome-wide association study in sweetpotato 399 409 EN Rumi Sasai Graduate School of Environmental and Life Science, Okayama University Hiroaki Tabuchi Kyusyu Okinawa Agricultural Research Center, National Agriculture and Food Research Organization Kenta Shirasawa Kazusa DNA Research Institute Kazuki Kishimoto Graduate School of Environmental and Life Science, Okayama University Shusei Sato Graduate School of Life Science, Tohoku University Yoshihiro Okada Kyusyu Okinawa Agricultural Research Center, National Agriculture and Food Research Organization Akihide Kuramoto Graduate School of Agriculture, Kyoto University Akira Kobayashi Kyusyu Okinawa Agricultural Research Center, National Agriculture and Food Research Organization Sachiko Isobe Kazusa DNA Research Institute Makoto Tahara Graduate School of Environmental and Life Science, Okayama University Yuki Monden Graduate School of Environmental and Life Science, Okayama University The southern root-knot nematode, Meloidogyne incognita, is a pest that decreases yield and the quality of sweetpotato [Ipomoea batatas (L.) Lam.]. There is a demand to produce resistant cultivars and develop DNA markers to select this trait. However, sweetpotato is hexaploid, highly heterozygous, and has an enormous genome (similar to 3 Gb), which makes genetic linkage analysis difficult. In this study, a high-density linkage map was constructed based on retrotransposon insertion polymorphism, simple sequence repeat, and single nucleotide polymorphism markers. The markers were developed using F-1 progeny between J-Red, which exhibits resistance to multiple races of M. incognita, and Choshu, which is susceptible to multiple races of such pest. Quantitative trait locus (QTL) analysis and a genome-wide association study detected highly effective QTLs for resistance against three races, namely, SP1, SP4, and SP6-1, in the Ib01-6 J-Red linkage group. A polymerase chain reaction marker that can identify genotypes based on single nucleotide polymorphisms located in this QTL region can discriminate resistance from susceptibility in the F-1 progeny at a rate of 70%. Thus, this marker could be helpful in selecting sweetpotato cultivars that are resistant to multiple races of M. incognita. No potential conflict of interest relevant to this article was reported.

The Japanese Society of Veterinary Science Acta Medica Okayama 0916-7250 81 8 2019 Risk assessment for hepatitis E virus infection from domestic pigs introduced into an experimental animal facility in a medical school 1191 1196 EN Hirohito Ogawa Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Haruko Hirayama Department of Animal Resources, Advanced Science Research Center, Okayama University Satsuki Tanaka Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Norio Yata Department of Animal Resources, Advanced Science Research Center, Okayama University Hikaru Namba Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nobuko Yamashita Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kenzo Yonemitsu Laboratory of Veterinary Microbiology, Joint Faculty of Veterinary Medicine, Yamaguchi University Ken Maeda Laboratory of Veterinary Microbiology, Joint Faculty of Veterinary Medicine, Yamaguchi University Katsumi Mominoki Department of Animal Resources, Advanced Science Research Center, Okayama University Masao Yamada Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Hepatitis E virus (HEV) is known to cause zoonotic infections from pigs, wild boars and deer. Domestic pigs have been used as an experimental animal model in medical research and training; however, the risks of HEV infection from pigs during animal experiments are largely unknown. Here, we retrospectively investigated the seroprevalence and detection rates of viral RNA in 73 domestic pigs (average 34.5 kg) introduced into an animal experimental facility in a medical school during 2012-2016. We detected anti-HEV immunoglobulin G antibodies in 24 of 73 plasma samples (32.9%), though none of the samples were positive for viral RNA. Plasma samples of 18 pigs were sequentially monitored and were classified into four patterns: sustained positive (5 pigs), sustained negative (5 pigs), conversion to positive (6 pigs) and conversion to negative (2 pigs). HEV genomes were detected in 2 of 4 liver samples from pigs that were transported from the same farm during 2016-2017. Two viral sequences of the overlapping open reading frame (ORF) 2/3 region (97 bp) were identical and phylogenetically fell into genotype 3. A 459-bp length of the ORF2 region of an amplified fragment from a pig transported in 2017 was clustered with the wbJYG1 isolate (subgenotype 3b) with 91.5% (420/459 bp) nucleotide identity. Based on our results, we suggest that domestic pigs introduced into animal facilities carry a potential risk of HEV infection to researchers, trainees and facility staff. Continuous surveillance and precautions are important to prevent HEV infection in animal facilities. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 12019712 91 2019 Collagen adhesion gene is associated with blood stream infections caused by methicillin-resistant Staphylococcus aureus 22 31 EN Yasunori Iwata Division of Infection Control, Kanazawa University Kenji Satou Faculty of Electrical and Computer Engineering, Kanazawa University Kengo Furuichi Division of Nephrology, Kanazawa Medical University School of Medicine Ikuko Yoneda Division of Nephrology, Kanazawa University Takuhiro Matsumura Department of Bacteriology, Kanazawa University Masahiro Yutani Graduate School of Environmental and Life Science, Okayama University Yukako Fujinaga Graduate School of Environmental and Life Science, Okayama University Atsushi Hase Faculty of Electrical and Computer Engineering, Kanazawa University Hidetoshi Morita Graduate School of Environmental and Life Science, Okayama University Toshiko Ohta University of Tsukuba Yasuko Senda Division of Infection Control, Kanazawa University Yukiko Sakai-Takemori Division of Infection Control, Kanazawa University Taizo Wada Division of Infection Control, Kanazawa University Shinichi Fujita Division of Infection Control, Kanazawa University Taito Miyake Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Disease Control and Homeostasis, Kanazawa University Haruka Yasuda Department of Nephrology and Laboratory Medicine, Kanazawa University Norihiko Sakai Division of Nephrology, Kanazawa University, Kanazawa, Japan; Division of Blood Purification, Kanazawa University Shinji Kitajima Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Disease Control and Homeostasis, Kanazawa University Tadashi Toyama Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Disease Control and Homeostasis, Kanazawa University Yasuyuki Shinozaki Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Disease Control and Homeostasis, Kanazawa University Akihiro Sagara Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Disease Control and Homeostasis, Kanazawa University Taro Miyagawa Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Disease Control and Homeostasis, Kanazawa University Akinori Hara Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Disease Control and Homeostasis, Kanazawa University Miho Shimizu Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Disease Control and Homeostasis, Kanazawa University Yasutaka Kamikawa Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Disease Control and Homeostasis, Kanazawa University Kazuho Ikeo Laboratory of DNA Data Analysis, National Institute of Genetics Shigeyuki Shichino Department of Molecular Preventive Medicine, University of Tokyo Satoshi Ueha Department of Molecular Preventive Medicine, University of Tokyo Takuya Nakajima Department of Molecular Preventive Medicine, University of Tokyo Kouji Matsushima Department of Molecular Preventive Medicine, University of Tokyo Shuichi Kaneko epartment of Disease Control and Homeostasis, Kanazawa University Takashi Wada Division of Nephrology, Kanazawa University, Kanazawa, Japan; Department of Nephrology and Laboratory Medicine, Kanazawa University Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection.<br/> Methods: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information.<br/> Results: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria.<br/> Conclusions: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection. (c) 2019 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 22144269 21 2019 Clinical and genetic aspects of mild hypophosphatasia in Japanese patients 100515 EN Katsuyuki Yokoi Department of Pediatrics, Fujita Health University School of Medicine Yoko Nakajima Department of Pediatrics, Fujita Health University School of Medicine Yasuko Shinkai Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University Yoshimi Sano Department of Plastic Surgery, Division of Pediatric Dentistry & Orthodontics, Fujita Health University of Medicine Mototaka Imamura Department of Plastic Surgery, Division of Pediatric Dentistry & Orthodontics, Fujita Health University of Medicine Tomoyuki Akiyama Department of Child Neurology, Okayama University Hospital Tetsushi Yoshikawa Department of Pediatrics, Fujita Health University School of Medicine Tetsuya Ito Department of Pediatrics, Fujita Health University School of Medicine Hiroki Kurahashi Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University Background: Hypophosphatasia (HPP) is a rare inborn error of metabolism that results from a dysfunctional tissue non-specific alkaline phosphatase enzyme (TNSALP). Although genotype-phenotype correlations have been described in HPP patients, only sparse information is currently available on the genetics of mild type HPP.<br/> Methods: We investigated 5 Japanese patients from 3 families with mild HPP (patients 1 and 2 are siblings; patient 4 is a daughter of patient 5) who were referred to Fujita Health University due to the premature loss of deciduous teeth. Physical and dental examinations, and blood, urine and bone density tests were conducted. Genetic analysis of the ALPL gene was performed in all patients with their informed consent.<br/> Results: After a detailed interview and examination, we found characteristic symptoms of HPP in some of the study cases. Mobile teeth or the loss of permanent teeth were observed in 2 patients, and 3 out of 5 patients had a history of asthma. The serum ALP levels of all patients were 30% below the lower limit of the age equivalent normal range. ALPL gene analysis revealed compound heterozygous mutations, including Ile395Val and Leu520Argfs in family 1, Val95Met and Gly491Arg in family 2, and a dominant missense mutation (Gly456Arg) in family 3. The 3D-modeling of human TNSALP revealed three mutations (Val95Met, Ile395Val and Gly456Arg) at the homodimer interface. Severe collisions between the side chains were predicted for the Gly456Arg variant.<br/> Discussion: One of the characteristic findings of this present study was a high prevalence of coexisting asthma and a high level serum IgE level. These characteristics may account for the fragility of tracheal tissues and a predisposition to asthma in patients with mild HPP. The genotypes of the five mild HPP patients in our present study series included 1) compound heterozygous for severe and hypomorphic mutations, and 2) dominant-negative mutations. All of these mutations were at the homodimer interface, but only the dominant-negative mutation was predicted to cause a severe collision effect between the side chains. This may account for varying mechanisms leading to different effects on TNSALP function. No potential conflict of interest relevant to this article was reported.

TAYLOR & FRANCIS Acta Medica Okayama 00498254 49 8 2018 Minor contribution of CYP3A5 to the metabolism of hepatitis C protease inhibitor paritaprevir in vitro 935 944 EN Su Nwe San Graduate School of Pharmaceutical Sciences, International University of Health and Welfare Jun Matsumoto Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University Yumi Saito Department of Pharmaceutical Sciences, School of Pharmacy , International University of Health and Welfare Masako Koike Department of Pharmaceutical Sciences, School of Pharmacy , International University of Health and Welfare Hiroaki Sakaue Department of Biochemistry, School of Pharmacy , Tokyo University of Pharmacy and Life Sciences Yoshinori Kato Department of Pharmaceutical Sciences, School of Pharmacy , International University of Health and Welfare Masachika Fujiyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University Noritaka Ariyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University Harumi Yamada Department of Pharmaceutical Sciences, School of Pharmacy , International University of Health and Welfare Paritaprevir (PTV) is a non-structural protein 3/4A protease inhibitor developed for the treatment of hepatitis C disease as a fixed dose combination of ombitasvir (OBV) and ritonavir (RTV) with or without dasabuvir. The aim of this study was to evaluate the effects of cytochrome P450 (CYP) 3A5 on in vitro PTV metabolism using human recombinant CYP3A4, CYP3A5 (rCYP3A4, rCYP3A5) and human liver microsomes (HLMs) genotyped as either CYP3A5*1/*1, CYP3A5*1/*3 or CYP3A5*3/*3. The intrinsic clearance (CLint, Vmax/Km) for the production of a metabolite from PTV in rCYP3A4 was 1.5 times higher than that in rCYP3A5. The PTV metabolism in CYP3A5*1/*1 and CYP3A5*1/*3 HLMs expressing CYP3A5 was comparable to that in CYP3A5*3/*3 HLMs, which lack CYP3A5. CYP3A4 expression level was significantly correlated with PTV disappearance rate and metabolite formation. In contrast, there was no such correlation found for CYP3A5 expression level. This study represents that the major CYP isoform involved in PTV metabolism is CYP3A4, with CYP3A5 having a minor role in PTV metabolism. The findings of the present study may provide foundational information on PTV metabolism, and may further support dosing practices in HCV-infected patients prescribed PTV-based therapy. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 09411291 49 3 2018 A single-nucleotide polymorphism in a gene modulating glucocorticoid sensitivity is associated with the decline in total lung capacity after lung transplantation. 268 274 EN Haruchika Yamamoto Department of General Thoracic Surgery and Breast and Endocrinological SurgeryOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Seiichiro Sugimoto Department of General Thoracic SurgeryOkayama University Hospital Shin Tanaka Department of General Thoracic Surgery and Breast and Endocrinological SurgeryOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Takeshi Kurosaki Department of Organ Transplant CenterOkayama University Hospital Shinji Otani Department of Organ Transplant CenterOkayama University Hospital Masaomi Yamane Department of General Thoracic Surgery and Breast and Endocrinological SurgeryOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Naruto Taira Department of Breast and Endocrinological SurgeryOkayama University Hospital Takahiro Oto Department of Organ Transplant CenterOkayama University Hospital Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological SurgeryOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences PURPOSE:</br> Glucocorticoids are used to prevent chronic lung allograft dysfunction (CLAD) after lung transplantation (LT). Our study was aimed at assessing the association between the glucocorticoid-induced transcript 1 gene (GLCCI1) variant, which modulates glucocorticoid sensitivity, and the postoperative lung function and development of CLAD after LT. </br> METHODS:</br> A total of 71 recipients of LT were genotyped for the GLCCI1 variant (rs37972) and divided into three groups: the homozygous mutant allele (TT) group, the heterozygous mutant allele (CT) group, and the wild-type allele (CC) group. The results of pulmonary function tests were compared with the postoperative baseline values. </br> RESULTS:</br> The total lung capacity (TLC) in the TT group was significantly lower than that in the CC group at 3 years after LT (P = 0.029). In the recipients of cadaveric LT, the TLC and forced expiratory volume in 1 s in the TT group were significantly lower than those in the CC groups, resulting in a significant worse CLAD-free survival at 3 years after LT (P = 0.016). </br> CONCLUSION:</br> The GLCCI1 variant was associated with a significant decrease of the TLC at 3 years after LT and the development of CLAD at 3 years, especially in patients undergoing cadaveric LT. No potential conflict of interest relevant to this article was reported.

Frontiers Research Foundation Acta Medica Okayama 1664-462X 577 2019 Development of Genome-Wide SNP Markers for Barley via Reference- Based RNA-Seq Analysis 577 EN Tsuyoshi Tanaka Breeding Informatics Research Unit, Division of Basic Research, Institute of Crop Science, National Agriculture and Food Research Organization (NARO), Goro Ishikawa Breeding Strategies Research Unit, Division of Basic Research, Institute of Crop Science, National Agriculture and Food Research Organization (NARO) Eri Ogiso-Tanaka Soybean and Field Crop Applied Genomics Research Unit, Division of Field Crop Research, Institute of Crop Science, National Agriculture and Food Research Organization (NARO) Takashi Yanagisawa Wheat and Barley Breeding Unit, Division of Wheat and Barley Research, Institute of Crop Science, National Agriculture and Food Research Organization (NARO) Kazuhiro Sato Group of Genome Diversity, Institute of Plant Science and Resources, Okayama University Marker-assisted selection of crop plants requires DNA markers that can distinguish between the closely related strains often used in breeding. The availability of reference genome sequence facilitates the generation of markers, by elucidating the genomic positions of new markers as well as of their neighboring sequences. In 2017, a high quality genome sequence was released for the six-row barley (Hordeum vulgare) cultivar Morex. Here, we developed a de novo RNA-Seq-based genotyping procedure for barley strains used in Japanese breeding programs. Using RNA samples from the seedling shoot, seedling root, and immature flower spike, we mapped next-generation sequencing reads onto the transcribed regions, which correspond to ∼590 Mb of the whole ∼4.8-Gbp reference genome sequence. Using 150 samples from 108 strains, we detected 181,567 SNPs and 45,135 indels located in the 28,939 transcribed regions distributed throughout the Morex genome. We evaluated the quality of this polymorphism detection approach by analyzing 387 RNA-Seq-derived SNPs using amplicon sequencing. More than 85% of the RNA-Seq SNPs were validated using the highly redundant reads from the amplicon sequencing, although half of the indels and multiple-allele loci showed different polymorphisms between the platforms. These results demonstrated that our RNA-Seq-based de novo polymorphism detection system generates genome-wide markers, even in the closely related barley genotypes used in breeding programs. No potential conflict of interest relevant to this article was reported.

Academic Press Acta Medica Okayama 09699961 50 2013 CACNA1A variants may modify the epileptic phenotype of Dravet syndrome 209 217 EN Iori Ohmori Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mamoru Ouchida Department of Molecular Genetics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Katsuhiro Kobayashi Yoshimi Jitsumori Akiko Mori Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hiroyuki Michiue Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teiichi Nishiki Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yoko Ohtsuka Hideki Matsui Department of Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Dravet syndrome is an intractable epileptic syndrome beginning in the first year of life. De novo mutations of SCN1A, which encode the Na(v)1.1 neuronal voltage-gated sodium channel, are considered the major cause of Dravet syndrome. In this study, we investigated genetic modifiers of this syndrome. We performed a mutational analysis of all coding exons of CACNA1A in 48 subjects with Dravet syndrome. To assess the effects of CACNA1A variants on the epileptic phenotypes of Dravet syndrome, we compared clinical features in two genotype groups: 1) subjects harboring SCN1A mutations but no CACNA1A variants (n=20) and 2) subjects with SCN1A mutations plus CACNA1A variants (n=20). CACNA1A variants detected in patients were studied using heterologous expression of recombinant human Ca(v)2.1 in HEK 293 cells and whole-cell patch-clamp recording. Nine CACNA1A variants, including six novel ones, were detected in 21 of the 48 subjects (43.8%). Based on the incidence of variants in healthy controls, most of the variants seemed to be common polymorphisms. However, the subjects harboring SCN1A mutations and CACNA1A variants had absence seizures more frequently than the patients with only SCN1A mutations (8/20 vs. 0/20, p=0.002). Moreover, the former group of subjects exhibited earlier onset of seizures and more frequent prolonged seizures before one year of age, compared to the latter group of subjects. The electrophysiological properties of four of the five novel Ca(v)2.1 variants exhibited biophysical changes consistent with gain-of-function. We conclude that CACNA1A variants in some persons with Dravet syndrome may modify the epileptic phenotypes. No potential conflict of interest relevant to this article was reported.

SCIENCEDOMAIN international Acta Medica Okayama 22310886 3 2 2013 Multi-locus Genotyping Reveals High Occurrence of Mixed Assemblages in Giardia duodenalis within a Limited Geographical Boundary 190 197 EN Avik Kumar Mukherjee Department of Parasitology, National Institute of Cholera and Enteric Diseases Sumallya Karmakar Department of Parasitology, National Institute of Cholera and Enteric Diseases Dibyendu Raj Department of Parasitology, National Institute of Cholera and Enteric Diseases Sandipan Ganguly Department of Parasitology, National Institute of Cholera and Enteric Diseases Aim: 　To determine the common genotypes of Giardia duodenalis causing diarrhea in the study region and to assess the extent of genetic polymorphism among them.　 Study Design: 　Stool samples were collected from the patients attending IDBG Hospital, Kolkata with diarrheal complaints through a systemic sampling technique and were screened for Giardia duodenalis. The G. duodenalis positive samples were subjected to molecular genotyping through ‘PCR - Direct DNA sequencing’ procedure. All the sequence data obtained were incorporated into MEGA 4 software for multiple alignment and validation followed by phylogenetic analysis. The genotyping data obtained are stored in Excel spreadsheets and incorporated into EpiInfo 3.1 for analyzing possible association of genotype outcome with common physical factors such as age, sex etc.　 Place and Duration of Study: 　Department of parasitology, National Institute of Cholera and Enteric Diseases, Kolkata, India from July 2009 to November 2011.　 Methodology: 　A total of 68 Giardia duodenalis positive stool samples were identified from the diarrhea patients attending IDBG hospital in the city and were subjected to multi-locus genotyping. Fragments of ß-giardin, Glutamate-dehydrogenase and Triosephosphate-isomerase genes of Giardia were amplified from those samples with specific primers and sequenced. All the sequences were analyzed using MEGA 4 software for obtaining the genotyping results. Results: Multi-locus genotyping identified 13 isolates as assemblage A and 41 as assemblage B, whereas 14 of them could not be assigned in a particular group. Detailed phylogenetic analysis revealed that multiple genotypes were observed in those 14 isolates depending upon the marker loci.　 Conclusion: 　The study could produce a preliminary idea about the G. duodenalis genotypes found in Kolkata city. High percentage of mixed assemblages in the study population also revealed the presence of genetic diversity among a small population of diarrheal patient within a limited geographical boundary. It has also hypothesized the possibility of inter-assemblage genetic exchange among Giardia. No potential conflict of interest relevant to this article was reported.

Elsevier Science Acta Medica Okayama 0264410X 32 supplment 1 2014 Hospital based surveillance and genetic characterization of rotavirus strains in children (<5 years) with acute gastroenteritis in Kolkata, India, revealed resurgence of G9 and G2 genotypes during 2011-2013 A20 A28 EN Satarupa Mullick National Institute of Cholera and Enteric Diseases Paulami Mandal National Institute of Cholera and Enteric Diseases Mukti Kant Nayak National Institute of Cholera and Enteric Diseases Souvik Ghosh Department of Hygiene, Sapporo Medical University School of Medicine Papiya De National Institute of Cholera and Enteric Diseases K. Rajendran National Institute of Cholera and Enteric Diseases Mihir K. Bhattacharya National Institute of Cholera and Enteric Diseases Utpala Mitra National Institute of Cholera and Enteric Diseases Thandavarayan Ramamurthy National Institute of Cholera and Enteric Diseases Nobumichi Kobayashi Department of Hygiene, Sapporo Medical University School of Medicine Mamta Chawla-Sarkar National Institute of Cholera and Enteric Diseases INTRODUCTION:　 India accounts for an estimated 457,000-884,000 hospitalizations and 2 million outpatient visits for diarrhea. In spite of the huge burden of rotavirus (RV) disease, RV vaccines have not been introduced in national immunization programme of India. Therefore, continuous surveillance for prevalence and monitoring of the circulating genotypes is needed to assess the disease burden prior to introduction of vaccines in this region.　 METHODS:　 During January 2011 through December 2013, 830 and 1000 stool samples were collected from hospitalized and out-patient department (OPD) patients, respectively, in two hospitals in Kolkata, Eastern India. After primary screening, the G-P typing was done by multiplex semi-nested PCR using type specific primers followed by sequencing. Phylogenetic analysis for the VP7 gene of 25 representative strains was done.　 RESULTS:　 Among hospitalized and OPD patients, 53.4% and 47.5% cases were positive for rotaviruses, respectively. Unlike previous studies where G1 was predominant, in hospitalized cases G9 rotavirus strains were most prevalent (40%), followed by G2 (39.6%) whereas G1 and G12 occurred at 16.4% and 5.6% frequency. In OPD cases, the most prevalent strain was G2 (40.3%), followed by G1, G9 and G12 at 25.5%, 22.8%, 9.3%, respectively. Phylogenetically the G1, G2 and G9 strains from Kolkata did not cluster with corresponding genotypes of Rotarix, RotaTeq and Rotavac (116E) vaccine strains.　 CONCLUSION:　 The study highlights the high prevalence of RV in children with gastroenteritis in Kolkata. The circulating genotypes have changed over the time with predominance of G9 and G2 strains during 2011-2013. The current G2, G9 and G1 Kolkata strains shared low amino acid homologies with current vaccine strains. Although there is substantial evidence for cross protection of vaccines against a variety of strains, still the strain variation should be monitored post vaccine introduction to determine if it has any impact on vaccine effectiveness. No potential conflict of interest relevant to this article was reported.

Frontiers Media S.A. Acta Medica Okayama 1664302X 7 2016 Phenotypic and Genetic Heterogeneity in Vibrio cholerae O139 Isolated from Cholera Cases in Delhi, India during 2001-2006 1250 EN Raikamal Ghosh Division of Bacteriology, National Institute of Cholera and Enteric Diseases Naresh C. Sharma Maharishi Valmiki Infectious Diseases Hospital Kalpataru Halder Infectious Diseases and Immunology Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology Rupak K. Bhadra Infectious Diseases and Immunology Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology Goutam Chowdhury Division of Bacteriology, National Institute of Cholera and Enteric Diseases Gururaja P. Pazhani Division of Bacteriology, National Institute of Cholera and Enteric Diseases Sumio Shinoda Collaborative Research Center of Okayama University for Infectious Diseases in India, National Institute of Cholera and Enteric Diseases Asish K. Mukhopadhyay Division of Bacteriology, National Institute of Cholera and Enteric Diseases G. Balakrish Nair Center for Human Microbial Ecology, Translational Health Science and Technology Institute Thadavarayan Ramamurthy Center for Human Microbial Ecology, Translational Health Science and Technology Institute Incidence of epidemic Vibrio cholerae serogroup O139 has declined in cholera endemic countries. However, sporadic cholera caused by V. cholerae O139 with notable genetic changes is still reported from many regions. In the present study, 42 V. cholerae O139 strains isolated from 2001 to 2006 in Delhi, India, were retrospectively analyzed to understand their phenotype and molecular characteristics. The majority of isolates were resistant to ampicillin, furazolidone and nalidixic acid. Though the integrative conjugative element was detected in all the O139 isolates, the 2004–2006 isolates remained susceptible to co-trimoxazole, chloramphenicol, and streptomycin. Cholera toxin genotype 1 was present in the majority of the O139 isolates while few had type 3 or a novel type 4. In the cholera toxin encoding gene (ctx) restriction fragment length polymorphism, the majority of the isolates harbored three copies of CTX element, of which one was truncated. In this study, the ctx was detected for the first time in the small chromosome of V. cholerae O139 and one isolate harbored 5 copies of CTX element, of which 3 were truncated. The ribotype BII pattern was found in most of the O139 isolates. Three V. cholerae O139 isolated in 2001 had a new ribotype BVIII. Pulsed-field gel electrophoresis analysis revealed clonal variation in 2001 isolates compared to the 2004–2006 isolates. Molecular changes in V. cholerae O139 have to be closely monitored as this information may help in understanding the changing genetic features of this pathogen in relation to the epidemiology of cholera. No potential conflict of interest relevant to this article was reported.

PLOS Acta Medica Okayama 11 2 2017 Comparative genome analysis of VSP-II and SNPs reveals heterogenic variation in contemporary strains of Vibrio cholerae O1 isolated from cholera patients in Kolkata, India e0005386 EN Daisuke Imamura Collaborative Research Center of Okayama University for Infectious Diseases in India Masatomo Morita Department of Bacteriology I, National Institute of Infectious Diseases Tsuyoshi Sekizuka Pathogen Genomics Center, National Institute of Infectious Diseases Tamaki Mizuno Collaborative Research Center of Okayama University for Infectious Diseases in India Taichiro Takemura Vietnam Research Station, Institute of Tropical Medicine, Nagasaki University Tetsu Yamashiro Vietnam Research Station, Institute of Tropical Medicine, Nagasaki University Goutam Chowdhury Division of Bacteriology, National Institute of Cholera and Enteric Diseases Gururaja P. Pazhani Division of Bacteriology, National Institute of Cholera and Enteric Diseases Asish K. Mukhopadhyay Division of Bacteriology, National Institute of Cholera and Enteric Diseases Thandavarayan Ramamurthy Translational Health Science and Technology Institute Shin-ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Makoto Kuroda Pathogen Genomics Center, National Institute of Infectious Diseases Sumio Shinoda Collaborative Research Center of Okayama University for Infectious Diseases in India Makoto Ohnishi Department of Bacteriology I, National Institute of Infectious Diseases Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epicenter for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 73 1 2019 The Role of Kallikrein-Related Peptidases in Atopic Dermatitis 1 6 EN Shin Morizane Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Review 10.18926/AMO/56452 Excessive protease activity is a characteristic abnormality that affects the epidermal barrier in patients with atopic dermatitis (AD). Kallikrein-related peptidases (KLKs) are excessively expressed in AD lesions, and it is suggested that the abnormal action of KLKs is involved in the skin barrier dysfunction in AD. In other words, overexpressed KLKs disrupt the normal barrier function, and due to that breakdown, external substances that can become antigens of AD easily invade the epidermis, resulting in dermatitis, coupled with the induction of Th2 cytokines. Further investigations are required to elucidate the role of KLKs in AD; this knowledge could contribute to the design of new therapeutic and prophylactic drugs for AD. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 72 4 2018 Mixed HCV Infection of Genotype 1B and Other Genotypes Influences Non-response during Daclatasvir + Asunaprevir Combination Therapy 401 406 EN Nozomu Wada Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fusao Ikeda Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Chizuru Mori Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koichi Takaguchi Department of Internal Medicine, Kagawa Prefectural Central Hospital Shin-ichi Fujioka Department of Internal Medicine, Okayama Saiseikai General Hospital Haruhiko Kobashi Department of Internal Medicine, Okayama Red Cross Hospital Yoichi Morimoto Department of Gastroenterology and Hepatology, Kurashiki Central Hospital Kazuya Kariyama Department of Liver Disease Center, Okayama City Hospital Kosaku Sakaguchi Department of Internal Medicine, Fukuyama City Hospital Noriaki Hashimoto Department of Internal Medicine, Mihara Red Cross Hospital Akio Moriya Department of Gastroenterology, Mitoyo General Hospital Mitsuhiko Kawaguchi Department of Internal Medicine, Kawaguchi Medical Clinic Hirokazu Miyatake Department of Internal Medicine, Hiroshima City Hospital Hiroaki Hagihara Department of Gastroenterology, Sumitomo Besshi Hospital Junichi Kubota Department of Internal Medicine, Tajiri Hospital Hiroki Takayama Department of Gastroenterology, Tsuyama Central Hospital Yasuto Takeuchi Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tetsuya Yasunaka Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akinobu Takaki Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshiaki Iwasaki Health Service Center, Okayama University Hiroyuki Okada Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/56178 Daclatasvir (DCV) + asunaprevir (ASV) combination therapy has become available for patients with hepatitis C virus (HCV) serogroup 1 infection. We studied the efficacy of this therapy by focusing on the factors associated with sustained virological responses (SVR) including resistance-associated variants (RAVs) and mixed infection of different HCV genotypes. We enrolled 951 HCV serogroup 1-positive patients who received this combination therapy at our hospital or affiliated hospitals. The presence of RAVs in non-structural (NS) regions 3 and 5A was analyzed by direct sequencing. HCV genotypes were determined by PCR with genotype-specific primers targeting HCV core and NS5B regions. SVR was achieved in 91.1% of patients. Female sex, age > 70 years, and RAVs were significantly associated with non-SVR (p<0.01 for all). Propensity score-matching results among the patients without RAVs regarding sex, age, and fibrosis revealed that mixed HCV infection determined by HCV NS5B genotyping showed significantly lower SVR rates than 1B-mono infection (p=0.02). Female sex and RAVs were significant factors associated with treatment failure of this combination therapy for patients with HCV serogroup 1 infection. Mixed HCV infection other than 1B-mono infection would be useful for predicting treatment failure. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 72 3 2018 Analysis of All 34 Exons of the SPINK5 Gene in Japanese Atopic Dermatitis Patients 275 282 EN Shin Morizane Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mamoru Ouchida Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ko Sunagawa Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Saeko Sugimoto Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mina Kobashi Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Sugihara Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hayato Nomura Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhide Tsuji Nishigawara Dermatology Clinic Atsushi Sato Sato Dermatology Clinic Yoshihiro Miura Miura Dermatology Clinic Hiroaki Hattori Hattori Dermatology and Allergy Clinic Kotaro Tada Tada Dermatology Clinic Wook-Kang Huh Dr. Huh's Dermatology Clinic Akemi Seno Department of Dermatology, Mitoyo General Hospital Keiji Iwatsuki Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/56073 Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a large multidomain serine protease inhibitor that is expressed in epidermal keratinocytes. Nonsense mutations of the SPINK5 gene, which codes for LEKTI, cause Netherton syndrome, which is characterized by hair abnormality, ichthyosis, and atopy. A single nucleotide polymorphism (SNP) of SPINK5, p.K420E, is reported to be associated with the pathogenesis of atopic dermatitis (AD). We studied all 34 exons of the SPINK5 gene in Japanese 57 AD patients and 50 normal healthy controls. We detected nine nonsynonymous variants, including p.K420E; these variants had already been registered in the SNP database. Among them, p.R654H (n=1) was found as a heterozygous mutation in the AD patients, but not in the control. No new mutation was detected. We next compared the data of the AD patients with data from the Human Genetic Variation Database provided by Kyoto University; a significant difference was found in the frequency of the p.S368N genotype distribution. PolyPhen-2 and SIFT, two algorithms for predicting the functional effects of amino acid substitutions, showed significant scores for p.R654H. Therefore, R654H might be a risk factor for epidermal barrier dysfunction in some Japanese AD patients. No potential conflict of interest relevant to this article was reported.

Cambridge Univ. Press for the Society for General Microbiology Acta Medica Okayama 0022-1317 93 7 2012 Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines 1422 1431 EN Midori Takeda Department of Tumor Virology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masanori Ikeda Department of Tumor Virology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuo Ariumi Department of Tumor Virology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takaji Wakita Department of Virology II, National Institute of Infectious Disease Nobuyuki Kato Department of Tumor Virology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences 　A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann-Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication. No potential conflict of interest relevant to this article was reported.

Elsevier Science Acta Medica Okayama 0168-1702 167 1 2012 Identification of host genes showing differential expression profiles with cell-based long-term replication of hepatitis C virus RNA 74 85 EN Hiroe Sejima Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kyoko Mori Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yasuo Ariumi Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Masanori Ikeda Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Nobuyuki Kato Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences 　Persistent hepatitis C virus (HCV) infection frequently causes hepatocellular carcinoma. However, the mechanisms of HCV-associated hepatocarcinogenesis and disease progression are unclear. Although the human hepatoma cell line, HuH-7, has been widely used as the only cell culture system for robust HCV replication, we recently developed new human hepatoma Li23 cell line-derived OL, OL8, OL11, and OL14 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates. OL, OL8, OL11, and OL14 cells were cultured for more than 2 years. We prepared cured cells from OL8 and OL11 cells by interferon-γ treatment. The cured cells were also cultured for more than 2 years. cDNA microarray and RT-PCR analyses were performed using total RNAs prepared from these cells. We first selected several hundred highly or moderately expressed probes, the expression levels of which were upregulated or downregulated at ratios of more than 2 or less than 0.5 in each set of compared cells (e.g., parent OL8 cells versus OL8 cells cultured for 2 years). From among these probes, we next selected those whose expression levels commonly changed during a 2-year culture of genome-length HCV RNA-replicating cells, but which did not change during a 2-year culture period in cured cells. We further examined the expression levels of the selected candidate genes by RT-PCR analysis using additional specimens from the cells cultured for 3.5 years. Reproducibility of the RT-PCR analysis using specimens from recultured cells was also confirmed. Finally, we identified 5 upregulated genes and 4 downregulated genes, the expression levels of which were irreversibly altered during 3.5-year replication of HCV RNA. These genes may play roles in the optimization of the environment in HCV RNA replication, or may play key roles in the progression of HCV-associated hepatic diseases. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 0721-7714 36 4 2017 Selection of transformation-efficient barley genotypes based on TFA (transformation amenability) haplotype and higher resolution mapping of the TFA loci 611 620 EN Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Brigid Meints Department Crop and Soil Sciences, Washington State University Matthew J. Moscou The Sainsbury Laboratory Luis Cistue Department Genetica y Produccion Vegetal Begoña Echávarri Kazuhiro Sato Institute of Plant Science and Resources, Okayama University Patrick M. Hayes Department Crop and Soil Science, Oregon State University Key message: The genetic substitution of transformation amenability alleles from ‘Golden Promise’ can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars. Abstract: Barley (Hordeum vulgare) cv. ‘Golden Promise’ is one of the most useful and well-studied cultivars for genetic manipulation. In a previous report, we identified several transformation amenability (TFA) loci responsible for Agrobacterium-mediated transformation using the F2 generation of immature embryos, derived from ‘Haruna Nijo’ × ‘Golden Promise,’ as explants. In this report, we describe higher density mapping of these TFA regions with additional SNP markers using the same transgenic plants. To demonstrate the robustness of transformability alleles at the TFA loci, we genotyped 202 doubled haploid progeny from the cross ‘Golden Promise’ × ‘Full Pint.’ Based on SNP genotype, we selected lines having ‘Golden Promise’ alleles at TFA loci and used them for transformation. Of the successfully transformed lines, DH120366 came the closest to achieving a level of transformation efficiency comparable to ‘Golden Promise.’ The results validate that the genetic substitution of TFA alleles from ‘Golden Promise’ can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 71 1 2017 Human RAD 17 Polymorphism at Codon 546 Is Associated with the Risk of Colorectal Cancer 59 68 EN Yukiko Yasuda Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akiko Sakai Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Sachio Ito Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kaori Sasai Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nagahide Matsubara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mamoru Ouchida Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Katayama Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kenji Shimizu Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/54826 Human RAD17 acts as an activator of checkpoint signals in response to DNA damage. Here we evaluated the association of hRAD17 Leu546Arg (rs1045051), a missense single nucleotide polymorphism, with the risk of colorectal cancer (CRC) in relation to smoking and alcohol consumption habits in 212 CRC patients and 1,142 cancer-free controls in a case-control study conducted in Japan. The results showed that the hRAD17 Leu/Arg genotype compared to the Leu/Leu genotypes was significantly associated with the protective effect on CRC risk with the adjusted odds ratio (OR) of 0.68 [95% confidence interval (CI): 0.49−0.95, p=0.024], and the males with the Arg/Arg genotype had a greater risk of CRC compared to those with the Leu/Leu and Leu/Arg genotypes (OR=1.87, 95%CI 1.03−3.40, p=0.04). In stratified studies, the protective effect of the Leu/Arg genotype on CRC risk was markedly higher in the light smokers (< 20 pack years) (OR=0.61, 95%CI 0.40−0.94, p=0.024) and the rectal cancer patients (OR=0.49, 95%CI 0.31−0.78, p=0.003). The risk of the Arg/Arg genotype was associated with heavy smoking (≥ 20 pack-years) (OR=2.24, 95%CI 1.09−4.61, p=0.03). These findings suggest that the genetic variant of hRAD17 Leu546Arg polymorphism has a significant effect on CRC susceptibility in Japanese. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 70 2 2016 Determination of Oncogenic Human Papillomavirus (HPV) Genotypes in Anogenital Cancers in Myanmar 103 110 EN Mu Mu Shwe Hlaing Myat Thu Khin Saw Aye Aye Aye Myint Mya Thida Khin Shwe Mar Khin Khin Oo Khin Sandar Aye Shigeru Okada Kyaw Zin Thant Original Article 10.18926/AMO/54189 Molecular and epidemiologic investigations suggest a causal role for human papillomavirus (HPV) in anogenital cancers. This study identified oncogenic HPV genotypes in anogenital cancers among men and women in a 2013 cross-sectional descriptive study in Myanmar. In total, 100 biopsy tissues of histologically confirmed anogenital cancers collected in 2008-2012 were studied, including 30 penile and 9 anal cancers from Yangon General Hospital and 61 vulvar cancers from Central Women's Hospital, Yangon. HPV-DNA testing and genotyping were performed by polymerase chain reaction-restriction fragment length polymorphism. Overall, 34% of anogenital cancers were HPV-positive. HPV was found in 44.4% of anal (4/9), 36.1% of vulvar (22/61), and 26.7% of penile (8/30) cancers. The most frequent genotypes in anal cancers were HPV 16 (75%) and 18 (25%). In vulvar cancers, HPV 33 was most common (40.9%), followed by 16 (31.8%), 31 (22.7%), and 18 (4.6%). In penile cancers, HPV 16 (62.5%) was most common, followed by 33 (25%) and 18 (12.5%). This is the first report of evidencebased oncogenic HPV genotypes in anogenital cancers among men and women in Myanmar. This research provides valuable information for understanding the burden of HPV-associated cancers of the anus, penis, and vulva and considering the effectiveness of prophylactic HPV vaccination. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2015 Virus genotypes and responses of serum-specific antibodies in children with primary mumps and mumps reinfection EN Rika Sakata No potential conflict of interest relevant to this article was reported.

PUBLIC LIBRARY SCIENCE Acta Medica Okayama 1932-6203 8 8 2013 New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication e72519 EN Youki Ueda Midori Takeda Kyoko Mori Hiromichi Dansako Takaji Wakita Hye-Sook Kim Akira Sato Yusuke Wataya Masanori Ikeda Nobuyuki Kato BACKGROUND: Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed. 　 METHODOLOGY/PRINCIPAL FINDINGS: 　 　 Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.　 　 CONCLUSIONS/SIGNIFICANCE:　 　 We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2015 Effectiveness of Extending Treatment Duration in Therapy with Pegylated Interferon and Ribavirin for Genotype 2 Hepatitis C Virus Infection EN Shintarou Nanba No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 69 4 2015 Effectiveness of Extending Treatment Duration in Therapy with Pegylated Interferon and Ribavirin for Genotype 2 Hepatitis C Virus Infection 237 244 EN Shintarou Nanba Fusao Ikeda Shin-ichi Fujioka Yasuyuki Araki Kouichi Takaguchi Noriaki Hashimoto Hiroyuki Seki Akinobu Takaki Yoshiaki Iwasaki Kazuhide Yamamoto Original Article 10.18926/AMO/53560 The effectiveness of extending treatment duration as response guided therapy was previously reported for chronic hepatitis C (CHC) genotype 1, but is still controversial for genotype 2. The present study is a retrospective cohort study to investigate the effectiveness of extending treatment duration in therapy with pegylated interferon and ribavirin for patients with CHC genotype 2 by focusing on the timing at which patients obtained undetectable HCV RNA. A total of 306 patients who obtained undetectable HCV RNA by week 24 of treatment and completed 24 weeks of treatment were enrolled. Rapid virological response (RVR) to standard therapy was achieved by 122 patients (51ｵ), and 89ｵ of them obtained sustained virological response (SVR), while 69ｵ of non-RVR patients achieved SVR. Non-RVR patients with undetectable HCV RNA at week 8, and insufficient adherence＜80ｵ pegylated interferon and ribavirin during the first 24 weeks, significantly improved their SVR rate by extended therapy. Among patients receiving extended therapy, drug adherences did not differ between SVR and non-SVR patients, indicating that extending treatment duration might compensate for insufficient antiviral effects due to insufficient drug adherences. This finding might be useful in creating a guideline for extending treatment duration for patients with CHC genotype 2. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2015 New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication EN Yuki Ueda No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 29 6 2014 Human Rho guanine nucleotide exchange factor 11 gene is associated with schizophrenia in a Japanese population 552 558 EN Yutaka Mizuki Manabu Takaki Yuko Okahisa Shinji Sakamoto Masafumi Kodama Hiroshi Ujike Yosuke Uchitomi ObjectiveThe human Rho guanine nucleotide exchange factor 11 (ARHGEF11) gene is one of the candidate genes for type 2 diabetes mellitus (T2DM). ARHGEF11 is mapped to chromosome 1q21, which has susceptible risk loci for T2DM and schizophrenia. We hypothesized that ARHGEF11 contributes to the pathogenesis of schizophrenia. MethodWe selected eight single nucleotide polymorphisms of ARHGEF11 that had significant associations with T2DM for a case-control association study of 490 patients with schizophrenia and 500 age-matched and sex-matched controls. ResultsWe did not find any differences in allelic, genotypic associations, or minor allele frequencies with schizophrenia. Analysis of the rs6427340-rs6427339 haplotype and the rs822585-rs6427340-rs6427339 haplotype combination provided significant evidence of an association with schizophrenia (global permutations p=0.00047 and 0.0032, respectively). C-C of the rs6427340-rs6427339 haplotype and A-C-C of the rs822585-rs6427340-rs6427339 haplotype carried higher risk factors for schizophrenia (permutation p=0.0010 and 0.0018, respectively). A-C-T of the rs822585-rs6427340-rs6427339 haplotype had a possible protective effect (permutation p=0.031). ConclusionThese results provide new evidence that ARHGEF11 may constitute a risk factor for schizophrenia. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2014 Hepatitis C Virus-specific T-cell Response Correlates with Hepatitis Activity and Donor IL28B Genotype Early after Liver Transplantation EN Ryuichiro Tsuzaki No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 69 1 2015 Trend of Human Papillomavirus Genotypes in Cervical Neoplasia Observed in a Newly Developing Township in Yangon, Myanmar 51 58 EN Mu Mu Shwe Kyi Kyi Nyunt Shigeru Okada Teruo Harano Hlaing Myat Thu Hla Myat Mo Mo Mo Mo Win Khin Khin Oo KhinThet Wai Khin Saw Aye Myo Khin Original Article 10.18926/AMO/53122 Persistent infection with oncogenic types of human papillomavirus (HPV) is the most important risk factor associated with cervical cancer. This study detected the oncogenic HPV genotypes in cervical neoplasia in relation to clinicopathological findings using a cross-sectional descriptive method in 2011 and 2012. Cervical swabs and colposcopy-directed cervical biopsy tissues were collected from 108 women (median age 45 years;range 20-78) showing cervical cytological changes at Sanpya General Hospital, Yangon, Myanmar. HPV DNA testing and genotyping were performed by polymerase chain reaction and restriction fragment length polymorphism. HPV was identified in women with cervical intraepithelial neoplasia (CIN) 1 (44.4%), CIN2 (63.2%), CIN3 (70.6%), and squamous cell carcinoma (SCC) (74.1%). The association between cervical neoplasia and HPV positivity was highly significant (p＝0.008). Most patients infected with HPV were between 40-49 years of age, and the youngest were in the 20- to 29-year-old age group. The most common genotype was HPV 16 (65.6%) with the following distribution:70% in CIN1, 41.7% in CIN2, 91.7% in CIN3, and 60% in SCC. HPV-31 was the second-most frequent (21.9%):30% in CIN1, 33.3% in CIN2, 8.3% in CIN3, and 15% in SCC. The third-most frequent-genotype was HPV-18 (7.8%):8.3% in CIN1, and 20% in SCC. Another genotype was HPV-58 (4.7%):16.7% in CIN1 and 5% in SCC. The majority of CIN/SCC cases were associated with HPV genotypes 16, 31, 18, and 58. If oncogenic HPV genotypes are positive, the possibility of cervical neoplasia can be predicted. Knowledge of the HPV genotypes distribution can predict the effectiveness of the currently used HPV vaccine. No potential conflict of interest relevant to this article was reported.

BioMed Central Ltd. Acta Medica Okayama 1471-2407 13 2013 Effects of lifestyle and single nucleotide polymorphisms on breast cancer risk: a case-control study in Japanese women EN Taeko Mizoo Naruto Taira Keiko Nishiyama Tomohiro Nogami Takayuki Iwamoto Takayuki Motoki Tadahiko Shien Junji Matsuoka Hiroyoshi Doihara Setsuko Ishihara Hiroshi Kawai Kensuke Kawasaki Youichi Ishibe Yutaka Ogasawara Yoshifumi Komoike Shinichiro Miyoshi Background: Lifestyle factors, including food and nutrition, physical activity, body composition and reproductive factors, and single nucleotide polymorphisms (SNPs) are associated with breast cancer risk, but few studies of these factors have been performed in the Japanese population. Thus, the goals of this study were to validate the association between reported SNPs and breast cancer risk in the Japanese population and to evaluate the effects of SNP genotypes and lifestyle factors on breast cancer risk. Methods: A case-control study in 472 patients and 464 controls was conducted from December 2010 to November 2011. Lifestyle was examined using a self-administered questionnaire. We analyzed 16 breast cancer-associated SNPs based on previous GWAS or candidate-gene association studies. Age or multivariate-adjusted odds ratios (OR) and 95% confidence intervals (95% CI) were estimated from logistic regression analyses. Results: High BMI and current or former smoking were significantly associated with an increased breast cancer risk, while intake of meat, mushrooms, yellow and green vegetables, coffee, and green tea, current leisure-time exercise, and education were significantly associated with a decreased risk. Three SNPs were significantly associated with a breast cancer risk in multivariate analysis: rs2046210 (per allele OR = 1.37 [95% CI: 1.11-1.70]), rs3757318 (OR = 1.33[1.05-1.69]), and rs3803662 (OR = 1.28 [1.07-1.55]). In 2046210 risk allele carriers, leisure-time exercise was associated with a significantly decreased risk for breast cancer, whereas current smoking and high BMI were associated with a significantly decreased risk in non-risk allele carriers. Conclusion: In Japanese women, rs2046210 and 3757318 located near the ESR1 gene are associated with a risk of breast cancer, as in other Asian women. However, our findings suggest that exercise can decrease this risk in allele carriers. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0944-1174 48 3 2013 The impact of patatin-like phospholipase domain-containing protein 3 polymorphism on hepatocellular carcinoma prognosis 405 412 EN Yasuto Takeuchi Fusao Ikeda Yuki Moritou Hiroaki Hagihara Tetsuya Yasunaka Kenji Kuwaki Yasuhiro Miyake Hideki Ohnishi Shinichiro Nakamura Hidenori Shiraha Akinobu Takaki Yoshiaki Iwasaki Kazuhiro Nouso Kazuhide Yamamoto The single nucleotide polymorphism (SNP) rs738409 in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is associated with hepatic fat accumulation and disease progression in patients with non-alcoholic fatty liver disease and alcoholic liver disease (ALD). This study was conducted to determine whether PNPLA3 rs738409 SNPs affect development and prognosis of hepatocellular carcinoma (HCC) in patients with various liver diseases. We enrolled 638 consecutive Japanese patients newly diagnosed with HCC between 2001 and 2010: 72 patients with hepatitis B virus (HBV), 462 with hepatitis C virus (HCV), and 104 with non-B non-C (NBNC). NBNC patients exhibited large tumors of advanced TNM stages at HCC diagnosis, and had significantly poorer prognosis than HBV or HCV patients (P < 0.001 and < 0.001, respectively; log-rank test). The G/G genotype of PNPLA3 rs738409 SNP had significantly higher distribution in NBNC patients (P < 0.001) and was significantly associated with higher body mass index (BMI) and an increased aspartate aminotransferase to platelet ratio index. No significant differences were observed in survival with differences in PNPLA3 SNP genotypes among the patients, although ALD patients with the G/G genotype of PNPLA3 SNP and low BMI had significantly poorer survival than those with high BMI (P = 0.028). The G/G genotype of PNPLA3 rs738409 SNP was more frequently distributed, and associated with BMI and fibrosis among NBNC-HCC patients but not among HBV or HCV patients. These genotypes might affect HCC prognosis in ALD patients, but not in HBV, HCV, or NAFLD patients. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0095-1137 52 2 2014 Necessity for Reassessment of Patients with Serogroup 2 Hepatitis C Virus (HCV) and Undetectable Serum HCV RNA 544 548 EN Yuki Moritou Fusao Ikeda Yasuto Takeuchi Hiroyuki Seki Shintaro Nanba Yoshiaki Iwasaki Kazuhide Yamamoto We encountered a patient positive for anti-hepatitis C virus (HCV) whose serum HCV RNA was undetectable with the Roche AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM) version 1 but showed a high viral load with the Abbott RealTime HCV assay (ART). Discrepancies in the detectability of serum HCV RNA were investigated among 891 consecutive patients who were positive for anti-HCV. Specific nucleotide variations causing the undetectability of HCV RNA were determined and confirmed by synthesizing RNA coding those variations. Serum samples with the discrepancies were also reassessed by CAP/CTM version 2. Among the 891 anti-HCV-positive patients, 4 patients had serum HCV RNA levels that were undetectable by CAP/CTM version 1 despite having levels of > 5 log IU/ml that were detected by ART. All four patients had HCV genotype 2a and high titers of anti-HCV. Sequencing of the HCV 5' noncoding regions revealed 2 common variations, A at nucleotide (nt) 145 and T at nt 151. Synthesized RNAs of the HCV 5' noncoding region with standard (NCR145G151C) and variant nucleotides at nt 145 and nt 151 were quantified with CAP/CTM. RNAs of NCR145G151C and NCR145G151T were quantifiable with CAP/CTM version 1, while those of NCR145A151T and NCR145A151C went undetected. The substitution from G to A at nt 145 specifically conferred this undetectability, while this undetectability was reverted in synthesized HCV RNA with correction of this variation. Reassessment of these samples by CAP/CTM version 2 resulted in similar levels of HCV RNA being detected by ART. We conclude that HCV patients with undetectable HCV RNA by CAP/CTM version 1 should be reassessed for viral quantification. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 126 3 2014 平成25年度岡山医学会賞 総合研究奨励賞（結城賞） 187 190 EN Hiromasa Yamamoto 受賞対象論文: Yamamoto H, Higasa K, Sakaguchi M, Shien K, Soh J, Ichimura K, Furukawa M, Hashida S, Tsukuda K, Takigawa N, Matsuo K, Kiura K, Miyoshi S, Matsuda F, Toyooka S：Novel germline mutation in the transmembrane domain of HER2 in familial lung adenocarcinomas. J Natl Cancer Inst (2014) 106, djt338 No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 68 5 2014 Hepatitis C Virus-specific T-cell Response Correlates with Hepatitis Activity and Donor IL28B Genotype Early after Liver Transplantation 291 302 EN Ryuichiro Tsuzaki Akinobu Takaki Takahito Yagi Fusao Ikeda Kazuko Koike Yoshiaki Iwasaki Hidenori Shiraha Yasuhiro Miyake Hiroshi Sadamori Susumu Shinoura Yuzo Umeda Ryuichi Yoshida Daisuke Nobuoka Masashi Utsumi Eiichi Nakayama Toshiyoshi Fujiwara Kazuhide Yamamoto Original Article 10.18926/AMO/52898 It is not known how the immune system targets hepatitis C virus (HCV)-infected HLA-mismatched hepatocytes under immune-suppressed conditions after orthotopic liver transplantation (OLT). In addition, the relationship between the HCV-specific immune response and IL28B variants as predictors of HCV clearance has not been well-characterized. We determined the IL28B polymorphisms for 57 post-OLT HCV carriers, and we assessed the HCV-specific immune responses by measuring the peripheral blood mononuclear cell-derived HCV-specific interferon-gamma (IFN-γ) response using an enzyme-linked immunospot assay. At 1-3 years after OLT, patients with no active hepatitis showed higher total spots on the immunospot assay. At＞3 years after OLT, patients with resolved HCV showed higher levels of core, NS3, NS5A, and total spots compared to the chronic hepatitis patients. The IL28B major genotype in the donors correlated with higher spot counts for NS5A and NS5B proteins at 1-3 years after OLT. In the post-OLT setting, the HCV-specific immune response could be strongly induced in patients with no active hepatitis with an IL28B major donor or sustained virological response. Strong immune responses in the patients with no active hepatitis could only be maintained for 3 years and diminished later. It may be beneficial to administer IFN treatment starting 3 years after OLT, to induce the maximum immunological effect. No potential conflict of interest relevant to this article was reported.

Elsevier B.V. Acta Medica Okayama 0168-1656 185 2014 A rapid and enhanced DNA detection method for crop cultivar discrimination 57 62 EN Yuki Monden Kazuto Takasaki Satoshi Futo Kousuke Niwa Mitsuo Kawase Hiroto Akitake Makoto Tahara In many crops species, the development of a rapid and precise cultivar discrimination system has been required for plant breeding and patent protection of plant cultivars and agricultural products. Here, we successfully evaluated strawberry cultivars via a novel method, namely, the single tag hybridization (STH) chromatographic printed array strip (PAS) using the PCR products of eight genomic regions. In a previous study, we showed that genotyping of eight genomic regions derived from FaRE1 retrotransposon insertion site enabled to discriminate 32 strawberry cultivars precisely, however, this method required agarose/acrylamide gel electrophoresis, thus has the difficulty for practical application. In contrast, novel DNA detection method in this study has some great advantages over standard DNA detection methods, including agarose/acrylamide gel electrophoresis, because it produces signals for DNA detection with dramatically higher sensitivity in a shorter time without any preparation or staining of a gel. Moreover, this method enables the visualization of multiplex signals simultaneously in a single reaction using several independent amplification products. We expect that this novel method will become a rapid and convenient cultivar screening assay for practical purposes, and will be widely applied to various situations, including laboratory research, and on-site inspection of plant cultivars and agricultural products. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 68 4 2014 Prevalence and Outcomes of Acute Hepatitis B in Okayama, Japan, 2006-2010 243 247 EN Nozomu Wada Tetsuya Yasunaka Fusao Ikeda Sohji Nishina Masaaki Korenaga Keisuke Hino Shin-ichi Fujioka Toshiya Osawa Tatsuya Itoshima Miwa Kawanaka Gotaro Yamada Kazuya Kariyama Hiroki Takayama Junichi Kubota Yoichi Morimoto Takaaki Mizushima Haruhiko Yamashita Hiroaki Tanioka Yuji Negoro Junichi Toshimori Haruhiko Kobashi Atsushi Hirano Yasuo Itano Akinobu Takaki Kazuhide Yamamoto Original Article 10.18926/AMO/52790 Hepatitis B virus (HBV) is one of the major viruses causing acute hepatitis. Recently, the incidence of acute hepatitis with genotype A has been increasing in Japan. The aim of this study was to investigate acute hepatitis B (AHB) in Okayama prefecture, with special attention to HBV genotype A. AHB patients who visited one of 12 general hospitals in Okayama prefecture between 2006 and 2010 were retrospectively analyzed. Over the course of the study period, 128 patients were diagnosed with AHB. Sexual transmission was supposed in the majority of patients (78 patients, 61%), including 59 (76%) having sex with heterosexual partners. The genotypes of HBV were assessed in 90 patients (70%), of whom 27 patients were infected with genotype A, 5 with genotype B, and 58 with genotype C. The prevalence of genotype A was significantly higher among male patients (28.7%), aged 20-29 (35.6%, p＜0.01), among men who had sex with men (100%, p＜0.005), and among patients having sex with unspecified partners (44.8%, p＜0.005). Genotype A was not a significant factor associated with delayed HBsAg disappearance. Caution should be exercised with regard to sexually transmissible diseases in order to slow the pandemic spread of AHB due to genotype A. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 68 2 2014 Prevalence of High-Risk Human Papillomavirus (HR-HPV) Infection among Women with Normal and Abnormal Cervical Cytology in Myanmar 79 87 EN Mu-Mu-Shwe Teruo Harano Shigeru Okada Aye-Aye-Win Khin-Saw-Aye Hlaing-Myat-Thu Mo-Mo-Win Khin-Khin-Oo Myo-Khin Original Article 10.18926/AMO/52404 This study aimed to determine the prevalence of normal and abnormal cervical cytology in women who attended the cervical cancer screening clinic of the Department of Medical Research in Lower Myanmar, and to determine the proportion of high-risk (HR) human papillomavirus (HPV) infection and HPV genotypes in women with normal and abnormal cervical cytology. A total of 1,771 women were screened from 2010 to 2011. Among them, 762 women (43.0%) had a normal smear, and 866 (48.9%) and 87 (4.9%) were diagnosed with inflammatory smears and atypical squamous cells of undetermined significance (ASCUS), respectively. Diagnoses of low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) numbered 42 (2.3%) and 11 (0.6%) respectively. Three cases of squamous cell carcinoma (SCC) (0.2%) were detected. Cervical swabs were collected from 96 women with abnormal cervical cytology and 20 with normal cytology. HR-HPV DNA testing was performed by polymerase chain reaction (PCR) with pU1M/pU2R primers. HR-HPV were identified in 35.5% (22/62) of inflammatory smears, 60% (6/10) of ASCUS, 86.7% (13/15) of LSIL, 50% (3/6) of HSIL, 100% (3/3) of SCC and 5% (1/20) of normal cytology. In PCR-positive cases, HPV genotyping was analyzed by the cleaved amplification polymorphism method. The most prevalent HPV genotypes were HPV-16 (60.4%) followed by HPV-31 (14.6%), HPV-18 (12.5%) and HPV-58 (12.5%). Women with abnormal cervical cytology were 10 times more likely to be HR-HPV positive than those with normal cytology (p＝0.0001). This study suggests that the implementation of a cervical cytology screening program and routine vaccination against HPV in preadolescent and adolescent groups are needed to reduce the burden of HPV-associated cervical cancer. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1471-2407 11 2011 Hepatitis B virus core promoter mutations G1613A and C1653T are significantly associated with hepatocellular carcinoma in genotype C HBV-infected patients EN Masashi Tatsukawa Akinobu Takaki Hidenori Shiraha Kazuko Koike Yoshiaki Iwasaki Haruhiko Kobashi Shin-Ichi Fujioka Kohsaku Sakaguchi Kazuhide Yamamoto Background: Hepatitis B virus (HBV) is a major cause of hepatocarcinogenesis. To identify mutations relevant to hepatocellular carcinoma (HCC) development, we compared the full genome sequences of HBV from the sera of patients with and without HCC. Methods: We compared the full genome sequences of HBV isolates from 37 HCC patients (HCC group 1) and 38 patients without HCC (non-HCC group 1). We also investigated part of the core promoter region sequences from 40 HCC patients (HCC group 2) and 68 patients without HCC. Of the 68 patients who initially did not have HCC, 52 patients remained HCC-free during the follow-up period (non-HCC group 2), and 16 patients eventually developed HCC (pre-HCC group 2). Serum samples collected from patients were subjected to PCR, and the HBV DNA was directly sequenced. Results: All patients had genotype C. A comparison of the nucleotide sequences of the HBV genome between HCC group 1 and non-HCC group 1 revealed that the prevalence of G1613A and C1653T mutations in the core promoter region was significantly higher in the HCC group. These mutations tended to occur simultaneously in HCC patients. Multivariate analysis with group 2 revealed that the presence of HCC was associated with aging and the double mutation. Future emergence of HCC was associated with aging and the presence of a single G1613A mutation. Conclusions: G1613A and C1653T double mutations were frequently found in patients with HCC. A single G1613A mutation was associated with future emergence of HCC. These mutations may serve as useful markers in predicting HCC development. No potential conflict of interest relevant to this article was reported.

Biomed Central Ltd Acta Medica Okayama 1471-2164 10 2009 Development and implementation of high-throughput SNP genotyping in barley EN Timothy J. Close Prasanna R. Bhat Stefano Lonardi Yonghui Wu Nils Rostoks Luke Ramsay Arnis Druka Nils Stein Jan T. Svensson Steve Wanamaker Serdar Bozdag Mikeal L. Roose Matthew J. Moscou Shiaoman Chao Rajeev K. Varshney Peter Szuecs Kazuhiro Sato Patrick M. Hayes David E. Matthews Andris Kleinhofs Gary J. Muehlbauer Joseph DeYoung David F. Marshall Kavitha Madishetty Raymond D. Fenton Pascal Condamine Andreas Graner Robbie Waugh Background: High density genetic maps of plants have, nearly without exception, made use of marker datasets containing missing or questionable genotype calls derived from a variety of genic and non-genic or anonymous markers, and been presented as a single linear order of genetic loci for each linkage group. The consequences of missing or erroneous data include falsely separated markers, expansion of cM distances and incorrect marker order. These imperfections are amplified in consensus maps and problematic when fine resolution is critical including comparative genome analyses and map-based cloning. Here we provide a new paradigm, a high-density consensus genetic map of barley based only on complete and error-free datasets and genic markers, represented accurately by graphs and approximately by a best-fit linear order, and supported by a readily available SNP genotyping resource. Results: Approximately 22,000 SNPs were identified from barley ESTs and sequenced amplicons; 4,596 of them were tested for performance in three pilot phase Illumina GoldenGate assays. Data from three barley doubled haploid mapping populations supported the production of an initial consensus map. Over 200 germplasm selections, principally European and US breeding material, were used to estimate minor allele frequency (MAF) for each SNP. We selected 3,072 of these tested SNPs based on technical performance, map location, MAF and biological interest to fill two 1536-SNP "production" assays (BOPA1 and BOPA2), which were made available to the barley genetics community. Data were added using BOPA1 from a fourth mapping population to yield a consensus map containing 2,943 SNP loci in 975 marker bins covering a genetic distance of 1099 cM. Conclusion: The unprecedented density of genic markers and marker bins enabled a high resolution comparison of the genomes of barley and rice. Low recombination in pericentric regions is evident from bins containing many more than the average number of markers, meaning that a large number of genes are recombinationally locked into the genetic centromeric regions of several barley chromosomes. Examination of US breeding germplasm illustrated the usefulness of BOPA1 and BOPA2 in that they provide excellent marker density and sensitivity for detection of minor alleles in this genetically narrow material. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1344-7610 59 4 2009 Mapping of QTL for intermedium spike on barley chromosome 4H using EST-based markers 383 390 EN Fahimeh Shahinnia Badraldin Ebrahim Sayed-Tabatabaei Kazuhiro Sato Mohammad Pourkheirandish Takao Komatsuda The lateral spikelets of two-rowed barley are reduced in size and sterile, but in six-rowed barley all three spikelets are fully fertile. The trait is largely controlled by alleles at the vrs1 locus on chromosome arm 2HL, as modified by the allele present at the I locus on chromosome arm 4HS. Molecular markers were developed to saturate the 4HS region by exploiting expressed sequence-tags, either previously mapped in barley to this region, or present in the syntenic region of rice chromosome 3. Collinearity between rice and barley was strong in the 4.8 cM interval BJ468164-AV933435 and the 10 cM interval AV942364-BJ455560. A major QTL for lateral spikelet fertility (the I locus) explained 44% of phenotypic variance, and was located in the interval CB873567-BJ473916. The genotyping of near-isogenic lines for I placed the locus in a region between CB873567 and EBmac635, and therefore the most likely position of the I locus was proximal to CB873567 in a 5.3 cM interval between CB873567-BJ473916. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2013 Hepatitis B virus core promoter mutations G1613A and C1653T are significantly associated with hepatocellular carcinoma in genotype C HBV-infected patients EN Masashi Tatsukawa No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 68 1 2014 Impact of Comorbid Hepatic Steatosis on Treatment of Chronic Hepatitis C in Japanese Patients and the Relationship with Genetic Polymorphism of IL28B, PNPLA3 and LDL Receptor 17 22 EN Yuki Moritou Fusao Ikeda Yoshiaki Iwasaki Nobuyuki Baba Kouichi Takaguchi Tomonori Senoh Takuya Nagano Yasuto Takeuchi Tetsuya Yasunaka Hideki Ohnishi Yasuhiro Miyake Akinobu Takaki Kazuhiro Nouso Kazuhide Yamamoto Original Article 10.18926/AMO/52139 The impact of hepatic steatosis on interferon therapy for patients with chronic hepatitis C (CHC) has been associated with single-nucleotide polymorphisms (SNP) of IL28B, patatin-like phospholipase domain-containing protein 3 (PNPLA3), and low-density lipoprotein (LDL) receptor. Whether this holds true for Japanese patients, however, remains unresolved. The present study prospectively enrolled 226 Japanese patients with CHC, and investigated the impact of hepatic steatosis and its related SNPs, including rs8099917 of IL28B, rs738409 of PNPLA3, and rs14158 of LDL receptor, on outcomes of peg-interferon and ribavirin therapy. In multivariate logistic regression analysis, significant factors affecting the severity of hepatic steatosis were high body mass index and the minor alleles of IL28B SNP (p＝0.020 and 0.039, respectively). The risk alleles of PNPLA3 SNP also showed weak association (p＝0.059). Severe steatosis and the minor alleles of IL28B SNP were significantly associated with null or partial virological response in patients with HCV genotype 1, as were female gender, and low LDL cholesterol (p＝0.049, and ＜0.001, respectively). The SNP genotype of PNPLA3 and LDL receptor did not have a significant impact on therapeutic outcomes. With respect to the SNP sites examined, the SNP of PNPLA3 has a weak association with severe hepatic steatosis, but not with the outcome of interferon therapy. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2013 Distribution and molecular characterization of Porphyromonas gulae carrying a new fimA genotype EN Yoshie Yamasaki No potential conflict of interest relevant to this article was reported.

Ohara Instituts für landwirtschaftliche Biologie Acta Medica Okayama 12 2 1964 EXPERlMENTAL STUDIES ON NATURAL SELECTION FOR TlME OF HEADING AND ITS INNER FACTORS IN SOME BARLEY HYBRID POPULATIONS II. Genotypic Constitutions of Selection Lines Derived from the Populations Grown at Different Locations 197 215 EN Shozo Yasuda No potential conflict of interest relevant to this article was reported.

Endocrine Soc Acta Medica Okayama 0021-972X 97 7 2012 Serum Vaspin Concentrations Are Closely Related to Insulin Resistance, and rs77060950 at SERPINA12 Genetically Defines Distinct Group with Higher Serum Levels in Japanese Population E1202 E1207 EN Sanae Teshigawara Jun Wada Kazuyuki Hida Atsuko Nakatsuka Jun Eguchi Kazutoshi Murakami Motoko Kanzaki Kentaro Inoue Takahiro Terami Akihiro Katayama Izumi Iseda Yuichi Matsushita Nobuyuki Miyatake John F. McDonald Kikuko Hotta Hirofumi Makino Context: Vaspin is an adipokine with insulin-sensitizing effects identified from visceral adipose tissues of genetically obese rats. Objective: We investigated genetic and nongenetic factors that define serum concentrations of vaspin. Design, Setting and Participants: Vaspin levels were measured with RIA in Japanese subjects with normal fasting plasma glucose (NFG; n = 259) and type 2 diabetes patients (T2D; n = 275). Single nucleotide polymorphisms (SNP) at SERPINA12 (vaspin) gene locus were discovered, and five SNP were genotyped in the subjects with varied body mass index (n = 1138). Results: The level of serum vaspin in 93% of the samples was found to vary from 0.2 to nearly 2 ng/ml in NFG subjects (n = 259) and from 0.2 to nearly 3 ng/ml in T2D patients (n = 275) (Vaspin(Low) group), whereas a significant subpopulation (7%) in both groups displayed much higher levels of 10-40 ng/ml (Vaspin(High) group). In the Vaspin(Low) group, serum vaspin levels in T2D were significantly higher than healthy subjects (0.99 +/- 0.04 vs. 0.86 +/- 0.02 ng/ml; P < 0.01). Both in T2D and genotyped Japanese population, serum vaspin levels closely correlated with homeostasis model of assessment for insulin resistance rather than anthropometric parameters. By genotyping, rs77060950 tightly linked to serum vaspin levels, i.e. CC (0.6 +/- 0.4 ng/ml), CA (18.4 +/- 9.6 ng/ml), and AA (30.5 +/- 5.1 ng/ml) (P < 2 x 10(-16)). Putative GATA-2 and GATA-3 binding consensus site was found at rs77060950. Conclusions: Serum vaspin levels were related to insulin resistance, and higher levels of serum vaspin in 7% of the Japanese population are closely linked to minor allele sequence (A) of rs77060950. (J Clin Endocrinol Metab 97: E1202-E1207, 2012) No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 125 1 2013 肺癌と分子標的薬 57 66 EN Katsuyuki Kiura Mitsune Tanimoto No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 124 3 2012 薬物相互作用 (25―Ｃ型肝炎治療薬テラプレビルにおける薬物相互作用） 259 263 EN Kazuhiko Yamaji Toshiaki Sendo No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 124 3 2012 高分化型ヒト肝癌由来細胞株“HuH-7” 231 238 EN Hidekazu Nakabayashi Kazuhisa Taketa 高分化型ヒト肝癌由来細胞株“HuH-7”は，1982年にCancer Researchにその樹立を報告した．HuH-7は，当時の岡山大学医学部附属癌源研究施設病理部門（故佐藤二郎教授）の下で樹立し，これまで多くの研究分野で利用され，世界的に有名な肝癌細胞株となっている．本稿では，有用性の高い分化機能を有するヒト肝癌細胞株HuH-7について，肝細胞癌の腫瘍マーカーであるα-fetoprotein（AFP）を中心に，この細胞株を用いた研究分野に関する詳細を紹介する． No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1932-6203 6 4 2011 Environmental Stress-Dependent Effects of Deletions Encompassing Hsp70Ba on Canalization and Quantitative Trait Asymmetry in Drosophila melanogaster EN Kazuo H. Takahashi Phillip J. Daborn Ary A. Hoffmann Toshiyuki Takano-Shimizu Hsp70 genes may influence the expression of wing abnormalities in Drosophila melanogaster but their effects on variability in quantitative characters and developmental instability are unclear. In this study, we focused on one of the six Hsp70 genes, Hsp70Ba, and investigated its effects on within-and among-individual variability in orbital bristle number, sternopleural bristle number, wing size and wing shape under different environmental conditions. To do this, we studied a newly constructed deletion, Df(3R)ED5579, which encompasses Hsp70Ba and nine non-Hsp genes, in the heterozygous condition and another, Hsp70Ba(304), which deletes only Hsp70Ba, in the homozygous condition. We found no significant effect of both deletions on within-individual variation quantified by fluctuating asymmetry (FA) of morphological traits. On the other hand, the Hsp70Ba(304)/Hsp70Ba(304) genotype significantly increased among-individual variation quantified by coefficient of variation (CV) of bristle number and wing size in female, while the Df(3R)ED5579 heterozygote showed no significant effect. The expression level of Hsp70Ba in the deletion heterozygote was 6 to 20 times higher than in control homozygotes, suggesting that the overexpression of Hsp70Ba did not influence developmental stability or canalization significantly. These findings suggest that the absence of expression of Hsp70Ba increases CV of some morphological traits and that HSP70Ba may buffer against environmental perturbations on some quantitative traits. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2012 Phenotype–phenotype and genotype–phenotype correlations in patients with idiopathic superior oblique muscle palsy EN Sayuri Okubo No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0022-1317 91 9 2010 Complete genome constellation of a caprine group A rotavirus strain reveals common evolution with ruminant and human rotavirus strains 2367 2373 EN Souvik Ghosh Mohammed Mahbub Alam Muzahed Uddin Ahmed Rafiqul Islam Talukdar Shyamal Kumar Paul Nobumichi Kobayashi This study reports the first complete genome sequence of a caprine group A rotavirus (GAR) strain, GO34. The VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes of strain GO34, detected in Bangladesh, were assigned to the G6-P[1]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genotypes, respectively. Strain GO34 was closely related to the VP4, VP6–7 and NSP4–5 genes of bovine GARs and the NSP1 gene of GO34 to an ovine GAR. Strain GO34 shared low nucleotide sequence identities (<90 %) with VP2–3 genes of other GARs, and was equally related to NSP3 genes of human, ruminant and camelid strains. The VP1, VP6 and NSP2 genes of strain GO34 also exhibited a close genetic relatedness to human G2, G6, G8 and G12 DS-1-like GARs, whereas the NSP1 of GO34 was also closely related to human G6P[14] strains. All these findings point to a common evolutionary origin of GO34 and bovine, ovine, antelope, guanaco and human G6P[14] GARs, although phylogenetically GO34 is not particularly closely related to any other rotavirus strains known to date. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0304-4017 171 1-2 2010 Molecular characterization and assessment of zoonotic transmission of Cryptosporidium from dairy cattle in West Bengal, India 41 47 EN Shahbaz Manzoor Khan Chanchal Debnath Amiya Kumar Pramanik Lihua Xiao Tomoyoshi Nozaki Sandipan Ganguly Few studies in the past have examined the genetic diversity and zoonotic potential of Cryptosporidium in dairy cattle in India. To assess the importance of these animals as a source of human Cryptosporidium infections, fecal samples from 180 calves, heifers and adults and 51 farm workers on two dairy farms in West Bengal, India were genotyped by PCR-RFLP analysis of the 18S rRNA gene of Cryptosporidium followed by DNA sequencing of the PCR products. Phylogenetic analysis was carried out on the DNA sequences obtained in the study and those available in GenBank. The overall prevalence of Cryptosporidium in cattle was 11.7% though the infection was more prevalent in younger calves than in adult cattle. The occurrence of Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium andersoni in cattle followed an age-related pattern. A Cryptosporidium suis-like genotype was also detected in a calf. Farm workers were infected with Cryptosporidium hominis, C. parvum and a novel C. bovis genotype. These findings clearly suggest that there is a potential risk of zoonotic transmission of Cryptosporidium infections between cattle and humans on dairy farms in India. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0304-4017 178 3-4 2011 Molecular evidence for zoonotic transmission of Giardia duodenalis among dairy farm workers in West Bengal, India 342 345 EN Shahbaz Manzoor Khan Chanchal Debnath Amiya Kumar Pramanik Lihua Xiao Tomoyoshi Nozaki Sandipan Ganguly No study in the past has examined the genetic diversity and zoonotic potential of Giardia duodenalis in dairy cattle in India. To assess the importance of these animals as a source of human G. duodenalis infections and determine the epidemiology of bovine giardiasis in India, fecal samples from 180 calves, heifers and adults and 51 dairy farm workers on two dairy farms in West Bengal, India were genotyped by PCR-RFLP analysis of the β-giardin gene of G. duodenalis followed by DNA sequencing of the nested PCR products. The overall prevalence of G. duodenalis in cattle was 12.2% (22/180), the infection being more prevalent in younger calves than in adult cattle. Zoonotic G. duodenalis Assemblage A1 was identified in both calves and workers although the most prevalent genotype detected in cattle was a novel Assemblage E subgenotype. These findings clearly suggest that there is a potential risk of zoonotic transmission of G. duodenalis infections between cattle and humans on dairy farms in India. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1567-1348 11 1 2011 Full genomic analysis of a simian SA11-like G3P[2] rotavirus strain isolated from an asymptomatic infant: Identification of novel VP1, VP6 and NSP4 genotypes 57 63 EN Souvik Ghosh Zipporah Gatheru James Nyangao Noriaki Adachi Noriko Urushibara Nobumichi Kobayashi We report here the full genomic analysis of a simian SA11-like G3P[2] group A rotavirus (GAR) strain, B10, isolated from an asymptomatic infant in Kenya in 1987. By nucleotide sequence identities and phylogenetic analyses, the VP7–VP4–VP2–VP3–NSP1–NSP2–NSP3–NSP5 genes of strain B10 exhibited maximum genetic relatedness to those of the different isolates of simian strain SA11, and were assigned to the G3–P[2]–C5–M5–A5–N5–T5–H5 genotypes, respectively. On the other hand, the VP1, VP6 and NSP4 genes of strain B10 did not belong to any of the established GAR genotypes, and therefore, were assigned to new genotype numbers R8, I16 and E13, respectively, by the Rotavirus Classification Working Group. These observations suggested that strain B10 might have originated from reassortment event/s involving simian SA11-like strains and GAR strains from unknown animal host species (possibly other wild animals) preceding transmission to humans. Alternatively, considering the lack of data on simian GARs, it might be also possible that the VP1, VP6 and NSP4 genes of strain B10 are those of unknown simian strains, and that strain B10 might be a typical simian strain that was directly transmitted to humans. Therefore, either hypothesis pointed towards a rare instance of possible direct transmission of GARs from an animal host (possibly a monkey or some other wild animal) to humans. This was corroborated by the presence of different species of wild animals including non-human primates, and unhygienic conditions at the sampling site. To our knowledge, the present study is the first report on the detection of a simian SA11-like G3P[2] GAR strain in humans. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 0304-8608 155 2 2009 Molecular characterization of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of bovine group B rotaviruses: identification of a novel VP4 genotype 159 167 EN S Ghosh N Kobayashi S Nagashima M Chawla-Sarkar T Krishnan B Ganesh TN Naik Studies on bovine group B rotaviruses (GBRs) are limited. To date, only the VP6 gene of a single bovine GBR strain and the VP7 and NSP5 genes of a few bovine GBR strains have been sequenced and analyzed. In the present study, using a single-primer amplification method, we have determined the full-length nucleotide sequences of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of three bovine GBR strains from eastern India. In all six of these genes, the bovine GBR strains shared high genetic relatedness among themselves but exhibited high genetic diversity with cognate genes of human, murine and ovine GBRs. Interestingly, as with group A rotaviruses, the bovine GBR VP1, VP2, VP6 and NSP2 genes appeared to be more conserved than the VP4 and NSP1 genes among strains of different species. The present study provides important insights into the genetic makeup and diversity of bovine GBRs, and also identifies a novel GBR VP4 genotype. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 66 2 2012 Different Responses to 5-fluoraouracil in Mutagenicity and Gene Expression between Two Human Lymphoblastoid Cell Lines with or without TP53 Mutation 119 129 EN Hiroaki Oka Mamoru Ouchida Takuya Kondo Fumio Morita Kenji Shimizu Original Article 10.18926/AMO/48262 Human lymphoblastoid TK6 and WTK-1 cells are widely used to detect mutagens in vitro. TK6 cells have wild-type TP53 alleles, while WTK-1 cells have one allele of mutated TP53. Both cells were treated with 5-fluorouracil (5-FU), and gene mutation assay and micronucleus assay were performed to clarify the differential response related to the TP53 gene status. The effects of 5-FU on gene expression were assessed by microarray and quantitative RT-PCR analyses. In WTK-1 cells, 5-FU increased the frequency of cells with micronucleus and mutation. In TK6 cells, frequency of cells with micronucleus was increased but the mutation frequency was not. The cytotoxicity induced by 5-FU was more prominent in TK6 cells than in WTK-1 cells. Analysis of gene expression showed that the genes involved in the TP53 pathway were up-regulated in TK6 cells but not in WTK-1 cells. The differential responses to 5-FU between these cell lines appeared to be due to the difference in the TP53 gene status, thus providing a molecular basis for the bioassays using these cell lines in the toxicology field. Our results indicate that the clinical efficacy of 5-FU chemotherapy may depend on the TP53 genotype. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 2186-7755 101 2012 ‘ファースト’雄性不稔突然変異体 (T -4) を種子親とした軟果皮中玉トマトF(1) 系統 (MS-II) の特性 19 24 EN Junko Toya Masaharu Masuda Kenji Murakami Breeding for a soft pericarp in medium-sized tomato fruit was conducted by crossing the male sterile mutant (T-4) of the large-fruited 'First' and a small-fruited pure line with a soft pericarp (S). Pericarp characteristics of the F(1) hybrid (named MS-II) were compared with the parents and two similar medium-fruited tomato cultivars, 'Red ore' and 'Frutica'. Pericarp firmness in MS-II was lower as compared with that of both T-4 and S. Differences in pericarp firmness among MS-II, 'Red ore' and 'Frutica' were dependent on truss. In the first truss, MS-II developed fruits with a softer pericarp than 'Red ore', but with a firmer pericarp than 'Frutica'. In the second and third trusses, pericarp firmness of the fruit in MS-II tended to be lower than those of the other two cultivars. The thickness of the exocarp cuticle in MS-II was lower than that in 'Red ore', but was no different to that in 'Frutica'. Thus genotypic differences in pericarp firmness among MS-II, 'Red ore' and 'Frutica' seem to be derived from differences in the degree of cutin development in the epidermal perimeter. A thinner cuticle can explain pericarp softness in the fruits above the second truss in MS-II. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 65 5 2011 Single Nucleotide Polymorphism WRN Leu1074Phe Is Associated with Prostate Cancer Susceptibility in Chinese Subjects 315 323 EN Lei Wang Haruki Kaku Peng Huang Kexin Xu Kai Yang Jiheng Zhang Ming Li Liping Xie Xiaofeng Wang Akiko Sakai Masami Watanabe Yasutomo Nasu Kenji Shimizu Hiromi Kumon Yanqun Na Original Article 10.18926/AMO/47013 Deficiencies in the human DNA repair gene WRN are the cause of Werner syndrome, a rare autosomal recessive disorder characterized by premature aging and a predisposition to cancer. This study evaluated the association of WRN Leu1074Phe (rs1801195), a common missense single nucleotide polymorphism in WRN, with prostate cancer susceptibility in Chinese subjects. One hundred and forty-seven prostate cancer patients and 111 male cancer-free control subjects from 3 university hospitals in China were included. Blood samples were obtained from each subject, and the single nucleotide polymorphism WRN Leu1074Phe was genotyped by using a Snapshot assay. The results showed that WRN Leu1074Phe was associated with the risk of prostate cancer in Chinese men and that the TG/GG genotype displayed a decreased prevalence of prostate cancer compared with the TT genotype (OR＝0.58, 95%CI:0.35-0.97, p＝0.039). Through stratified analysis, more significant associations were revealed for the TG/GG genotype in the subgroup with diagnosis age <_ 72 yr (OR＝0.27, 95%CI:0.12-0.61, p＝0.002) and in patients with localized diseases (OR＝0.36, 95%CI:0.19-0.70, p＝0.003). However, no statistically significant difference was found in the subgroup with age ＞72 yr or in patients with advanced diseases. We concluded that the genetic variant Leu1074Phe in the DNA repair gene WRN might play a role in the risk of prostate cancer in Chinese subjects. No potential conflict of interest relevant to this article was reported.

Academic Press Inc Elsevier Science Acta Medica Okayama 0006-291X 412 2 2011 Tumor suppressor REIC/Dkk-3 interacts with the dynein light chain, Tctex-1 391 395 EN Kazuhiko Ochiai Masami Watanabe Hideo Ueki Peng Huang Yasuyuki Fujii Yasutomo Nasu Hirofumi Noguchi Takeshi Hirata Masakiyo Sakaguchi Nam-ho Huh Yuji Kashiwakura Haruki Kaku Hiromi Kumon Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. HuH-7 hepatoma-derived cells are widely used as the only cell-based HCV replication system for HCV research, including drug assays. Recently, using different hepatoma Li23-derived cells, we developed an HCV drug assay system (ORL8), in which the genome-length HCV RNA (O strain of genotype 1b) encoding renilla luciferase replicates efficiently. In this study, using the HuH-7-derived OR6 assay system that we developed previously and the ORL8 assay system, we evaluated 26 anti-HCV reagents, which other groups had reported as anti-HCV candidates using HuH-7-derived assay systems other than ORB. The results revealed that more than half of the reagents showed different anti-HCV activities from those in the previous studies, and that anti-HCV activities evaluated by the ORB and ORL8 assays were also frequently different. In further evaluation using the HuH-7-derived AH1R assay system, which was developed using the AH1 strain of genotype 1b, several reagents showed different anti-HCV activities in comparison with those evaluated by the OR6 and ORL8 assays. These results suggest that the different activities of anti-HCV reagents are caused by the differences in cell lines or HCV strains used for the development of assay systems. Therefore, we conclude that plural HCV assay systems developed using different cell lines or HCV strains are required for the objective evaluation of anti-HCV reagents. No potential conflict of interest relevant to this article was reported.

Academic Press Inc Elsevier Science Acta Medica Okayama 0006-291X 409 4 2011 Plural assay systems derived from different cell lines and hepatitis C virus strains are required for the objective evaluation of anti-hepatitis C virus reagents 663 668 EN Youki Ueda Kyoko Mori Yasuo Ariumi Masanori Ikeda Nobuyuki Kato Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. HuH-7 hepatoma-derived cells are widely used as the only cell-based HCV replication system for HCV research, including drug assays. Recently, using different hepatoma Li23-derived cells, we developed an HCV drug assay system (ORL8), in which the genome-length HCV RNA (O strain of genotype 1b) encoding renilla luciferase replicates efficiently. In this study, using the HuH-7-derived OR6 assay system that we developed previously and the ORL8 assay system, we evaluated 26 anti-HCV reagents, which other groups had reported as anti-HCV candidates using HuH-7-derived assay systems other than ORB. The results revealed that more than half of the reagents showed different anti-HCV activities from those in the previous studies, and that anti-HCV activities evaluated by the ORB and ORL8 assays were also frequently different. In further evaluation using the HuH-7-derived AH1R assay system, which was developed using the AH1 strain of genotype 1b, several reagents showed different anti-HCV activities in comparison with those evaluated by the OR6 and ORL8 assays. These results suggest that the different activities of anti-HCV reagents are caused by the differences in cell lines or HCV strains used for the development of assay systems. Therefore, we conclude that plural HCV assay systems developed using different cell lines or HCV strains are required for the objective evaluation of anti-HCV reagents. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 66 6 1954 遺伝神経病の遺伝子に就ての考察 1117 1120 EN Yakichi Ocho I have mentioned the irregularity of heredity of nervous diseases and tried to account for it. The apparent complexity of human pathogenic gene is caused by considering a number of mutations which have different origins as disease unit. Generally speaking, pathogenic gene may be regarded as normal gene whose functions are degenerated or lost through mutation. So instead of Holmes's formula c=f(G. E), I have found my own, c=f[(P. N. gM)E](P: pathogenic gene, N: normal gene, gM. genotypic milieu). Don't P and M stand rather in the relation of compensation than in that of dominance? Thus in the same disease we find some gene dominant and some ressesive, Expressivity of skipping or hetero and homo may be looked upon as mere difference of quantity. The individual difference of the extent of the site of the pathologic changes is vast, ranging from "formes frustes" to "homo lethal". About these phenomena I have tried to give a systematic and consecutive explanation. As disease examples, I have mentioned, including ones of my own examination, Huntington's chorea, Friedreich's ataxia, athetose double, dystrophia progressiva, etc. No potential conflict of interest relevant to this article was reported.

BioMed Central Acta Medica Okayama 2 2002 High-susceptibility of photosynthesis to photoinhibition in the tropical plant Ficus microcarpa L. f. cv. Golden Leaves EN Shunichi Takahashi Ayumu Tamashiro Yasuko Sakihama Yasusi Yamamoto Yoshinobu Kawamitsu Hideo Yamasaki <p><b>Background:</b> The tropical plant Ficus microcarpa L. f. cv. Golden Leaves (GL) is a high-light sensitive tropical fig tree in which sun-leaves are yellow and shade-leaves are green. We compared the response of photosynthetic activities to strong light between GL and its wild-type (WT, Ficus microcarpa L. f.).<br /></p> <p><b>Results:</b> Field measurements of maximum photosystem II (PSII) efficiency (F_v/F_m) of intact sunleaves in GL showed that photo synthetic activity was severely photoinhibited during the daytime (F_v/F_m= 0.46) and subsequently recovered in the evening (F_v/F_m = 0.76). In contrast, WT did not show any substantial changes of F_v/F_m values throughout the day (between 0.82 and 0.78). Light dependency of the CO2 assimilation rate in detached shade-leaves of GL showed a response similar to that in WT, suggesting no substantial difference in photosynthetic performance between them.Several indicators of photoinhibition, including declines in PSII reaction center protein (D1)content, F_v/Fm value, and O2 evolution and CO2 assimilation rates, all indicated that GL is much more susceptible to photoinhibition than WT. Kinetics of PAM chlorophyll a fluorescence revealed that nonphotochemical quenching (NPQ) capacity of GL was lower than that of WT.<br /></p> <p><b>Conclusion:</b> We conclude that the photosynthetic apparatus of GL is more highly susceptible to photoinhibition than that of WT.</p> No potential conflict of interest relevant to this article was reported.

Wiley-Liss, Inc. Acta Medica Okayama 1552-4841 135B 1 2005 A functional glutathione S-transferase P1 gene polymorphism is associated with methamphetamine-induced psychosis in Japanese population 5 9 EN Tasuku Hashimoto Kenji Hashimoto Daisuke Matsuzaka Eiji Shimizu Yoshimoto Sekine Inada Yoshiya Nakao Iwata Mutsuo Harano Tokutaro Komiyama Mitsuhiko Yamada Ichiro Sora Hiroshi Ujike Masaomi Iyo Several lines of evidence suggest that oxidative stress plays a role in the mechanisms of action of methamphetamine (MAP) in the human brain. Given the role of glutathione S-transferases (GSTs) in the protection against oxidative stress, genes encoding the GSTs have been considered as candidates for association studies of MAP abuse. This study was undertaken to investigate the role of the functional polymorphism of GSTP1 gene exon 5 (Ile105Val) in the pathogenesis of MAP abuse. Genotyping for GSTP1 gene polymorphism exon 5 (Ile105Val) in 189 MAP abusers and 199 normal controls was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Association between GSTP1 gene polymorphism and clinical features (prognosis of psychosis (transient-type and prolonged-type), spontaneous relapse (positive and negative), and poly-substance abuse) of MAP abusers was evaluated. Significant differences in the frequency of both alleles (P = 0.026, odds ratio: 1.70, 95% confidence intervals (CI) 1.06-2.72) and genotypes (P = 0.029) between MAP abusers and controls were detected. In particular, a significant difference in both genotype frequency (P = 0.013) and allele frequency (P = 0.014, odds ratio: 1.84, 95% CI 1.13-2.97) between MAP abusers with psychosis (transient-type and prolonged-type) and controls was detected. Our findings suggest that the polymorphism (Ile 105Val) on exon 5 of the GSTP1 gene may contribute to a vulnerability to psychosis associated with MAP abuse in Japanese population. No potential conflict of interest relevant to this article was reported.

Wiley-Liss, Inc. Acta Medica Okayama 1552-4841 132B 1 2005 Association study between brain-derived neurotrophic factor gene polymorphisms and methamphetamine abusers in Japan 70 73 EN Kanako Itoh Kenji Hashimoto Eiji Shimizu Yoshimoto Sekine Norio Ozaki Toshiya Inada Mutsuo Harano Nakao Iwata Tokutaro Komiyama Mitsuhiko Yamada Ichiro Sora Kenji Nakata Hiroshi Ujike Masaomi Iyo Several lines of evidence suggest that genetic factors might contribute to drug abuse vulnerability. Recent genomic scans for association demonstrated that the brain-derived neurotrophic factor (BDNF) gene was associated with drug abuse vulnerability. In this study, we analyzed association of two BDNF gene single nucleotide polymorphisms (SNPs), 132C>T (C270T named formerly) in the noncoding region of exon V and 196G >A (val66met) in the coding region of exon XIIIA, with methamphetamine (MAP) abuse in Japan. No significant differences were found in the frequency of the genotype or allele in these two SNPs between MAP abusers and controls (132C>T in exon V: genotype, p = 0.586, allele, p = 0.594; 196G>A (val66met) in exon XIIIA: genotype, p = 0.889, allele, p = 0.713). Furthermore, there was no difference between clinical parameters (e.g. prognosis psychosis, spontaneous relapse, or poly-substance abuse) and the two SNPs of BDNF gene. These results suggest that the two SNPs (132C>T in exon V and 196G>A (val66met) in exon XIIIA) of the BDNF gene may not be associated with Japanese MAP abusers. No potential conflict of interest relevant to this article was reported.

BioMed Central Acta Medica Okayama 3 2003 No association between the sigma receptor type 1 gene and schizophrenia: results of analysis and meta-analysis of case-control studies EN Naohiko Uchida Hiroshi Ujike Kenji Nakata Manabu Takaki Akira Nomura Takeshi Katsu Yuji Tanaka Takaki Imamura Ayumu Sakai Shigetoshi Kuroda <p><b>Background:</b> Several lines of evidence have supported possible roles of the sigma receptors in the etiology of schizophrenia and mechanisms of antipsychotic efficacy. An association study provided genetic evidence that the sigma receptor type 1 gene (SIGMAR1) was a possible susceptibility factor for schizophrenia, however, it was not replicated by a subsequent study. It is necessary to evaluate further the possibility that the SIGMAR1 gene is associated with susceptibility to schizophrenia. Methods: A case-control association study between two polymorphisms of the SIGMAR1 gene, G-241T/C-240T and Gln2Pro, and schizophrenia in Japanese population, and meta-analysis including present and previous studies.<br /> <b>Results:</b>There was no significant association of any allele or genotype of the polymorphisms with schizophrenia. Neither significant association was observed with hebephrenic or paranoid subtype of schizophrenia. Furthermore, a meta-analysis including the present and previous studies comprising 779 controls and 636 schizophrenics also revealed no significant association between the SIGMAR1 gene and schizophrenia.<br /> <b>Conclusion:</b> In view of this evidence, it is likely that the SIGMAR1 gene does not confersusceptibility to schizophrenia.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 61 1 2007 A homosexual japanese man with acute hepatitis due to hepatitis B virus genotype ae, concurrent with amebic colitis 35 39 EN Yasuhiro Miyake Yoshiaki Iwasaki Shin Ishikawa Masashi Tatsukawa Toru Nawa Jun Kato Akinobu Takaki Haruhiko Kobashi Kohsaku Sakaguchi Yasushi Shiratori Case Report 10.18926/AMO/32914 We report herein a case with acute hepatitis due to hepatitis B virus genotype Ae, concurrent with amebic colitis. A 39-year-old homosexual Japanese man was admitted to our hospital with jaundice. Laboratory tests showed an elevation of transaminase and positivity for hepatitis B surface antigen and IgM-type antibody to hepatitis B core antigen. The hepatitis B virus genotype was determined to be Ae. Furthermore, a mud-like stool with blood and mucous had sometimes been noted during the past 3 years, and amebic colitis was shown by colonofi berscopy during hospitalization. The patient was diagnosed with acute hepatitis B, concurrent with amebic colitis, and was successfully treated with lamivudine and metronidazole. In Japanese patients with acute hepatitis B virus genotype A infection, homosexual activity tends to be high. Furthermore, in Japanese homosexual men, amebiasis has been increasing. Thus, in Japanese patients with acute hepatitis B, a determination of genotype should be performed in order to investigate the route of transmission of hepatitis B virus, and a search for amebiasis should be performed in patients with acute hepatitis due to hepatitis B virus genotype A. Furthermore, education of homosexual men regarding hepatitis B virus, hepatitis B virus vaccination, and amebiasis is urgently required. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 61 3 2007 Polymorphisms in the tumor necrosis factor-alpha gene in Turkish women with pre-eclampsia and eclampsia 153 160 EN Ayfer Pazarbasi Mülkiye Kasap Ali Írfan Güzel Halil Kasap Meliz Onbasioglu Burcu Özbakir Ayse Demirkazik Fatma Tuncay Özgünen Evrim Gürtunç Original Article 10.18926/AMO/32904 The genetic background predisposing pregnant women to pre-eclampsia/eclampsia (PE/E) is still unknown. The aim of the current study was to investigate whether there is an association between the TNF-alpha-308 and 850 polymorphisms and PE or eclampsia. In this study, 40 cases of eclampsia, 113 cases of PE and 80 normotensive control cases were genotyped for the TNF-alpha-G-308A and C-850 polymorphisms. At position 308, the replacement of Guanine with Adenosine was denoted as TNF2. We found a significant difference between the TNF2 allele frequencies of the eclamptic, pre-eclamptic and normotensive controls. TNF2 (AA) polymorphism frequency was significantly higher among the eclamptics and pre-eclamptics (control : 5%, PE : 13.3%, E : 12.9%). A significantly different genotype distribution of C-850T polymorphism was observed between the PE/E and control groups, with the frequency of the variant TT genotype being significantly reduced in the preeclamptics (PE : 17% ; E : 17.5%) when compared with the control group (24.3%). We have demonstrated an association between TNF-alpha polymorphisms and pre-eclampsia susceptibility. However, it is not known whether C-850T polymorphism has a functional effect on the TNF-alpha gene. In addition, it was not possible to determine whether this polymorphism promotes the progression from PE to eclampsia because of no statistically significant difference between eclampsia and the controls. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 57 5 2003 Anti-viral actions and viral dynamics in the early phase of three different regimens of interferon treatment for chronic hepatitis C: differences between the twice-daily administration of interferon-beta treatment and the combination therapy with interferon-alpha plus ribavirin. 217 225 EN Hirofumi Nakajima Hiroyuki Shimomura Yoshiaki Iwasaki Fusao Ikeda Fumi Umeoka Piao Chengyu Hideaki Taniguchi Yasuhiro Ohnishi Shin-jiro Takagi Shin-ichi Fujioka Yasushi Shiratori Article 10.18926/AMO/32825 <p>To improve the efficacy of interferon (IFN) treatment for chronic hepatitis C, we have proposed the twice-daily administration of IFN-beta as a promising induction therapy. In this study, we demonstrated differences between the clearance of circulating HCV-RNA and the induction of anti-viral actions during the first 2 weeks of treatment. Nine patients with a high viral load and genotype 1b were randomly assigned to 3 groups: group A received 3MU of IFN-beta twice a day at intervals of 5 and 19 h; group B received 3MU of IFN-beta twice a day at intervals of 10 and 14 h; group C received 6MU of IFN-alpha once a day with ribavirin. The expression of OAS2, PKR, and MxA in peripheral blood mononuclear cells (PBMCs) were quantified by real-time polymerase chain reaction method. The viral clearance showed a bi-phasic pattern, and those in the second phase of groups A and B were significantly steeper than that of group C. The peak level of OAS2 during the first phase was correlated with the first phase decay. The MxA expression tended to be higher in group A and B than in group C. The expression of these 3 proteins tended to decrease at day 6 in group C, but increase in groups A and B. These might make differences in the viral decay during the second phase</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 57 2 2003 Analysis of short tandem repeat (STR) polymorphisms by the powerplex 16 system and capillary electrophoresis: application to forensic practice. 59 71 EN Osamu Okamoto Yuji Yamamoto Sachiyo Inagaki Kei Yoshitome Takaki ishikawa Kiyomi Imabayashi Satoru Miyaishi Hideo Ishizu Article 10.18926/AMO/32819 <p>Allele and genotype frequencies for 15 short tandem repeat (STR) polymorphisms--D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA--in a Japanese population were estimated. No deviations of the observed allele frequency from Hardy-Weinberg equilibrium expectations were found for any of the systems studied. Between 2 new pentanucleotide STR loci, Penta E and Penta D, for which there is only limited data regarding the allelic distribution in Japanese, the Penta E locus was found to be highly polymorphic and exhibited a tri- or tetra-modal distribution pattern having allelic peaks with 5, 11, 15 and 20 repeats. The distribution was significantly different from that of the other ethnic groups. Statistical parameters of forensic importance, the power of discrimination (PD), observed and expected heterozygosity values (H), polymorphism information content (PIC), power of discrimination (PD), matching probability (pM), power of exclusion (PE), and typical paternity index (PI), were calculated for the loci. These parameters indicated the usefulness of the loci in forensic personal identification and paternity testing among Japanese. The systems Penta E, FGA, D18S51 and D8S1179 were the most informative. This method was successfully applied to forensic personal identification and paternity testing among Japanese, thereby confirming its efficacy for forensic practice.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 24 1 1970 Autosomal polymorphism in Donryu strain rats 81 91 EN Hiroshi Masuji Article 10.18926/AMO/32794 <p>1) Normal karyotype of Donryu strain rat was determined according to the classification of KURITA et al. (8). Namely, the number of chromosomes was 42 in diploid cells, and chromosomes were divided into 3 groups (A, B and C) according to the position of centromere. A.group was consisted of 7 pairs of metacentric chromosomes, B-group 4 pairs of sub· meta.subtelocentrics and C.group 10 pairs of telocentrics and Y. 2) Among all chromosome pairs a pair of the longest telocentric chromosomes (C.l), 4 pairs of all the B.group, and the Y chromosome were recognizable. 3) The presence of polymorphism was demonstrated in the smallest submetacentric chromosomes (BA),: namely, (I) a homologous submeta. centric pair, (II) a homologous subtelocentric pair and (III) a heteromor. phic submeta and subtelocentric pair which seemed to be a hybrid from (I) and (II). To distinguish the polymorphism in their genotype from phenotype was impossible. 4) Animals with type III B·4 chromosomes were produced by type I and type II animals. 5) By checking the chromosomes of the inbred Donryu strain rats maintained over 40 generations by brother.sister mating at Nihon Rat Co., polymorphism in BA chromosomes was also recognized.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 58 3 2004 Analysis of HCV genotypes from blood donors shows three new HCV type 6 subgroups exist in Myanmar. 135 142 EN Toshiyuki Shinji Yi Yi Kyaw Katsunori Gokan Yasuhito Tanaka Koji Ochi Nobuchika Kusano Takaaki Mizushima Shin-ichi Fujioka Hidenori Shiraha Aye Aye Lwin Yasushi Shiratori Masashi Mizokami Myo Khin Masayuki Miyahara Shigeru Okada Norio Koide Article 10.18926/AMO/32110 The prevalence of hepatitis C virus (HCV) genotypes in Myanmar in comparison with the rest of Southeast Asia is not well known. Serum samples were obtained from 201 HCV antibody-positive volunteer blood donors in and around the Myanmar city of Yangon. Of these, the antibody titers of 101 samples were checked by serial dilution using HCV antibody PA test II and Terasaki microplate as a low-cost method. To compare antibody titers by this method and RNA identification, we also checked HCV-RNA using the Amplicor 2.0 test. Most high-titer groups were positive for HCV-RNA. Of the 201 samples, 110 were successfully polymerase chain reaction (PCR) amplified. Among them, 35 (31.8%) were of genotype 1, 52 (47.3%) were of genotype 3, and 23 (20.9%) were of type 6 variants, and phylogenetic analysis of these type 6 variants revealed that 3 new type 6 subgroups exist in Myanmar. We named the subgroups M6-1, M6-2, and M6-3. M6-1 and M6-2 were relatively close to types 8 and 9, respectively. M6-3, though only found in one sample, was a brand-new subgroup. These subtypes were not seen in Vietnam, where type 6 group variants are widely spread. These findings may be useful for analyzing how and when these subgroups were formed. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 55 3 2001 Molecular virology of hepatitis C virus. 133 159 EN Nobuyuki Kato Review 10.18926/AMO/32025 <p>Hepatitis C virus (HCV), discovered in 1989, is the major causative agent of parenteral non-A, non-B hepatitis worldwide. Following the development of a method of diagnosing HCV infection, it became apparent that HCV frequently causes chronic hepatitis. Persistent infection with HCV is implicated in liver cirrhosis and hepatocellular carcinoma. Current worldwide estimations suggest that more than 170 million people have been infected with HCV, an enveloped positive single-stranded RNA (9.6-kilobases) virus belonging to the Flaviviridae. The HCV genome shows remarkable sequence variation, especially in the hypervariable region 1 of the E2 protein-encoding region, and globally, HCV appears to be distributed with more than 30 genotypes. Complicated &#34;quasispecies&#34; and frequent mutations of viral genomes have also emerged. The HCV genome encodes a large polyprotein precursor of about 3,000 amino acid residues, and this precursor protein is cleaved by the host and viral proteinases to generate at least 10 proteins in the following order: NH2-core-envelope (E1)-E2-p7-nonstructural protein 2 (NS2)-NS3-NS4A-NS4B-NS5A-NS5B-COOH. These viral proteins not only function in viral replication but also affect a variety of cellular functions. Although several explanations have been proposed, the mechanisms of HCV infection and replication in targeted cells, the mechanism of persistent viral infection, and the pathogenesis of hepatic diseases (hepatitis or hepatocellular carcinoma) are all poorly understood. A major reason why these mechanisms remain unclear is the lack of a good experimental HCV replication system. Although several classical trials using cultured cells have been reported, several new, more promising experimental strategies (generations of infectious cDNA clone, replicon, animal models, etc.) are currently being designed and tested, in order to resolve these problems. In addition, new therapies for chronic hepatitis have also been developed. The enormous body of information collected thus far in the field of HCV research is summarized below, and an overview of the current status of HCV molecular virology of HCV is provided.&#60;/P&#62;</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 59 5 2005 A new HLA-DRB1 genotyping method using single nucleotide polymorphism (SNP) analysis with multiplex primer extension reactions and its application to mixed samples. 179 194 EN Kiyomi Imabayashi Yuji Yamamoto Sachiyo Inagaki Yusuke Doi Kei Yoshitome Satoru Miyaishi Hideo Ishizu Article 10.18926/AMO/31971 <p>We have improved on conventional methods for HLA-DRB1 genotyping and devised a new method that is simple, cost-effective, and adequately applicable to routine forensic practice. This method consists of group-specific polymerase chain reaction (PCR) of the exon 2 region of the HLA-DRB1 gene and simultaneous detection of single nucleotide polymorphisms (SNPs) at multiple sites using multiplex primer extension reactions. With this method, we successfully detected HLA-DRB1 genotypes from the following materials: the peripheral blood of 142 donors, 6 aged saliva stains of known DRB1 genotype stored for 5-10 years at room temperature, 10 aged bloodstains of unknown DRB1 genotype stored for 29 years at room temperature, and minimal bloodstains and saliva stains from 3 donors of known DRB1 genotypes. Furthermore, we were able to type DRB1 alleles of the minor component in mixed samples at a proportion of 1/1,000 or 1/10,000. In a criminal case, DRB1 alleles detected from mixed bloodstains on a sword found at the scene enabled us to explain the case. This method is expected to be useful for forensic medicine.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 63 6 2009 Longevity-associated NADH Dehydrogenase Subunit-2 237 Leu/Met Polymorphism Modulates the Effects of Daily Alcohol Drinking on Yearly Changes in Serum Total and LDL Cholesterol in Japanese Men 331 338 EN Ryuji Makita Akatsuki Kokaze Tadahiro Ohtsu Mamoru Ishikawa Naomi Matsunaga Kanae Karita Masao Yoshida Nobukazu Tanaka Minoru Yamamoto Junichi Hayashi Yutaka Takashima Kiyoshi Kitamoto Original Article 10.18926/AMO/31825 <p>Reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 2 237 leucine/methionine (ND2-237 Leu/Met) polymorphism, is reportedly associated with longevity in the Japanese population. The ND2-237Met genotype may exert resistance to atherogenic diseases, such as myocardial infarction or cerebrovascular disorders. To investigate whether ND2-237 Leu/Met polymorphism is associated with yearly changes in serum lipid levels, we conducted a longitudinal study of 107 healthy Japanese male subjects. Analysis of covariance revealed that the interaction between the ND2-237 Leu/Met genotypes and habitual drinking was significantly associated with yearly changes in serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDLC) levels (p0.036 and p0.006, respectively). In multiple regression analysis, daily drinking was significantly and positively associated with yearly changes in serum LDLC levels in men with ND2-237Met (p0.026). After adjusting for covariates, yearly changes in serum LDLC levels were significantly lower in non-daily drinkers with ND2-237Met than in those with ND2-237Leu (p0.047). These results suggest that ND2-237Met has a beneficial impact on yearly changes in serum LDLC in non-daily drinkers but not in daily drinkers.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 56 1 2002 Catechol-O-methyltransferase and Parkinson's disease. 1 6 EN Chun-Hwi Tai Ruey-meei Wu Article 10.18926/AMO/31725 <p>Parkinson's disease (PD) is one of the main causes of neurological disability in the elderly. Levodopa is the gold standard for treating this disease, but chronic levodopa therapy is complicated by motor fluctuation and dyskinesia. The catechol-O-methyltransferase (COMT) inhibitors represent a new class of antiparkinsonian drugs. When coadministered with levodopa/decarboxylase inhibitor, 2 COMT inhibitors, tolcapone and entacapone have been shown to improve the clinical benefit of levodopa. COMT activity is genetically polymorphic, and individuals with the low activity (COMT(L/L)) genotype have a thermolabile COMT protein; studies suggest that this genotype is less common in Asians than in Caucasians. Differences in COMT activity may determine the individual response to levodopa and result in ethnic differences in PD susceptibility. Our recent study suggests that the COMTL allele can interact with the MAOB gene to increase the occurrence of PD in Taiwanese. In order to understand this new class of antiparkinsonian drugs, we review their basic properties, pharmacology, and clinical efficacy. The frequency distribution of COMT genetic polymorphisms among different populations and its implications in the etiology and drug response is also discussed.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 52 2 1998 Relationship of serum markers of hepatitis B and C virus replication in coinfected patients. 113 118 EN Hideyuki Tsuji Hiroyuki Shimomura Kozo Fujio Masaki Wato Junichi Kondo Toshimi Hasui Yasushi Ishi Shin-ichi Fujioka Takao Tsuji Article 10.18926/AMO/31311 <p>To evaluate viral interference between hepatitis B and C, we studied coinfected patients serologically and molecular biologically. Twenty-seven patients positive for hepatitis B surface antigen (HBsAg) and anti-hepatitis C virus (HCV) antibody, were classified into Groups BC-L and BC-H according to DNA-polymerase activity (less or greater than 100 cpm, respectively). Patients with hepatitis B or C alone were also enrolled as controls. HCV-RNA was detected more often in Group BC-L than in Group BC-H. Genotype 1b of HCV was determined in 75% of Group BC-H, 87.5% of Group BC-L, and 70.7% of hepatitis C-only patients. Activity of DNA-polymerase in coinfected patients was lower in patients positive for HCV-RNA as compared with those negative. HBsAg titers tended to be lower in coinfected patients than in patients with hepatitis B virus (HBV) alone. In conclusion, in coinfection, HBV may suppress the replication of HCV and HCV appears to reduce the expression of HBsAg and probably suppresses HBV replication.&#60;/P&#62;</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 52 6 1998 Improvement of sensitivity in HLA-DRB1 typing by semi-nested PCR-RFLP. 289 296 EN Seiichi Inoue Yuji Yamamoto Osamu Okamoto Hiroki Murakami Satoru Miyaishi Hideo Isizu Article 10.18926/AMO/31305 <p>A sensitive method of HLA-DRB1 typing was devised using a semi-nested polymerase chain reaction (PCR) followed by a restriction fragment length polymorphism (RFLP) analysis (semi-nested PCR-RFLP method). The first-round amplification (30 cycles) of the semi-nested PCR was performed using DRB generic primer pairs and the second round of PCRs (20 cycles) were performed using DRB1 group-specific primers. The products of the second round PCRs were digested with restriction endonucleases for the typing of HLA-DRB1 alleles. By this method, HLA-DRB1 typing was possible from 10 pg of genomic DNA extracted from lymphocytes and from 0.5 microliter of 1,000 times diluted blood without DNA extraction. HLA-DRB1 alleles could be typed from a 2-mm long bloodstained cotton thread prepared from 10 times diluted blood and from a 2-mm thread of whole blood bloodstains stored at room temperature for 2 years. From the mixture of blood of two individuals with different genotypes, DRB1 alleles of the minor component were detected down to 1/1,000 of the major component. This semi-nested PCR-RFLP method is useful for HLA-DRB1 typing from extremely small amounts of DNA and from mixed samples.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 52 4 1998 Haptoglobin genotyping by allele-specific polymerase chain reaction amplification 173 181 EN Akemi Yano Yuji Yamamoto Satoru Miyaishi Hideo Ishizu Article 10.18926/AMO/31301 <p>We performed haptoglobin (Hp) genotyping by polymerase chain reaction using allele-specific primer-pairs. The major six genotypes of Hp were identified using this method. Among Japanese individuals living in Ehime and Okayama Prefectures, the allele frequencies were estimated to be Hp2 = 0.723 and Hp1s = 0.277. Genotyping of Hp was possible with 0.3 ng of DNA and with 0.125 microliter of blood. It was also possible with whole blood left at room temperature for a month and also with the bloodstains left at room temperature for three years. In the heated blood samples, both alleles, Hp2 and Hp1s, were detected in those heated at 100 degrees C for 2 h. In bloodstains, Hp2 and Hp1s were detected in samples heated at 100 degrees C for 2 h and 120 degrees C for 30 min. In addition, the genotype could be detected in samples other than blood such as saliva, hair roots, tissue sections and dental pulps. The present method for Hp genotyping is expected to become a useful method in forensic analysis.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 48 6 1994 Intrafamilial clustering of genotypes of hepatitis C virus RNA. 293 297 EN Michiko Takahashi Gotaro Yamada Toshihiko Doi Masahiro Takatani Fumitoshi Kishi Rieko Miyamoto Hiroshi Yoshizawa Hiroaki Okamoto Takao Tsuji Article 10.18926/AMO/31098 <p>Hepatitis C virus (HCV)-RNA in the blood was measured by polymerase chain reaction (PCR) in 37 subjects from eight families in which 2 or more persons tested seropositive for antibodies against C100-3 or CP9. HCV-RNA was positive in 17 of 37 subjects. Two or more HCV-RNA-positive subjects were observed in six of the families. Intrafamilial HCV infection was studied by determining the HCV-RNA type (I, II, III or IV) by PCR using type-specific primers. In two families, all of the subjects showed type III infection, and in three other families, all of the subjects showed type II infection, with different types of HCV infections being observed in only one family. The HCV type was uniform in all but one. These findings suggest a possibility of intrafamilial infection between husbands and wives and between members of the same household.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 60 6 2006 Three Type 6 Hepatitis C Virus Subgroups among Blood Donors in the Yangon Area of Myanmar Are Identified as Subtypes 6m and 6n, and a Novel Subtype by Sequence Analysis of the Core Region. 345 349 EN Toshiyuki Shinji Aye Aye Lwin Katsunori Gokan Mikako Obika Hiromasa Ryuko Myo Khin Shigeru Okada Norio Koide Short Communication 10.18926/AMO/30719 Previously, using phylogenetic analysis of NS5b sequences, we found that three type 6 variant subgroups (M6-1, M6-2 and M6-3) exist in Myanmar. According to the new nomenclature of hepatitis C, M6-1 and M6-2 belong to subtypes 6m and 6n, respectively, but M6-3 is unassigned. In this study, we sequenced and phylogenetically analyzed the core region of these type 6 variant subgroups. Serum samples assigned as 6m or 6n by NS5b sequence were also identifi ed as 6 m or 6n by core region analysis. The M6-3 (sample name MYAN-3E-3) remained unassigned to a subgroup based on its core region analysis. The fi ndings of this study suggest that either the core region or the NS5b region can be analyzed for HCV subtype classifi cation. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 50 1 1996 IgA2 genotyping by polymerase chain reaction (PCR) using allele-specific amplification primers. 1 9 EN Shingo Takata Yuji Yamamoto Hideo Ishizu Article 10.18926/AMO/30512 <p>A method of genotyping IgA2 alleles in the human immunoglobulin alpha 2 heavy chain constant region (C alpha 2 gene) was developed by using the polymerase chain reaction (PCR). By this method, the genotype was determined by discriminating base substitution in the 3'-flanking region of alleles, A2m*1 and A2m*2, which manifest A2m serum types, by nested PCR using allele-specific primers. Three types, IgA2*1/IgA2*1, IgA2*2/IgA2*1, and IgA2*2/IgA2*2, were detected from DNA extracted from lymphocytes. Genotyping was possible from 100 pg of DNA by this method. The estimated allele frequency in 318 Japanese subjects was 0.561 for IgA2*1 and 0.439 for IgA2*2. Analysis of 29 cases of paternity tests suggested that the data follow Mendel's law of inheritance. This genotype could also be detected in whole blood, blood stains, saliva stains, and various organs and tissues. These results suggest the usefulness of the present method for paternity testing and individual identification in forensic medicine.</p> No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 49 3 1995 Virological and serological characterization of asymptomatic blood donors positive for anti-hepatitis C virus antibody. 137 144 EN Hideyuki Tsuji Hiroyuki Shimomura Masaki Wato Junichi Kondo Takao Tsuji Article 10.18926/AMO/30409 <p>To study the virological and serological characteristics of asymptomatic hepatitis C virus (HCV) carriers, 165 blood donors positive for antibody against HCV proteins by the second generation assay, were analyzed for their clinical backgrounds, serological reactivity against antigens derived from HCV by recombinant immunoblot assay, and the amount and genotype of HCV by the polymerase chain reaction. Compared with blood donors having abnormal levels of alanine aminotransferase (ALT), sera from the donors with normal levels of ALT reacted less frequently against NS4 antigens (anti-5-1-1: 34.4% vs. 54.5%, P = 0.0609; anti-c100-3: 34.4% vs. 56.1%, P &#60; 0.05). Also the positivity for antibodies against these antigens were more frequent in sera from donors with genotype 1b HCV-RNA than other genotypes (anti-5-1-1: 61.0% vs. 23.5%, P &#60; 0.01; anti-c 100-3: 61.0% vs. 26.5%, P &#60; 0.01). The prevalence of each genotype in blood donors with normal ALT levels was different from that in patients with advanced liver disease (P &#60; 0.05), genotype 1b being less and genotype 2a being more frequent. The number of HCV-RNA copies/0.5 ml in donors with normal ALT was 10(7.9 +/- 1.0) (n = 27) and that in patients with chronic liver disease was 10(7.4 +/- 0.8) (n = 116), the difference being statistically significant (P &#60; 0.05). In conclusion, the results of this study suggest that asymptomatic blood donors carrying HCV have the serological and virological characteristics different from the patients with advanced liver disease.</p> No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 89 5-6 1977 岡山地方におけるパプトグロビン型の分布について 721 724 EN Hideo Ishizu The distribution of haptoglobin (Hp) types of 600 healthy adults in Okayama district was examined by the method of Smithies', and the following results were obtained. 1) The phenotype distribution of Hp types in Okayama district was found to be: Hp 1-1, 39 (6.5%); Hp 2-1, 234 (39.0%); and Hp 2-2, 327 (54.5%), and the calculated genotype frequencies are Hp(1)=0.260 and Hp(2)=0.760. 2) No relationship between the distribution of Hp types and sex, ABO blood groups or Rh(D) blood groups was observed. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 103 9-10 1991 Ciclosporin 投与におけるリンパ球中濃度及びNK活性 1065 1073 EN Fumiyuki Inoue Ciclosporin is an effective immunosuppressant for kidney transplantation, although it has the side effect of nephrotoxicity. Ordinarily, the optimal dosage of Ciclosporin is determined by the serum trough level. However, it might be better to make the determination from the concentration in immunocompetent cells. In this report, the serum level of Ciclosporin was compared with the lymphocyte concentration along with the inhibiting activity to NK cells. In in vivo administration, Ciclosporin levels in serum and lymphocytes increased in proportion to the dosage. The suppression of NK activity correlated with the dosage. The suppression of NK activity ceased within 4 days after cessation of administration. In in vitro administration, the Ciclosporin level in lymphocytes increased in proportion to the dosage. The suppression of NK activity correlated with its level in the lymphocytes. Ciclosporin successfully suppressed NK activity against both K562 cells and skin cells. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 105 7-8 1993 PCR-RFLP 法による HLA-DNA-DQ タイピングと腎移植 715 731 EN Keiichi Hirakawa Two methods of HLA-DNA typing (PCR-RFLP method and PCR-SSO method) were performed on HLA-DQ in living related, living unrelated and cadaveric renal transplants. These two DNA typing methods allowed more accurate and more detailed typing than the conventional typing method. The effect of DNA histocompatibility of DQA 1 and DQB 1, both typed by the PCR-RFLP method, on clinical outcome and surviving graft rate of renal transplant patients was analyzed. There was no correlation found between the number of mismatches between donor and recipient of DQA 1 typing in cadaveric renal transplants, of DQB 1 typing in cadaveric, living unrelated and living related transplants and the clinical outcome nor the surviving graft rate of renal transplant patients. Both the clinical outcome and the surviving graft rate in the group in which DQ 5 mismatch between donor and recipient was positive were statistically poorer than that in the group in which DQ 5 mismatch was negative. This result suggests the existance of DQ 5 mismatch between donor and recipient greatly influences graft survival after renal transplantation. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 108 3-6 1996 循環，呼吸，免疫動態よりみた胸部食道癌に対する1期的手術と分割手術との比較検討 83 95 EN Tomoyoshi Muramatsu There are two major surgical procedures for excision of esophageal carcinoma and reconstruction of the esophagus: the one-stage procedure and the two-stage procedure. In the present study, we evaluated the two methods by comparing the cardiovascular, respiratory and immune parameters of 10 patients who underwent one-stage procedure with those of 10 other patients who underwent two-stage procedure. To estimate cardiovascular function, we measured the left ventricular stroke work index (LVSWI)-pulmonary capillary wedge pressure (PCWP). Most of the patients treated by the one-stage procedure showed a significant decrease in LVSWI-PCWP, whereas the index of patients treated by the two-stage procedure did not change much. When we assessed the respiratory system by forced vital capacity (FVC) and peak expiratory flow (PEF), the patients treated by the two-stage procedure recovered much faster and better than those receiving the one-stage procedure. Natural killer (NK) activity in lymphocytes was also measured as a marker of the immuno-reactive system. Although most patients show a drop in NK activity one week after major surgery, NK activity did not demonstrate a significant change in any of the patients who underwent the two-stage procedume. Thus, our systemic comparison of the three different parameters demonstrated better results after the two-stage procedure and we recommend it over the one-stage procedure, especially for aged and high risk patients. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 114 3 2003 インターフェロン治療後C型慢性肝炎患者の長期予後　―ウイルス学的著効例，生化学的著効例と無効例の比較― 275 281 EN Masanobu Miyake To evaluate the prognosis of the sustained biochemical responder after interferon (IFN) therapy, we retrospectively studied 252 chronic hepatitis C patients who were treated with IFN. Patients were divided into four groups: group A, sustained virological responders (n=84); group B,sustained biochemical but not virological responders (n=43); group C, incomplete responders (n=64); group D, non responders (n=61). The levels of several liver function tests were evaluated at the end of the observation period (4.2±1.6 years, mean±SD) compared with those at just before IFN therapy. The levels of cholinesterase, albumin, γ-globulin, zinc sufate turbidity test, platelet count and clearance rate of indocyanine green test improved in group A (p<0.05), became worse in group D (p<0.05) and did not change in group B. The incidence of hepatocellular carcinoma was significantly higher in group D than in group B (p<0.01);Kaplan-Meier method, log-rank test). The hazard ratio for hapatocarcinogenesis of the patients in group A and B was significantly lower than that in group C and D (hazard ratio: 0.27, range of 0.08-0.98; p=0.046) adjusted for age, gender, stage and total alcohol consumption. These results suggest that the progress of liver disease and liver carcinogenesis was more suppressed in sustained biochemical responders than in non reponders. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 00301558 119 2 2007 B型肝炎ウイルスGenotype/subgenotype 205 207 EN Toshihiro Higashi No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 00301558 120 1 2008 Ⅵ　肝がんの外科的治療と再発治療の変遷 63 67 EN Takahito Yagi No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 00301558 120 1 2008 法医生物学におけるイムノアッセイの応用 43 48 EN Satoru Miyaishi No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2007 Hepatitis C Virus Genotype Distribution in Myanmar: Predominance of Genotype 6 and Existence of New Genotype 6 Subtype EN Lwin Aye Aye Aim: This study was performed to determine the prevalence and distribution of hepatitis C virus (HCV) genotypes in Myanmar. Methods: A total of 1333 peripheral blood samples were collected from four different border cities of Myanmar. The anti-HCV antibody-positive serum samples were identified. HCV was genotyped by reverse transcriptase polymerase chain reaction, direct DNA sequencing and phylogenetic analysis on the partial core genome. Results: The overall prevalence of HCV infection was 11.6% (154/1333). Regionally, it was 13.5% (47/349) in the north-eastern city, 12.8% (64/501) in the north-western city, 4.2% (16/380) in the southern city and 26.2% (27/103) in the western city. HCV was genotyped in 145/154 (94.2%) samples. Genotype 6 was the most prevalent genotype in this study (71/145, 49%), followed by genotype 3 (57/145, 39.3%), genotype 1 (16/145, 11%), and genotype 2 (1/145, 0.7%). Genotype 6 was mostly found in the northern cities and genotype 3 in the southern and western cities of Myanmar. Multiple HCV genotypes/subtypes were successfully characterized as 1a, 1b, 2a, 3a, 3b, 6m, 6n, and a new 6 subtype. Among them, subtype 6n was the most predominant subtype (38.6%), followed by subtype 3b (29.7%), 3a (9.6%), 6m (9%), 1b (6.9%), 1a (4.1%), new 6 subtype (1.4%) and 2a (0.7%). Subtype 6n was more widely distributed in the northern cities whereas subtype 3b was more common in the western city. The newly discovered genotype 6 subtype was from the northern cities. Conclusions: The results indicate there are regional differences of HCV genotype distribution in Myanmar. There is a distinct geographic variation from other South-East Asian countries in terms of the existence of the new genotype 6 subtype. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2007 Loss or down-regulation of HLA class I expression at the allelic level in freshly isolated leukemic blasts EN Kozo Masuda Loss or down-regulation of human leukocyte antigen (HLA) class I expression has been demonstrated in a variety of solid tumors. To date, such altered HLA expression has not been studied extensively in freshly isolated leukemic blasts. If it occurs, leukemic cells could escape T-cell surveillance as a consequence. Genotypes of nine leukemic cell lines were determined using a polymerase chain reaction for HLA classes I and II. Cells were also examined for HLA beta2-microglobulin, and allele-specific HLA protein expression using flow cytometry. Next, 44 samples of freshly isolated leukemic blasts from 43 patients with malignant hematological diseases were examined for allele-specific HLA expression using flow cytometry. Microsatellite analysis was performed to determine heterozygosity in the HLA region on chromosome 6. Genotype analysis for HLA class I together with microsatellite analysis demonstrated loss of HLA haplotype in HL-60 cells. No loss of HLA haplotype was observed in 44 samples of freshly isolated leukemic blasts. As reported previously, flow cytometric analysis rarely demonstrated loss or down-regulation of HLA expression at initial diagnosis (3/39; 7.7%); however, this was evident in two of five cases in relapse (40.0%), which contrasts with previous reports. In one patient with acute leukemia, HLA-A2 cell surface expression was present at initial diagnosis, lost at relapse, and completely restored after 48 h of culture in the presence of interferon-gamma. These results suggest loss of allele-specific HLA expression may be involved in the pathogenesis of relapse in patients with leukemia. The findings should be valuable in designing new strategies for clinical immunotherapy. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2007 Nucleotide change of codon 38 in the X gene of hepatitis B virus genotype C is associated with an increased risk of hepatocellular carcinoma EN Ryosuke Muroyama BACKGROUND/AIMS: The hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, the HBV X gene, which encodes the pleiotropic transactivator HBx, has also been associated with the development of HCC. In this study, we investigated whether nucleotide changes in the X gene of genotype C are associated with the development of HCC. METHODS/RESULTS: We sequenced the X gene in age- and sex-matched 39 HBV-infected patients with HCC and 36 HBV-infected patients without HCC. A novel nucleotide change that resulted in a proline to serine substitution at codon 38 in HBx (codon-38 change) was preferentially found in patients with HCC. Then, sera were collected from a new group of age- and sex-matched 52 patients with HCC and 51 patients without HCC. In this cohort also, the codon-38 change was associated with HCC. Multiple logistic regression analysis showed the prevalence of the codon-38 change was significantly associated with HCC in all patients (P=0.001, odds ratio: 4.89). CONCLUSION: The codon-38 change in genotype C is an independent risk factor for the development of HCC and may serve as a useful molecular marker for predicting the clinical outcomes in patients infected with HBV. No potential conflict of interest relevant to this article was reported.

岡山大学資源生物科学研究所 Acta Medica Okayama 0916-930X 3 1 1995 オオムギの近辺野生種と栽培種における未熟胚由来カルスからの再分化能の比較 55 62 EN Kazuhide Rikiishi Kazuyoshi Takeda Shozo Yasuda The variation in shoot regeneration obility of calli derived from immature embryos was examined in 95 wild strains, 82 of which were of Hordeum spontaneum and 13 of which were H. agriocrithon, and 87 cultivated varieties collected from various countries or regions of the world. In 85 strains of the wild species, a number of calli regenerated shoots, and their proportion ranged from 1.2% to 75.7%. The average percentage of shoot regenerating calli was 21.7% among the strains that formed calli, 11.5% of which regenerated green and 10.2% albino shoots. On average, 21.4% and 23.9% calli regenerated shoots in H. spontaneum and H. agriocrithon, respectively and there was no significant difference between these values. A significant difference in the percentage of shoot regenerating calli was found among six variants (dawense, ishnatherum, laguncliforme, paradoxon, proskowetzii, spontaneum) which were comprised in H. spontaneum. In 73 varieties of cultivated species, there were shoots regenerating calli likely to wild species, and their proportion ranged from 3.2% to 85.5%. The average percentage of shoot regenerating calli was 25.4%, 22.0% of which regenerated green and 3.4% of which regenerated albino shoots. There was a significant difference in percentage of green shoots regenerating calli against shoots regenerating ones between the wild (53.0%) and cultivated species 886.6%). The two kinds of non-brittle rachis genotypes, Bt bt2 and bt Bt2 are one of the key characters distinguishing the oriental and occidental types of cultivated barley. The average percentages of shoot regenerating calli were 16.2% and 32.3% for the genotypes Bt bt2 and bt Bt2, respectively, suggesting that there is a geographical variation in the shoot regeneration ability of calli in the cultivated species. By contrast, the oriental and occidental strains of wild species showed no difference in the shoot regeneration ability of calli. The geographical variation of shoot regeneration ability differed significantly between wild and cultivated species. This suggests that the geographical variation of shoot regeneration ability occurred after the cultivation of the barley was established. No potential conflict of interest relevant to this article was reported.

岡山大学資源生物科学研究所 Acta Medica Okayama 0916-930X 3 1 1995 Selection Effectiveness for the Resistance to Net Blotch in Barley 43 53 EN Kazuhiro Sato Kazuyoshi Takeda Selection effectiveness for the resistance to net blotch was estimated by using two sets of F2 and F3 populations derived from the crosses between resistant and susceptible parents. In every F2 and F3 population, disease ratings showed a continuous distribution. As many F3 lines with intermediate resistance had a smaller variance and homozygous genotype, the resistance might be controlled by a few genes. The heritabilities of the disease rating were estimated　by correlation coefficients and regression coefficients between each F2 plant and the descended F3 lines. Another estimate for heritability was calculated by the selection differential in the F2 plants and genetic gain in the F3 lines. Despite the different level of resistance in the resistant parents of the two crosses, the three kinds of heritabilities estimated were similar and ranged from 0.6 to 0.8. Because of the fewer number of genes controlling the disease resistance and the higher heritabilities, selection in a early generation may be effective for net blotch resistance in barlcy. No potential conflict of interest relevant to this article was reported.

岡山大学資源生物科学研究所 Acta Medica Okayama 0916-930X 2 1 1994 オオムギ品種における完熟胚および未熟胚由来カルスの再分化能の比較 33 42 EN Kazuhide Rikiishi Shozo Yasuda The callus forming ability and regenerating ability of the calli derived from mature and immature embryos of 132 barley varieties were examined. These materials were taken from a world-wide collection preserved at the Barley Germplasm Center of Okayama University. The callus forming ability varied widely according to genotype in both mature and immature embryos, but the varieties collected from Ethiopia showed low callus forming ability. Calli derived from mature embryos generally did not regenerate shoots, except for three Japanese varieties. The frequency of shoot regeneration from the calli derived from immature embryos was somewhat higher than that from those derived from mature embryos. Many of the Korean and Japanese varieties had a high shoot regenerating ability. However, few of the varieties from Ethiopia and Southwest Asia had a high shoot regenerating ability. No correlation was observed btween root regenerating ability and shoot regenerating ability of the varieties. No correlation was observed between callus proliferation and root regenerating ability between calli derived from mature and immature embryos. We could not find any difference in the shoot regenerating ability btween the two-rowed and six-rowed genotypes. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 0474-0254 85 1 1996 オクラホマ州の硬質冬小麦の育種における遺伝子型と環境の交互作用と遺伝子型の環境反応性の指標化について 99 108 EN Makoto Tahara The Wheat breeding program at Oklahoma State University(OSU) is introduced with reference to genotype by environment interactions and linear regression analyses. Oklahoma is the second largest producer of hard red winter wheat in the US. The breeding porgram is conducted by the wheat breeding personnel of the Agronomy Department in collaboration with plant pathologists, entomologists and biochemists of OSU and wheat geneticists of the US Department of Agriculture. The main-stream breeding procedures are F2 or F3 progeny methods, which are modifications of pedigree and bulk breeding methods. The procedures for source population development,selection practice and field trials are discussed. The major objective of the projict is to develop wheat varieties with supperior yield and yield stability. Drought stress is a serious constraint to wheat crop and frequently causes substantial yield reduction in Okrahoma. Other major obstacles to wheat production are disease and insect damage which include leaf rust, mosaic diseses, septoria and green bug. Research and breeding activities to overcome thesse obstacles are briefly reviewed.Genotype by environment interactions are commonly found and cause serious problems in identifying superior genotype over a wide range of environments in the wheat breeding program. Linear regression analyses and other yield stability parameters are proposed to characterize genotype responses to varying environments. The grain yield data from cultivar trials during 1971-1982 were analyzed by an analysis of variance method and linear regression method. The analysis of varince indicated substantial genotype by environment interactions. The linear regression analyses could adequately explain much of the interaction and provided parameters to compare yield responses of genotypes over environments. Other stability parameters were also estimated and their relationships were discussed. The linear regression analyses revealed that selection toward higher average yield over environments favored genotypes adapted for high yeilding environments. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 0474-0254 93 1 2004 Epidemiological Aspects of the Japanese Tobamovirus Strain, Pepper Mild Motte Virus(PMMoV) Infecting the L2 resistance Genotype of Green Pepper(Capsicum annuum L.) 19 27 EN Kazuhiro Toyoda Yasufumi Hikichi Shigeharu Takeuchi Tomohisa Kuroda Ako Okumura Yoshiko Nasu Tetsuro Okuno Kazumi Suzuki To understand the epidemiological aspects of tobamovirus infecting the L resistance genotypes of green pepper, fifteen isolates were collected from geographically different fields and were chracterized by their biological properties. All isolates infected L1 and L2 plants systemically, but were localized in L3 and L4 plants. The symptomatology on several test plants and the reactivity to an antiserum showed that they were identical to that of a Japanese strain of pepper mild mottle virus (PMMoV-J). The viral infection was also confirmed by a reverse transcription and polymerase chain reaction (RT-PCR) with oligonucleotide primers that amplity the coat protein gene of PMMoV-RNA. On the other hand, the RT-PCR allowed us to detect PMMoV in seeds of some commercial cultivars of green pepper. Viruses isolated from the seeds could infect L2 plants systemically. Further analysis of the nucleotide sequence of the predicted coat protein gene revealed that the isolates from the commercial seeds were identical to that of PMMoV-J. These results indicated that the L2 resistance-breaking tobamovirus has prevailed in fields of green pepper in Japan. and that infected seeds may be one of the initial sources of the viral infection. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 0474-0254　 96 1 2007 Effect of Nutrient Levels and Mineral Composition on the Occurrence of Yellow-leaf-spot in Chrysanthemum 43 48 EN Akinori Oki Tanjuro Goto Kaori Nagasuga Atsushi Yamasaki 　Yellow-leaf-spot, a physiological abnormality occurring in leaves of several chrysanthemum (Chrysanthemum ×morifolium) cultivars harvested from September to October, is a very serious problem in Japan, of which causes have not been well established. Water stress, high temperature, high irradiation or nutrient stresses are possible physiological factors which may lead to yellow-leaf-spot. In the present study, effects of nutrient levels and mineral composition on the occurrence of yellow-leaf-spot were investigated. ‘Seikou-no-makoto’ and ‘Seikou-no-masaru’ plants were grown in 5 nutrient solutions (N 0, 60, 120, 180, 240 ppm based on Enshi-shoho). In ‘Seikou-no-masaru’ no yellow-leaf-spot occurred. However, in ‘Seikou-no-makoto’, the nodal position with spotted leaves and rate of yellow-leaf-spot increased as nutrient levels increased. ‘Seikou-no-makoto’ plants were supplied with 6 different nutrient solutions containing 3 times N, P, K, Ca, Mg or Fe in 1/3 concentration of Enshi-shoho solution for 3 or 14 days. The nodal position with spotted leaves and rate of yellow-leaf-spot was not affected by mineral composition. The nodal position with spotted leaves and rate of yellow-leaf-spot increased with increasing days of application. Both cultivare were supplied with 7 different nutrient solutions with lacked N, P, K, Ca, Mg, Fe or only microelement (no mineral) in 1/2 Enshi-shoho solution for 10 days. In ‘Seikou-no-masaru’, no yellow-leaf-spot occurred. It occurred only in ‘Seikou-nomakoto’. yellow-leaf-spot occurred in control, P, K, Ca, Mg, Fe deficiency and no mineral, but only slightly in all cases. These results suggest that the occurrence of yellow-leaf-spot was dependent on genotype, and that excessive or deficiency specific elemental mineral stress had no significant effect. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2004 A new method for ABO genotyping using a multiplex single-base primer extension reaction and its application to forensic casework samples EN Yusuke Doi No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2005 A New HLA-DRB1 Genotyping Method Using Single Nucleotide Polymorphism (SNP) Analysis with Multiplex Primer Extension Reactions and Its Application to Mixed Samples EN Kiyomi Imabayashi No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1991 マウス胚性幹細胞の培養とその応用に関する発生工学的研究 EN Tomoyuki Tokunaga Embryonic stem (ES) cells isolated from mouse preimplantation embryos can be maintained as a pluripotent cell line in culture. Since ES cells are capable of contributing to a wide variety tissues including the germ line, when they are injected into host embryos, they are considered to be the useful tool for the production of transgenic mice. However, these abilities of ES cells tend to lose during cul ture in long term, or in the process in transfection with foreign gene and selection process. The purposes of the present study is 1) to isolate new ES cell lines, 2) to examine the culture conditions for maintenance of them, 3) to develop efficient method for production of viable germ line chimaeras and 4) to demonstrate the production of transgenic mice. I. Studies on the isolation of embryonic stem cell lines from mouse blastocysts and inner cell masses in the mouse The mouse blastocysts or inner cell masses (ICMs)isolated were cultured on feeder cell layer of the primary embryonal fibroblast cells inactivated with mitomycin C treatment. Totally, 11 ES cell lines were isolated from 2l0 blastocysts or ICMs obtained from sevral different mouse strains. There was no difference in the efficiency of the ES cell line isolation among the mouse strains and between blastocysts and ICMs. Colonies of these cells showed formed EC cell like morphology forming compact islands of cells wi th unclear cell borders. Three of ES cell lines, TT-12, TT-B4 and F1/1, had diploid chromosomes with the proportions of 78%, 78% and 72% respectively. In order to determine the differentiation potential of the ES cells in vitro TT-12 and F1/1 cells were cultured in suspentlon without feeder cell layer. They differentiated spontaneously into the cystic embryoid bodies which contained variety of tissues including endoderm and ectoderm like cells, indicating pluripotency of the cells in vitro. II. Studies on the culture conditions to maintain embryonic stem cells. The proligeration activity and undifferentiation conditon of F1/1 cells, which were isolated from a blastocyst obtained from C57BL/6 female mated with CBA male, were examined when they were cultured with different feeder cell layers or with Leukemia inhibitory factor (LIF). The proportions od colony formation and colonies with positive for alkaline phosphatase were higher when F1/1 cells were cultured for 3 to 7 days with primary embryonal fibroblast cells than with the STO cells. These feeder cell layers are indispensable to inhibit the differentiation of F1/1 cells during culture. When F1/1 cells were cultured through 15 passages with LIF without feeder cell layer, they were maintained undifferentiated and 68% of them had normal karyotype (38+XY). However, no young were obtained when chimaeric embryos produced between 8 cell embryos and F1/1 cells maintained with LIF were trasferred to the recipients, while 50 to 88% of young obtained from chimaeric embyos with F1/1 cells maintained in culture with feeder cells were chimaeras. Although many ES cell lines have been isolated from several mouse strains, the cell lines to produce germ line chimaeras have been limited to the l29 strain origin. The present study demonstrated that F1/1 cells isolated from a blastocyst having the C57BL/6xCBA genotype could produce germ line chimaeras by injecting the cells into blastocysts and 8-cell embryos. The chimaera production rate using CD-1 blastocysts as the host was wery low (20%) as reported by other investigaters. However, when F1/1 cells were introduced into the perivitteline space of 8-cell embryos obtained from CD-1 strain mice, very high proportion (80%) of chimaeric mice be produced and the chimaeric mice showed extremely high chimaerism. In the breeding tests, germ line chimaeras were indicated in 89% of fertile male and most of them had spermatozoa derived only from F1/1 cells. No live youg were obtained when chimaeric eggs, which were produced by injecting F1/1 cells into parthenogenetic 8-cell eggs, were transferred. IV. Studies on the production of transgenic mice using ES cells F1/1 cells transformed with the neomycin phosphotransferase (neo) gene using electroporation methods were selected for G4l8 resistance and tested for its ability to produce chimaeric mice. Two types of wave forms, square wave and exponential decay, were used for electroporation. There was no difference between square wave and exponential decay in the efficiency of transformation of F1/1 cells. Southern blot hybridization analysis demonstrated that four of neomycin resistant ES cell lines had neo gene sequences in their genomic DNA but another two cell lines lacked it. The karyotype of them appeared to be stable, as it had still euploid chromosome constitution after transformation and the selection. Two of the lines contained significant populations of the cells with chromosomal abnomality. Overt coat color chimaeras were obtained and their germ lines were transmitted with all four neo resistant ES cell lines tested. Southern blot hybridization and PCR analysis of genomic DNA confirmed again that F(1) and F(2) generation of these germ line chimaeras were transmitted with neo gene stably. These results indicate that ES cells (F1/1) isolated from blastocyst of C57BL/6 female mated wi th a CBA male can be used as a vehicle for transgenesis. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1998 Haptoglobin(Hp) Genotyping by Allele Specific Polymerase Chain Reaction Amplification EN No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1996 IgA'2 Genotyping by Polymerase Chain Reaction (PCR) Using Allele-Specific Amplification Primers EN No potential conflict of interest relevant to this article was reported.