MDPI AG Acta Medica Okayama 2075-1729 16 10 2026 Purification, Structural Characterization, and Genomic Insights of an Anti-MRSA Peptide from Mangrove-Derived Brevibacillus sp. A34 1697 EN Thanyamai Thongin School of Pharmacy, Walailak University Namfa Sermkaew School of Pharmacy, Walailak University Apichart Atipairin School of Pharmacy, Walailak University Sucheewin Krobthong Center of Excellence in Natural Products Chemistry (CENP), Department of Chemistry, Faculty of Science, Chulalongkorn University Chanat Aonbangkhen Center of Excellence in Natural Products Chemistry (CENP), Department of Chemistry, Faculty of Science, Chulalongkorn University Jumpei Uchiyama Department of Infectious Diseases, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8558, Japan Nuttapon Songnaka School of Pharmacy, Walailak University The widespread prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in community and healthcare settings has driven the urgent search for novel antibacterial agents. Mangrove sediments are an underexplored source of antimicrobial-producing bacteria. This study characterized an antimicrobial fraction and the genome of the mangrove-derived isolate Brevibacillus sp. A34. The active fraction was purified via ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase chromatography. De novo MS/MS analysis assigned the octapeptide LLLLLLAH (905.19 Da), designated as A34 AMP, which was further confirmed by chemical synthesis. This active peptide inhibited all four tested S. aureus strains, including three MRSA strains, with a minimum inhibitory concentration (MIC) of 62 µg/mL. At 2× and 4× MIC, A34 AMP exhibited a concentration-dependent bactericidal effect within 20 h. Increased SYTOX Green uptake and morphological alterations supported a membrane permeabilization mechanism, and antimicrobial activity was retained following thermal, pH, protease, and surfactant treatments. Genome mining identified 23 putative biosynthetic gene clusters, including an 11-module nonribosomal peptide synthetase (NRPS) cluster linked to A34 AMP through three-dimensional adenylation-domain modeling. Collectively, these findings highlight A34 AMP from Brevibacillus sp. A34 as a promising bioactive anti-staphylococcal peptide scaffold. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2168-8184 17 2 2025 Elevated Plasma Progastrin-Releasing Peptide Levels Associated With the Diagnosis of Pulmonary Carcinoid Tumors: A Report of Two Cases e79601 EN Takashi Yamaoka Department of Respiratory Medicine, Japanese Red Cross Kobe Hospital Kiichiro Ninomiya Department of Allergy and Respiratory Medicine, Okayama University Hospital Taku Noumi Department of Respiratory Medicine, Japanese Red Cross Kobe Hospital Chiaki Matsumoto Department of Respiratory Medicine, Japanese Red Cross Kobe Hospital Keisuke Sugimoto Department of Respiratory Medicine, Japanese Red Cross Kobe Hospital Pulmonary carcinoids are the type of thoracic malignant tumors classified as neuroendocrine tumors. Case 1 involved a 59-year-old woman with a nodular shadow in the left upper pulmonary field observed on chest radiography. Chest computed tomography (CT) scan revealed a nodule in the left upper lobe and a soft tissue tumor near the left third rib. Given her elevated plasma progastrin-releasing peptide (ProGRP) level, a CT-guided biopsy was performed, confirming a diagnosis of pulmonary carcinoid with solitary bone metastasis. She subsequently underwent partial resection of the left upper lobe and the rib tumor. Case 2 involved a 67-year-old woman referred to our hospital after a chest CT scan revealed a nodule in the left lingular region. Her plasma ProGRP level was elevated, and a CT-guided biopsy confirmed a diagnosis of pulmonary carcinoid. As no lymph node metastases were detected, she underwent segmental resection of the left upper lobe. In both cases, plasma ProGRP levels decreased following surgical resection. These findings suggest that plasma ProGRP levels may serve as a useful diagnostic marker for pulmonary carcinoids. No potential conflict of interest relevant to this article was reported.

Royal Society of Chemistry (RSC) Acta Medica Okayama 1477-0520 2026 Stereoselective synthesis of β,β-diborylalanine (AlaBB) and its incorporation into oligopeptides EN Naoyuki Nitta Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kenshin Nakai Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Haruki Mizoguchi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Akira Sakakura Graduate School of Environmental, Life, Natural Science and Technology, Okayama University We report the stereoselective synthesis of β,β-diborylalanine (AlaBB), a compact amino acid scaffold designed to introduce a gem-diboronic acid motif as a branched connector in peptides. Pinacol-protected AlaBB derivatives, denoted as AlaBB(pin)2, were prepared from an Ellman imine, incorporated into short peptides, and converted to the corresponding gem-diboronic acid form. No potential conflict of interest relevant to this article was reported.

Royal Society of Chemistry (RSC) Acta Medica Okayama 1477-0520 2026 Stereoselective synthesis of β,β-diborylalanine (AlaBB) and its incorporation into oligopeptides EN Naoyuki Nitta Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kenshin Nakai Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Haruki Mizoguchi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Akira Sakakura Graduate School of Environmental, Life, Natural Science and Technology, Okayama University We report the stereoselective synthesis of β,β-diborylalanine (AlaBB), a compact amino acid scaffold designed to introduce a gem-diboronic acid motif as a branched connector in peptides. Pinacol-protected AlaBB derivatives, denoted as AlaBB(pin)2, were prepared from an Ellman imine, incorporated into short peptides, and converted to the corresponding gem-diboronic acid form. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1660-3397 24 9 2026 A Marine Brevibacillus-Derived Membrane-Lytic Peptide: Molecular Insight and Biophysical Characterization of a Novel AMP, FNL62-AMP 309 EN Namfa Sermkaew School of Pharmacy, Walailak University Apichart Atipairin School of Pharmacy, Walailak University Sucheewin Krobthong Center of Excellence in Natural Products Chemistry (CENP), Department of Chemistry, Faculty of Science, Chulalongkorn University Chanat Aonbangkhen Center of Excellence in Natural Products Chemistry (CENP), Department of Chemistry, Faculty of Science, Chulalongkorn University Yodying Yingchutrakul National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency Jumpei Uchiyama Department of Infectious Diseases, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nuttapon Songnaka School of Pharmacy, Walailak University This study characterizes the genomic identity, functional efficacy, and computational biophysics of FNL62-AMP, a novel antimicrobial peptide isolated from a phylogenomically distinct, newly identified Brevibacillus species. Production kinetics revealed a late-exponential phase onset of antibacterial activity with sustained potency against methicillin-resistant Staphylococcus aureus (MRSA). LC-MS/MS analysis identified the peptide sequence as NH2-LLLLFR-COOH. FNL62-AMP demonstrated excellent formulation resilience, retaining full anti-MRSA activity under high thermal stress (80 °C for 6 h) and showing robust resistance to generic trypsin and proteinase K proteolysis. Formulative co-incubation assays demonstrated charge-dependent compatibility, where nonionic Triton X-100 preserved baseline efficacy while ionic surfactants induced antagonism. In vitro time-kill kinetics, scanning electron microscopy, and SYTOX Green assays confirmed rapid, concentration-dependent bactericidal action driven by immediate membrane permeabilization. Molecular dynamics simulations successfully captured the spontaneous self-assembly of 64 FNL62-AMP monomers into a stable macro-aggregate. This consolidation process was quantitatively characterized by a simultaneous contraction in the radius of gyration (Rg), a sharp drop in solvent-accessible surface area (SASA), and a transitional plateau in mean squared displacement (MSD). Ultimately, the high thermal stability, structural resilience, and predictable surfactant compatibility of FNL62-AMP propose ways to be developed for lead optimization and druggability. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0953-8194 38 9 2026 Marine flatworms as windows into the evolution of bilaterian neuropeptide signalling e70259 EN Ryo Nakamura Ushimado Marine Institute, Faculty of Science, Okayama University Mayuko Hamada Ushimado Marine Institute, Faculty of Science, Okayama University Xavier Bailly Multicellular Marine Models Team, CNRS – Sorbonne University – Station Biologique de Roscoff Hirotaka Sakamoto Department of Biology, Faculty of Environmental, Life, Natural Science and Technology, Okayama University Tatsuya Sakamoto Ushimado Marine Institute, Faculty of Science, Okayama University The evolutionary origin(s) of neuropeptide signalling is still largely unknown. It has been hypothesised that signal transmission via neuropeptides played a crucial role in the regulation of nervous systems in the bilaterian ancestor. Identification, expression and functional analyses of neuropeptides and their receptors across different taxonomic groups are important for better understanding the origin(s) of bilaterian neuropeptide signalling. An example is flatworms belonging to the Platyhelminthes (spiralian protostomes) and Xenacoelomorpha (sister to all Bilateria or sister to Ambulacraria). Despite their simple morphology (i.e., a body plan lacking a coelom and circulatory system), they often possess brain-like central nervous systems and a number of bilaterian-conserved neuropeptides. This feature makes them useful models for analyses of neuropeptides and their links to nervous systems. Although previous studies have revealed orthologous relationships between vertebrate neuropeptides and those found in both marine Platyhelminthes and Xenacoelomorpha, our work suggests possible ancestral functions of vasopressin/oxytocin (VP/OT) peptides (platytocin) in these groups. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1356-9597 30 3 2025 Polished Rice Regulates Maturation but Not Survival of Secondary Cells in Drosophila Male Accessory Gland e70025 EN Shinichi Otsune Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Mirai Matsuka Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Chisato Shirakashi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Xuanshuo Zhang Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hideki Nakagoshi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University In Drosophila males, the accessory gland is responsive to nutrient signal-dependent regulation of fertility/fecundity. The accessory gland is composed of two types of binucleated epithelial cells, about 1000 main cells and 60 secondary cells (SCs). The transcription factors Defective proventriculus (Dve), Abdominal-B, and Ecdysone receptors (EcRs) are strongly expressed in adult SCs. In response to nutrient conditions during development, coordinated action between Dve and ecdysone signaling determines the optimal number of SCs and regulates their maturation. A downstream effector of ecdysone signaling, Ftz-F1, is crucial in this process. Another downstream effector, Polished rice (Pri), is small peptides of 11 or 32 amino acids. Here we show that pri is required for maturation of SCs and for male fecundity, whereas it is not involved in determination of the number of SCs. We provide evidence that Pri acts downstream of Ftz-F1 to regulate maturation but not survival of SCs. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 80 4 2026 The Impact of Migraine on Cerebral Artery Dissection 227 231 EN Kenichi Kashihara Okayama Neurology Clinic Toshikazu Hamaguchi Department of Neurology, Okayama Kyokuto Hospital Yasuko Takeda Okayama Neurology Clinic Original Article /10.18926/AMO/71065 The impact of migraines as a risk factor for cervicocerebral artery dissection was retrospectively investigated in patients who had a cervicocerebral artery dissection. We first evaluated the patients’ medical records and history of migraine and then calculated the proportion of patients with migraine as well as the relevant vascular risk factors. The prevalence of migraine with respect to the location of the cervicocerebral artery dissection was also identified. Sixty-two patients (37 men, 25 women) had both a cerebral artery dissection and a record of migraine. Among them, 93.5% presented with an isolated headache, and 6.5% presented with a related stroke. Regarding the locations of the artery dissection, 56 (90.3%) were in the intracranial vertebral artery (VA), three (4.8%) were in the internal carotid artery, and one case each (1.6%) was in an anterior cerebral artery, basilar artery (BA), and both the VA and BA. Among the patients with VA dissection, 42 (73.7%) presented with migraines. Among the four patients with dissection of an artery of anterior circulation, only one (25%) had a migraine history. The frequency of migraines tended to be higher in the patients with VA dissection compared to those with a dissection of arteries of anterior circulation. These data suggest that (i) migraine is a potent risk factor for cerebral artery dissection, especially intracranial VA dissection, and (ii) the intracranial VA may be more susceptible to dissection compared to arteries of anterior circulation. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2072-6643 18 11 2026 GLP-1 Receptor Agonists in the Rehabilitation of Patients with Heart Failure: Mechanisms, Clinical Evidence, and Future Perspectives 1688 EN Luh Oliva Saraswati Suastika Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuko K. Bando Department of Molecular Physiology and Cardiovascular Biology, Mie University Graduate School of Medicine Keiji Hoshino Division of Cardiology, Gunma Prefectural Cardiovascular Center Norimichi Koitabashi Division of Cardiology, Gunma Prefectural Cardiovascular Center Yukihiro Saito Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinsuke Yuasa Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazufumi Nakamura Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Heart failure (HF) remains associated with high morbidity and mortality, with heart failure with preserved ejection fraction (HFpEF) becoming increasingly prevalent and therapeutically challenging despite advances in pharmacological and rehabilitative care. Beyond their glucose-lowering effects, glucagon-like peptide-1 receptor agonists (GLP-1RAs) confer cardiometabolic benefits and may serve as effective adjuncts to cardiac rehabilitation (CR), particularly in obese patients with HFpEF. Obesity plays a central role in the pathophysiology of HFpEF, and GLP-1RAs promote weight loss, reduce insulin resistance and leptin signaling, and improve hemodynamic and metabolic abnormalities associated with HFpEF. Accumulating evidence suggests that the benefits of GLP-1RAs are phenotype-specific and more pronounced in patients with HFpEF than in patients with HF with reduced ejection fraction. Current clinical guidelines recommend GLP-1RAs for patients who have type 2 diabetes mellitus and established cardiovascular (CV) disease or are at high CV risk, with recent updates recognizing their potential benefits in patients with HFpEF and obesity. Cardiac rehabilitation, delivered through multidisciplinary programs, remains a cornerstone of HF management. Although caloric restriction and aerobic exercise can be beneficial in patients with HFpEF and obesity, these interventions alone are often insufficient. Sarcopenia is common in older patients with HFpEF and contributes to adverse outcomes, underscoring the importance of incorporating resistance training into CR programs. The most frequent adverse effects of GLP-1RAs are gastrointestinal events, which are generally mild to moderate but may lead to treatment discontinuation in some patients. Future studies should investigate the potential synergistic effects of GLP-1RAs and CR, clarify their long-term safety and efficacy in HF populations, and define their role beyond obese HFpEF phenotypes. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0021-9193 206 10 2024 Overexpression of diglucosyldiacylglycerol synthase leads to daptomycin resistance in Bacillus subtilis e00307-24 EN Ryogo Yamamoto Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazuya Ishikawa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yusuke Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazuyuki Furuta Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shin-Ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chikara Kaito Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The lipopeptide antibiotic daptomycin exhibits bactericidal activity against Gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanisms of bacterial resistance to daptomycin has gained great importance. In this study, we aimed to identify the genetic factors contributing to daptomycin resistance in Bacillus subtilis, a model Gram-positive bacterium. Our findings demonstrated that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis. Specifically, overexpression of ugtP resulted in increased levels of diglucosyldiacylglycerol (Glc2DAG) and decreased levels of acidic phospholipids cardiolipin and PG, as well as the basic phospholipid lysylphosphatidylglycerol. However, ugtP overexpression did not alter the cell surface charge and the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide. Furthermore, by serial passaging in the presence of daptomycin, we obtained daptomycin-resistant mutants carrying ugtP mutations. These mutants showed increased levels of Glc2DAG and a >4-fold increase in the minimum inhibitory concentration of daptomycin. These results suggest that increased Glc2DAG levels, driven by ugtP overexpression, modify the phospholipid composition and confer daptomycin resistance in B. subtilis without altering the cell surface charge of the bacteria. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0012-1606 520 2025 Tunicate-specific protein Epi-1 is essential for conferring hydrophilicity to the larval tunic in the ascidian Ciona 41 52 EN Kazu Kuroiwa Shimoda Marine Research Center, University of Tsukuba Kaoru Mita-Yoshida Shimoda Marine Research Center, University of Tsukuba Mayuko Hamada Ushimado Marine Institute, Okayama University Akiko Hozumi Shimoda Marine Research Center, University of Tsukuba Atsuo S. Nishino Department of Biology, Faculty of Agriculture and Life Science, Hirosaki University Yasunori Sasakura Shimoda Marine Research Center, University of Tsukuba Animals must avoid adhesion to objects in the environment to maintain their mobility and independence. The marine invertebrate chordate ascidians are characterized by an acellular matrix tunic enveloping their entire body for protection and swimming. The tunic of ascidian larvae consists of a surface cuticle layer and inner matrix layer. Hydrophilic substances coat the cuticle; this modification is thought to be for preventing adhesion. However, the molecule responsible for regulating this modification has not been clarified. We here found that the tunicate-specific protein Epi-1 is responsible for preventing adhesiveness of the tunic in the ascidian Ciona intestinalis Type A. Ciona mutants with homozygous knockouts of Epi-1 exhibited adhesion to plastic plates and to other individuals. The cuticle of the Epi-1 mutants was fragile, and it lost the glycosaminoglycans supplied by test cells, the accessory cells that normally attach to the tunic surface. Although it has an apparent signal peptide for membrane trafficking, we showed that the Epi-1 protein is localized to the cytosol of the epidermal cells. Our study suggests that the emergence of the tunicate-specific protein Epi-1 made the tunic less adhesive, providing a selective advantage for the last common tunicate ancestor. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2041-1723 17 1 2026 Structural insights into YheS-mediated release of SecM-arrested ribosome 4963 EN Kaishi Iso Department of Biological Sciences, Graduate School of Science, The University of Tokyo Toma Ikeda School of Life Science and Technology, Institute of Science Tokyo Kohei Yamasaki Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yushin Ando Department of Biological Sciences, Graduate School of Science, The University of Tokyo Fumiya K. Sano Department of Biological Sciences, Graduate School of Science, The University of Tokyo Tadaomi Furuta School of Life Science and Technology, Institute of Science Tokyo Hideki Taguchi School of Life Science and Technology, Institute of Science Tokyo Osamu Nureki Department of Biological Sciences, Graduate School of Science, The University of Tokyo Yuhei Chadani Faculty of Environmental, Life, Natural Science and Technology, Okayama University Yuzuru Itoh Department of Biological Sciences, Graduate School of Science, The University of Tokyo ATP-binding cassette subfamily F (ABCF) proteins interact with the ribosome to resolve translation defects near the peptidyl transferase center (PTC). In Escherichia coli, four ABCF proteins (EttA, Uup, YbiT, and YheS) selectively promote translation of distinct problematic nascent peptide sequences, but their molecular mechanisms remain unclear. Here, we present a 2.8 Å cryo-EM structure of the ribosome in complex with an ATPase-deficient mutant of YheS and investigate how it releases ribosomes arrested by the SecM nascent chain. YheS binds to the ribosomal E-site via the L1 stalk, and its P-site tRNA-interaction motif (PtIM) extends toward the PTC, displacing the CCA end of the P-site tRNA. Notably, the cryo-EM density corresponding to the SecM nascent chain within the exit tunnel is largely lost upon YheS binding. These observations suggest that YheS relieves peptide sequence-dependent stalling by perturbing nascent chain-tunnel interactions through P-site tRNA relocation. Steered molecular dynamics simulations provide qualitative support for this model. Together, our findings provide mechanistic insight into a mode of arrest release distinct from the translocon-mediated release mechanism. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1865-7257 19 3 2026 Successful management and 10-year survival of a rectal neuroendocrine tumor with rare systemic metastases via a non-portal venous pathway 491 498 EN Shigeru Horiguchi Department of Gastroenterology and Hepatology, Okayama University Hospital Kazuyuki Matsumoto Department of Gastroenterology and Hepatology, Okayama University Hospital Kazuya Miyamoto Department of Gastroenterology and Hepatology, Okayama University Hospital Akihiro Matsumi Department of Gastroenterology and Hepatology, Okayama University Hospital Hiroyuki Terasawa Department of Gastroenterology and Hepatology, Okayama University Hospital Yuki Fujii Department of Gastroenterology and Hepatology, Okayama University Hospital Kosei Takagi Department of Gastroenterological Surgery, Dentistry, and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Takashi Kuise Department of Gastroenterological Surgery, Okayama Red Cross Hospital Takehiro Tanaka Department of Pathology, Okayama University Hospital Motoyuki Otsuka Department of Gastroenterology and Hepatology, Okayama University Hospital Metastatic neuroendocrine tumors (NETs) typically involve the liver and lymph nodes. Metastases in the orbit, heart, and ovary are rare and present unique clinical challenges. We report a woman who was 70 years old at the time of initial presentation with liver metastases from a rectal neuroendocrine tumor. Following a right hepatectomy for liver metastases, she remained free of hepatic recurrence for 10 years. Notably, four years after the right hepatectomy, she developed new metastases in the left orbit, right ventricle, and left ovary, with diplopia as the sole clinical symptom. This clinical course suggests a distinct metastatic pathway associated with the lower rectum that bypasses the portal circulation, consistent with the dual drainage system of the rectal region. Management involved a strategic multimodal approach that includes systemic therapy with everolimus and lanreotide as well as targeted surgical resectioning of the cardiac and ovarian lesions. The orbital lesion achieved a complete response through systemic therapy alone, preserving visual function. Currently, 10 years after the initial treatment, the patient maintains an excellent performance status (ECOG PS 0) while receiving peptide receptor radionuclide therapy (PRRT). This case demonstrates that recognizing atypical metastatic pathways and employing strategic multimodal therapies can achieve long-term survival and functional preservation in rectal NETs. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1422-0067 27 2 2026 Possible Involvement of Hypothalamic Dysfunction in Long COVID Patients Characterized by Delayed Response to Gonadotropin-Releasing Hormone 832 EN Yuki Otsuka Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshiaki Soejima Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuhiro Nakano Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Atsuhito Suyama Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryosuke Takase Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kohei Oguni Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yohei Masuda Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Daisuke Omura Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasue Sakurada Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yui Matsuda Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toru Hasegawa Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroyuki Honda Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuki Tokumasu Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keigo Ueda Department of General Medicine, 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 Long COVID (LC) may involve endocrine dysfunction; however, the underlying mechanism remains unclear. To examine hypothalamic–pituitary responses in patients with LC, we conducted a single-center retrospective study of patients with refractory LC referred to our University Hospital who underwent anterior pituitary stimulation tests. Between February 2021 and November 2025, 1251 patients with long COVID were evaluated, of whom 207 (19%) had relatively low random ACTH or cortisol levels. Ultimately, 16 underwent anterior pituitary stimulation tests and were included. All tests were performed in an inpatient setting without exogenous steroids. Fifteen patients (six women, mean age 35.6 years) underwent corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), and gonadotropin-releasing hormone (GnRH) tests. All patients had mild acute COVID-19, eight had ≥2 vaccinations, and the mean interval from infection was 343 days. Frequent symptoms included fatigue (100%), insomnia (66.7%), headache (60.0%), anorexia/nausea (40.0%), and brain fog (40.0%). Mean early-morning cortisol and 24 h urinary free cortisol were 7.5 μg/dL and 41.0 μg/day, respectively. MRI showed an empty sella in one case. Peak hormonal responses were preserved (ΔACTH 247%, ΔTSH 918%, ΔPRL 820%, ΔFSH 187%, ΔLH 1150%); however, peaks were delayed beyond 60 min in ACTH (13%), LH (33%), and FSH (87%). Notably, significantly delayed elevations remained at 120 min in the responses of TSH (4.1-fold), PRL (1.8-fold), LH (9.3-fold), and FSH (2.8-fold), suggesting possible hypothalamic involvement, particularly in the gonadotropin responses. Additionally, serum IGF-I was lowered (−0.70 SD), while GH response (mean peak 35.5 ng/mL) was preserved by growth hormone-releasing peptide (GHRP)-2 stimulation. Low-dose hydrocortisone and testosterone were initiated for three patients. Although direct viral effects and secondary suppression have been proposed, our findings may suggest that, at least in part, the observed response characteristics are consistent with functional secondary hypothalamic dysfunction rather than irreversible primary injury. These findings highlight the need for objective endocrine evaluation before initiating hormone replacements. No potential conflict of interest relevant to this article was reported.

The Company of Biologists Acta Medica Okayama 0022-0949 229 2 2026 Insulin-like peptide has antagonistic pleiotropic effects on male combat traits and survival traits in an armed beetle jeb251318 EN Takumi Kato Graduate School of Science, Nagoya University Chiho Yoshimine Graduate School of Science, Tokyo Metropolitan University Haruna Fujioka Graduate School of Environmental, Natural Science and Technology, Okayama University Masako Katsuki Graduate School of Science, Nagoya University Kensuke Okada Graduate School of Environmental, Natural Science and Technology, Okayama University Yasukazu Okada Graduate School of Science, Nagoya University The expression of sexually selected traits, such as exaggerated weapons and ornaments, often entails trade-offs against life-history traits. While phenotypic trade-offs are well documented, the underlying molecular physiological mechanisms remain largely unexplored. In this study, we investigated the potential role of an insulin-like peptide, ILP2, in mediating the trade-off between sexually selected combat traits and survival traits in the broad-horned flour beetle, Gnatocerus cornutus. RNA interference (RNAi)-mediated knockdown (KD) of ILP2 during larval stages resulted in a reduction in the development of mandibular horns and overall body size. Interestingly, ILP2 KD males had increased lipid storage and enhanced starvation tolerance, indicating a shift in resource allocation from sexually selected traits to survival traits. Behaviorally, ILP2 KD males showed decreased locomotor activity and reduced aggression, leading to lower combat success. These findings suggest that ILP2 functions as a key mediator in the allocation of resources between combat and survival traits, highlighting its pleiotropic effects on morphology, metabolism and behavior. Our study provides novel insights into the molecular physiological mechanisms underlying life-history trade-offs associated with sexually selected traits. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 1574-6968 373 2026 Intercellular signal transduction within the mother cell compartment during Bacillus subtilis sporulation fnag055 EN Nobuki Kuwabara Department of Frontier Bioscience, Hosei University Masato Anzue Department of Frontier Bioscience, Hosei University Shin-ichi Miyoshi Research Center for Intestinal Health Science, Okayama University Tsutomu Sato Department of Frontier Bioscience, Hosei University Daisuke Imamura Research Center for Intestinal Health Science, Okayama University Intercellular signaling contributes to the spatiotemporal regulation of gene expression during sporulation in Bacillus subtilis. The mother cell transcription factor σE is initially produced as an inactive precursor protein pro-σE and activated by the processing enzyme SpoIIGA in response to the forespore-produced putative signaling molecule SpoIIR. However, the mechanism underlying the SpoIIR-mediated signal transduction remains poorly understood. In this study, we showed that the spoIIR-positive, spoIIGA-deleted strain was able to induce SpoIIGA-dependent pro-σE processing in co-cultured spoIIR-deleted, spoIIGA-positive strains. This signaling was dependent on SpoIIR expression and did not involve DNA transfer. Extracellular materials including secreted proteins and membrane vesicles were unlikely to be involved in this signaling pathway. Interestingly, cessation of co-incubation shaking enhanced the signaling, while the addition of membrane-solubilizing detergent abolished it. In addition, SpoIIR signaling did not necessitate release from the forespore membrane or extracellular translocation. A SpoIIR variant lacking the putative signal peptide-like hydrophobic domain produced solely in the mother cell compartment was still able to activate pro-σE. Overall, the study findings suggested that the forespore-produced SpoIIR is neither secreted nor externally translocated. Instead, SpoIIR appeared to be transferred into the mother cell compartment and interacts with the SpoIIGA cytoplasmic domain to trigger pro-σE processing. No potential conflict of interest relevant to this article was reported.

BMJ Acta Medica Okayama 2044-6055 16 3 2026 Protocol for an open-label, randomised, controlled trial to evaluate the efficacy and safety of sotatercept add-on therapy compared with pulmonary vasodilator-based standard of care for pulmonary vasodilator-resistant pulmonary arterial hypertension associated with unrepaired congenital shunts (atrial septal defect, ventricular septal defect or patent ductus arteriosus), including Eisenmenger syndrome: the SuMILE trial e113430 EN Keimei Yoshida Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University Kazuya Hosokawa Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University Takahiro Hiraide Department of Cardiology, Keio University School of Medicine Satoshi Akagi Department of Cardiovascular Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kentaro Ejiri Department of Cardiovascular Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yu Taniguchi Division of Cardiovascular Medicine, Kobe University Hospital Shiro Adachi Department of Cardiology, Nagoya University Hospital Takumi Inami Department of Cardiovascular Medicine, Kyorin University School of Medicine Naohiko Nakanishi Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine Masaharu Kataoka The Second Department of Internal Medicine, University of Occupational and Environmental Health Taijyu Satoh Department of Medical Science and Innovation, SiRIUS Institute of Medical Research, Tohoku University Shunsuke Tatebe Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine Toshiro Shinke Division of Cardiology, Department of Medicine, Showa Medical University Hospital Hideshi Tomita Periatric Heart Disease and Adult Congenital Heart Disease Center, Showa Medical University Hospital Yusuke Akazawa Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University Graduate School of Medicine Takashi Higaki Department of Pediatric and Adolescent Therapeutic and Developmental Education, Ehime University Graduate School of Medicine Koshiro Tagawa Center for Clinical and Translational Research, Kyushu University Hospital Ayako Ishikita Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University Soshun Asakawa Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University Kohtaro Abe Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University Introduction Eisenmenger syndrome and pulmonary arterial hypertension (PAH) due to unrepaired congenital shunts, including atrial septal defect (ASD), ventricular septal defect (VSD) and patent ductus arteriosus (PDA), remain life-threatening conditions despite advances in congenital heart disease (CHD) care. In this population, vasodilator-based therapies effective in other forms of PAH have shown limited benefit, and no disease-modifying treatment has been established. Sotatercept, an activin-signalling inhibitor, improved exercise capacity and haemodynamics in phase 2/3 PAH trials; however, patients with unrepaired CHD, including Eisenmenger syndrome, were excluded. The efficacy and safety of sotatercept in this population remain unknown.<br> Methods and analysis The SuMILE trial is a prospective, exploratory, multicentre, open-label, randomised, controlled trial conducted at 11 Japanese tertiary centres. 36 adults with vasodilator-resistant PAH due to unrepaired ASD, VSD or PDA, including Eisenmenger syndrome, will be randomised 2:1 to sotatercept add-on therapy plus vasodilator-based PAH therapy versus vasodilator-based PAH therapy alone. Sotatercept will be administered subcutaneously every 3 weeks in accordance with label-approved dose-modification rules for haemoglobin and platelet changes. The primary endpoint is the change in 6-min walk distance from baseline to week 24. Key clinical events will be independently adjudicated. Secondary endpoints include all-cause mortality or lung transplantation; pulmonary hypertension-related hospitalisation or initiation of parenteral prostacyclin and changes in WHO functional class, N-terminal pro-brain natriuretic peptide and emPHasis-10. Exploratory endpoints include genotype, right heart catheterisation and cardiac MRI parameters. The primary analysis will use ANCOVA, adjusting for baseline 6-min walk distance and randomisation stratum in the intention-to-treat population.<br> Ethics and dissemination The protocol has been reviewed and approved by the certified central review board (Kyushu University Hospital Clinical Ethics Review Board) and participating institutions. Written informed consent will be obtained from all participants. Findings will be disseminated through peer-reviewed journals, scientific conferences and trial registries.<br> Trial registration number Japan Registry of Clinical Trials no. 1071250069; ClinicalTrials.gov NCT07356778. Protocol version and date: V.1.3; 23 October 2025 No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 2211-7156 25 2026 Peptide nanomicelles for NIR light-dependent siRNA delivery 103265 EN Taufik Fatwa Nur Hakim Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Kindai University Shoumu Fujimoto Department of Applied Chemistry, Kindai University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University The peptide amphiphile PA8, derived from the GAVILRR peptide, was developed as a carrier for small interfering RNA (siRNA) delivery; however, its RNA interference (RNAi) efficacy was limited owing to predominant endocytotic uptake. In this study, the RNAi efficiency of PA8 nanomicelle/siRNA complexes was enhanced by modifying the nanomicelles with the photosensitizer DY750 and the tumor-homing peptide iRGD. The conjugation of DY750 to the nanomicelles facilitated endosomal escape of the nanomicelle/siRNA complexes, enabling the cytosolic release of siRNA. Additionally, the incorporation of iRGD improved RNAi delivery efficiency in the AsPC-1 pancreatic ductal adenocarcinoma cell line. PA8-DY750-iRGD nanomicelle complexes loaded with siRNA against polo-like kinase 1 (PLK1) achieved an 80% reduction in PLK1 mRNA levels in AsPC-1 cells and a moderate 28% knockdown in NCI-N87 gastric cancer cells. Notably, no RNAi effect was observed in noncancerous 1C3D3 pancreatic cells or HEK293T kidney cells, underscoring the selectivity of this system for AsPC-1 cells. These findings highlight the potential of PA8-DY750-iRGD nanomicelle complexes as a targeted therapeutic platform for specific cancers, particularly pancreatic cancer. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2045-2322 16 1 2026 Liquid–liquid phase separation by caged coacervating peptides 10464 EN Akinari Bando Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Kindai University Yuuki Kanazaki Department of Applied Chemistry, Kindai University Rika Tojo Department of Applied Chemistry, Kindai University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Liquid–liquid phase separation is an important biomolecular process in the formation of membraneless intracellular organelles that has inspired the development of artificial droplet systems. We developed caged coacervating peptides (CCPs) based on a histidine-rich squid beak protein sequence. The peptides were caged with a photodeprotectable (7-diethylaminocoumarin-4-yl)methoxycarbonyl group. The CCPs formed coacervates in the caged state and were partially dispersed upon blue-light irradiation. Photo-uncaging occurred rapidly, inducing coacervate dispersion. A mutant CCP with reduced π–π interactions exhibited efficient photo-dependent disassembly and enabled the encapsulation and release of a fluorescently labeled adenosine 5′-triphosphate (Bodipy-ATP) upon irradiation. These CCPs offer an efficient light-controlled approach for biomolecular encapsulation within coacervates and targeted drug delivery. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1422-0067 27 7 2026 CXCR2-Dependent Infiltration of Tumor-Associated Neutrophils Is Linked to Enhanced CD8+ T Cell Effector Function and Reduced Lung Metastasis in 4T1 Breast Cancer 3143 EN Tiantian Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Miao Tian Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Gakushi Nishida Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chunning Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Triple-negative breast cancer (TNBC) is characterized by prominent neutrophil infiltration; however, its significance remains controversial. Here, we investigated the role of neutrophil chemoattractant receptors in TNBC progression and metastasis. In contrast to wild-type (WT), Fpr1−/−, and Fpr2−/− mice, neutrophils were almost completely absent in 4T1 tumors from Cxcr2−/− mice, indicating a dominant role for CXCR2 in the recruitment of tumor-associated neutrophils, leading us to use Cxcr2−/− mice for further studies. Primary tumor growth was comparable between WT and Cxcr2−/− mice, whereas lung metastasis was significantly increased in Cxcr2−/− mice, with reduced expression of inflammatory cytokines, chemokines and cytotoxic molecules, including granzyme B and perforin, in primary tumors and metastatic lungs of Cxcr2−/− mice. In vitro, WT, but not Cxcr2−/−, neutrophils enhanced CD8+ T cell activation, partly via ICAM-1, and directly induced tumor cell death, supporting their anti-tumor function. To assess clinical relevance, transcriptomic data were analyzed. High neutrophil infiltration combined with elevated CXCR2 expression, and to a lesser extent CXCR1 expression, was associated with improved prognosis in patients with basal-like BC that largely overlaps with TNBC. Collectively, these findings suggest that CXCR2-mediated neutrophil recruitment exerts protective, anti-tumor effects and may represent a new prognostic marker for TNBC patients. No potential conflict of interest relevant to this article was reported.

American Society for Clinical Investigation Acta Medica Okayama 2379-3708 11 3 2025 Collagen-binding C-type natriuretic peptide enhances chondrogenesis and osteogenesis e198959 EN Kenta Hirai Department of Pediatrics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kenta Sawamura Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine Ryusaku Esaki Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine Ryusuke Sawada Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yuka Okusha Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Eriko Aoyama Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School Hiroki Saito Department of Orthopaedic Surgery, Kitasato University School of Medicine Kentaro Uchida Department of Orthopaedic Surgery, Kitasato University School of Medicine Takehiko Mima Department of Medical Technology, Faculty of Health Sciences, Ehime Prefectural University of Health Sciences Satoshi Kubota Department of Biochemistry and Molecular DentistryBacteriology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hirokazu Tsukahara Department of Pediatrics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Shiro Imagama Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine Masaki Matsushita Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yasuyuki Hosono Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences C-type natriuretic peptide (CNP) is known to promote chondrocyte proliferation and bone formation; however, CNP’s extremely short half-life necessitates continuous intravascular administration to achieve bone-lengthening effects. Vosoritide, a CNP analog designed for resistance to neutral endopeptidase, allows for once-daily administration. Nonetheless, it distributes systemically rather than localizing to target tissues, which may result in adverse effects such as hypotension. To enhance local drug delivery and therapeutic efficacy, we developed a potentially novel synthetic protein by fusing a collagen-binding domain (CBD) to CNP, termed CBD-CNP. This fusion protein exhibited stability under heat conditions and retained the collagen-binding ability and bioactivity as CNP. CBD-CNP localized to articular cartilage in fetal murine tibiae and promoted bone elongation. Spatial transcriptomic analysis revealed that the upregulation of chondromodulin expression may contribute to its therapeutic effects. Treatment of CBD-CNP mixed with collagen powder to a fracture site of a mouse model increased bone mineral content and bone volume compared with CNP-22. Intraarticular injection of CBD-CNP to a mouse model of knee osteoarthritis suppressed subchondral bone thickening. By addressing the limitations of CNP’s rapid degeneration, CBD-CNP leverages its collagen-binding capacity to achieve targeted, sustained delivery in collagen-rich tissues, offering a promising strategy for enhancing chondrogenesis and osteogenesis. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2079-6374 16 1 2026 Magnetic Detection of Cancer Cells Using Tumor-Homing Peptide-Modified Magnetic Nanoparticles 45 EN Shengli Zhou Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yuji Furutani Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kei Yamashita Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Sakuya Kako Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toshihiko Kiwa Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Magnetic nanoparticles (MNPs) provide a platform for target detection because of their magnetic responsiveness to alternating magnetic fields (AMFs). We developed a detection method using MNPs modified with tumor-homing peptides (THPs), PL1 and PL3, which selectively bind to protein components enriched in malignant tissues. THP-MNPs were synthesized using maleimide-PEG-NHS linkers and characterized using transmission electron microscopy. Human glioblastoma cancer U87MG and normal tissue-derived HEK293 cells were incubated with THP-MNPs, and the magnetic signals were measured using a high-temperature superconducting quantum interference device (SQUID) magnetometer under an AMF (1.06 kHz). Dark-field microscopy confirmed the preferential binding of THP-MNPs to U87MG cells. In the absence of cells, THP-MNPs exhibited AMF-dependent signal enhancement, which correlated with particle size reduction due to THP release. This increase was completely suppressed in the presence of U87MG cells, indicating a strong THP-mediated interaction. PL3-MNPs exhibited superior discrimination between malignant and non-malignant cells. These results demonstrate that SQUID-based magnetic measurements using THP-MNPs enable rapid and label-free cancer cell detection. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 2050-0904 13 2 2025 Maturity-Onset Diabetes of the Young (MODY) With HNF1B p.Glu105Lys Mutation Achieving Significant Insulin Reduction on Tirzepatide: A Case Report e70173 EN Mihiro Sue Department of Diabetology and Metabolism, NHO Okayama Medical Center Mayu Watanabe Department of Diabetology and Metabolism, NHO Okayama Medical Center Ayumi Inoue Department of Diabetology and Metabolism, NHO Okayama Medical Center Akihiro Katayama Department of Diabetology and Metabolism, NHO Okayama Medical Center Sanae Teshigawara Department of Internal Medicine, Diabetes Center, Okayama Saiseikai General Hospital Yuichi Matsushita Department of Diabetology and Metabolism, NHO Okayama Medical Center Masaya Takeda Department of Diabetology and Metabolism, NHO Okayama Medical Center Izumi Iseda Department of Diabetology and Metabolism, NHO Okayama Medical Center Jun Eguchi Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Kazuyuki Hida Department of Diabetology and Metabolism, NHO Okayama Medical Center This report describes the first case of maturity-onset diabetes in young (MODY) with HNF1B mutation started administration of the dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist, tirzepatide. A 26-year-old female with a 15-year history of diabetes mellitus was diagnosed with MODY, which is characterized by decreased insulin secretion. She was treated with tirzepatide, which significantly improved her glycemic management; insulin secretion increased the fasting serum C-peptide immunoreactivity from 0.36 to 1.09 ng/mL. The patient discontinued glimepiride, and her total daily insulin dose was reduced from 88 to 4 units. This report highlights the glucose-lowering effects of tirzepatide in a patient with MODY who has the HNF1B p.Glu105Lys mutation. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2399-3642 8 1 2025 A genome-wide association study identifies the GPM6A locus associated with age at onset in ALS 1720 EN Ryoichi Nakamura Department of Neurology, Aichi Medical University School of Medicine Genki Tohnai Division of ALS Research, Aichi Medical University School of Medicine Naoki Atsuta Department of Neurology, Aichi Medical University School of Medicine Yumi Matsuda Public Health Informatics Unit, Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine Satoru Morimoto Keio University Regenerative Medicine Research Center, Kawasaki Daisuke Ito Department of Neurology, Nagoya University Graduate School of Medicine Masahisa Katsuno Department of Neurology, Nagoya University Graduate School of Medicine Yuishin Izumi Department of Neurology, Tokushima University Graduate School of Biomedical Sciences Mitsuya Morita Division of Neurology, Department of Internal Medicine, Jichi Medical University Ikuko Iwata Department of Neurology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University Ichiro Yabe Department of Neurology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University Tomoko Nakazato Department of Neurology, Juntendo University School of Medicine Nobutaka Hattori Department of Neurology, Juntendo University School of Medicine Takehisa Hirayama Department of Neurology, Toho University Faculty of Medicine Osamu Kano Department of Neurology, Toho University Faculty of Medicine Asako Tamura Department of Neurology, Mie University Graduate School of Medicine Naoki Suzuki Department of Neurology, Tohoku University Graduate School of Medicine Masashi Aoki Department of Neurology, Tohoku University Graduate School of Medicine Kazumoto Shibuya Department of Neurology, Graduate School of Medicine, Chiba University Satoshi Kuwabara Department of Neurology, Graduate School of Medicine, Chiba University Masaya Oda Department of Neurology, Vihara Hananosato Hospital Rina Hashimoto Department of Neurology, NHO Higashinagoya National Hospital Ikuko Aiba Department of Neurology, NHO Higashinagoya National Hospital Tomohiko Ishihara Department of Neurology, Brain Research Institute, Niigata University Osamu Onodera Department of Neurology, Brain Research Institute, Niigata University Toru Yamashita Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroyuki Ishiura Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kota Bokuda Department of Neurology, Tokyo Metropolitan Neurological Hospital Toshio Shimizu Department of Neurology, Tokyo Metropolitan Neurological Hospital Yoshio Ikeda Department of Neurology, Gunma University Graduate School of Medicine Kazuko Hasegawa Division of Neurology, NHO Sagamihara National Hospital Fumiaki Tanaka Department of Neurology and Stroke Medicine, Yokohama City University Graduate School of Medicine Takanori Yokota Department of Neurology and Neurological Science, NucleoTIDE and PepTIDE Drug Discovery Center (TIDE), Institute of Science Tokyo Kazuaki Kanai Department of Neurology, Fukushima Medical University School of Medicine Yu-ichi Noto Department of Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine Ryuji Kaji Department of Neurology, Tokushima University Graduate School of Biomedical Sciences Hirohisa Watanabe Department of Neurology, Fujita Health University Tomoko Konishi Division of ALS Research, Aichi Medical University School of Medicine Mikiko Hasegawa Division of ALS Research, Aichi Medical University School of Medicine Hozuki Fukaya Division of ALS Research, Aichi Medical University School of Medicine Jun-ichi Niwa Department of Neurology, Aichi Medical University School of Medicine Manabu Doyu Department of Neurology, Aichi Medical University School of Medicine Yohei Okada Department of Neurology, Aichi Medical University School of Medicine Shiho Nakamura Keio University Regenerative Medicine Research Center, Kawasaki Fumiko Ozawa Keio University Regenerative Medicine Research Center, Kawasaki Hideyuki Okano Keio University Regenerative Medicine Research Center, Kawasaki Masahiro Nakatochi Public Health Informatics Unit, Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine Gen Sobue Division of ALS Research, Aichi Medical University School of Medicine Amyotrophic lateral sclerosis (ALS) exhibits considerable clinical variability, such as differences in age at onset (AAO). Multiple factors, including genetic factors, may underlie this variability; however, the specific determinants remain unclear. To identify genes affecting AAO, we have conducted a genome-wide association study in Japanese patients with ALS (discovery cohort: n = 1808; replication cohort: n = 207). Here, we show that the minor A allele of rs113161727 at the ADAM29-GPM6A locus is associated with a younger AAO in the discovery cohort (effect, -4.27 years; p = 4.60 × 10-8); this finding has been confirmed in the replication cohort (p = 0.0068) and meta-analysis (p = 1.08 × 10−9). Among 65 ALS patients with a SOD1 mutation, the AAO has been found to be 10.2 years younger in those with the A allele than in those without it (p = 0.002). This variant correlates with GPM6A upregulation in iPSC-derived motor neurons, suggesting GPM6A as a candidate AAO modifier. Overall, our study highlights the impact of genetic modifiers on ALS heterogeneity and provides a potential target for delaying disease onset. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1873-9601 19 4 2025 Interaction between nuclear‐translocated cellular communication network factor 2 and purine‐rich box 1 regulates the expression of fibrosis‐related genes e70051 EN Xuan Thi Nguyen Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masaharu Takigawa Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Okayama Japan Takashi Nishida Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Cellular communication network factor 2 (CCN2) with a nuclear localization signal-like peptide is known to promote fibrosis. However, translocation of CCN2 into the nucleus and its role in fibrosis remain unclear. We hypothesized that nuclear-translocated CCN2 is associated with purine-rich box 1 (PU.1), which is a transcription factor regulating the differentiation of myofibroblasts. Western blot analysis of the cytoplasmic and nuclear fractions of cell lysate and immunofluorescence analysis revealed that CCN2 was detectable in both the cytoplasm and nuclei of murine fibroblastic NIH3T3 cells. Additionally, chromatin immunoprecipitation (IP)-PCR and an electrophoretic mobility shift assay revealed that recombinant CCN2 protein bound to the regulatory region of Spi1, which encodes PU.1. Furthermore, IP-Western blot analysis showed that CCN2 interacted with PU.1. Finally, the forced expression of both Ccn2 and Spi1 significantly promoted the production of angiotensin II, and increased fibrosis-related molecules, such as Col1a1 and Acta2, at the gene and protein levels. These findings indicate that CCN2 translocated to the nucleus interacts with PU.1 and that the complex promotes the markers of myofibroblast differentiation, suggesting that CCN2 plays an important role in fibrosis via cooperation with PU.1, as a transcription co-factor. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0916-9636 48 11 2025 Efficacy and safety of esaxerenone with and without sodium–glucose cotransporter-2 inhibitor use in hypertensive patients with type 2 diabetes mellitus: a pooled analysis of five clinical studies 2924 2937 EN Hirohiko Motoki Department of Cardiovascular Medicine, Shinshu University School of Medicine Koichiro Kuwahara Department of Cardiovascular Medicine, Shinshu University School of Medicine Haruhito A. Uchida Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Kazuomi Kario Division of Cardiovascular Medicine, Department of Medicine, Jichi Medical University School of Medicine Tomohiro Katsuya Katsuya Clinic Tatsuo Shimosawa Department of Clinical Laboratory, School of Medicine, International University of Health and Welfare Kenichi Tsujita Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University Shoko Suzuki Data Intelligence Department, Daiichi Sankyo Co. Ltd. Tomohiro Suedomi Primary Medical Science Department, Daiichi Sankyo Co. Ltd. Takashi Taguchi Primary Medical Science Department, Daiichi Sankyo Co. Ltd. This pooled subanalysis of five multicenter, prospective, open-label, single-arm studies on esaxerenone aimed to evaluate the efficacy, organ-protective effects, and safety of esaxerenone in hypertensive patients with type 2 diabetes mellitus (T2DM), with and without concomitant sodium–glucose cotransporter-2 inhibitor (SGLT2i) therapy. In total, 283 and 279 patients were included in the safety (with SGLT2i, 148; without, 135) and full analysis sets (with SGLT2i; 145; without, 134), respectively. Significant changes in morning home systolic/diastolic blood pressure (SBP/DBP) from baseline to Week 12 were shown in the overall population (mean change: −11.9/−5.2 mmHg, both P < 0.001) and both SGLT2i and non-SGLT2i subgroups (−11.3/−4.8 and −12.5/−5.7 mmHg, respectively, all P < 0.001). Similar findings were observed in bedtime home and office SBP/DBP. The proportions of patients who achieved target home SBP/DBP < 135/85 mmHg were 71.2% (overall population) and 70.5% and 71.9% in the SGLT2i and non-SGLT2i subgroups, respectively. The urine albumin-to-creatinine ratio significantly improved from baseline to Week 12 in the overall population and SGLT2i subgroups (percentage change in geometric mean from baseline: −42.8%, −43.0%, and −42.6%, respectively, all P < 0.001). N-terminal pro-B-type natriuretic peptide levels improved in all groups. The incidence of serum potassium ≥5.5 mEq/L was 2.0% vs 5.2% in the SGLT2i vs non-SGLT2i subgroups. Esaxerenone demonstrated significant BP-lowering effects, and improved renal and cardiovascular parameters, regardless of SGLT2i use. Safety was consistent across groups, with the numerically lower incidence of serum potassium ≥5.5 mEq/L in the SGLT2i subgroup suggesting a potential mitigating effect of SGLT2is on the risk of hyperkalemia. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0916-9636 48 9 2025 Efficacy and safety of esaxerenone in hypertensive patients with chronic kidney disease, with or without type 2 diabetes mellitus: a pooled analysis of five clinical studies 2413 2426 EN Haruhito A. Uchida Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Hirohiko Motoki Department of Cardiovascular Medicine, Shinshu University School of Medicine Koichiro Kuwahara Department of Cardiovascular Medicine, Shinshu University School of Medicine Kazuomi Kario Division of Cardiovascular Medicine, Department of Medicine, Jichi Medical University School of Medicine Tomohiro Katsuya Katsuya Clinic Tatsuo Shimosawa Department of Clinical Laboratory, School of Medicine, International University of Health and Welfare Kenichi Tsujita Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University Shoko Suzuki Data Intelligence Department, Daiichi Sankyo Co., Ltd. Tomohiro Suedomi Primary Medical Science Department, Daiichi Sankyo Co., Ltd. Takashi Taguchi Primary Medical Science Department, Daiichi Sankyo Co., Ltd. Effective management of blood pressure (BP) and albuminuria are crucial for suppressing chronic kidney disease (CKD) progression and cardiovascular risks in hypertension. This pooled analysis evaluated the antihypertensive effects, organ-protective effects, and safety of esaxerenone in hypertensive patients with CKD by integrating five clinical studies of esaxerenone. Patients were divided based on type 2 diabetes mellitus (T2DM) status (with or without T2DM) and creatinine-based estimated glomerular filtration rate (eGFRcreat) (30 to <60 and ≥60 mL/min/1.73 m2). Significant changes in morning home BP from baseline at Week 12 were observed in the overall population (mean change −12.8/ − 5.4 mmHg), T2DM subgroups ( − 12.2/ − 4.5 and −14.5/ − 7.8 mmHg), and eGFRcreat subgroups ( − 12.5/ − 4.7 and −14.0/ − 6.9 mmHg) (all P < 0.001). Bedtime home and office BP showed similar tendencies. Urine albumin-to-creatinine ratio significantly improved from baseline at Week 12 in the overall population (mean change: −55.2%), T2DM subgroups ( − 56.5% and −52.0%), and eGFRcreat subgroups ( − 54.6% and −55.4%) (all P < 0.001). N-terminal pro-B-type natriuretic peptide levels significantly decreased in the overall population (percent change: −14.1%) and subgroup without T2DM ( − 25.3%). The incidence of serum potassium ≥5.5 mEq/L was lower in the subgroup with T2DM vs without T2DM (3.1% and 11.3%), potentially related to the use of sodium–glucose cotransporter 2 inhibitors. These findings highlight the sustained BP-lowering effect of esaxerenone throughout the day in hypertensive patients with CKD, irrespective of T2DM status, and its significant reduction in albuminuria. The data support the safety and efficacy of esaxerenone in this patient population, underscoring its potential as a valuable therapeutic option. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2045-2322 15 1 2025 Hypoglycemia and hyperinsulinemia induced by phenolic uremic toxins in CKD and DKD patients 5762 EN Yoshiyasu Tongu Tohoku University School of Medicine Tomoko Kasahara Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Yasutoshi Akiyama Laboratory of Oncology, Pharmacy Practice and Sciences, Tohoku University Graduate School of Pharmaceutical Sciences Takehiro Suzuki Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Hsin-Jung Ho Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Yotaro Matsumoto Laboratory of Oncology, Pharmacy Practice and Sciences, Tohoku University Graduate School of Pharmaceutical Sciences Ryota Kujirai Laboratory of Oncology, Pharmacy Practice and Sciences, Tohoku University Graduate School of Pharmaceutical Sciences Koichi Kikuchi Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Koji Nata Department of Medical Biochemistry, School of Pharmacy, Iwate Medical University Makoto Kanzaki Department of Biomedical Engineering, Tohoku University Kenshin Suzuki Tohoku University School of Medicine Shun Watanabe Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Chiharu Kawabe Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Yui Miyata Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Shun Itai Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Takafumi Toyohara Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Chitose Suzuki Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Tetsuhiro Tanaka Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshihisa Tomioka Laboratory of Oncology, Pharmacy Practice and Sciences, Tohoku University Graduate School of Pharmaceutical Sciences Takaaki Abe Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine Patients with end-stage renal disease have lower fasting plasma glucose and HbA1c levels, with significantly higher insulin levels. For a long time, it has been believed that this higher insulin level in renal failure is due to decreased insulin clearance caused by reduced renal function. However, here we reported that accumulation of the gut microbiota-derived uremic toxin, phenyl sulfate (PS) in the renal failure, increased insulin secretion from the pancreas by enhanced glucose-stimulated insulin secretion. Other endogenous sulfides compounds which accumulated as in the renal failure also increased glucose-stimulated insulin secretion from β-cell. With RNA-seq analyses and gene knock down, we demonstrated that insulin secretion evoked by PS was mediated by Ddah2. In addition, we also found that PS increased insulin resistance through lncRNA expression and Erk phosphorylation in the adipocytes. To confirm the relationship between PS and glucose metabolism in human, we recruited 2 clinical cohort studies (DKD and CKD) including 462 patients, and found that there was a weak negative correlation between PS and HbA1c. Because these trials did not measure fasting insulin level, we alternatively used the urinary C-peptide/creatinine ratio (UCPCR) as an indicator of insulin resistance. We found that PS may induce insulin resistance in patients with eGFR < 60 mL/min/1.73 m2. These data suggest that the accumulation of uremic toxins modulates glucose metabolism and induced insulin resistance in CKD and DKD patients. Considering HbA1c as a reflection of chronic hyperglycemia and UCPCR as a reflection of chronic hyperinsulinemia, our findings indicate that PS is negatively associated with hyperglycemia independent of CKD, and positively associated with hyperinsulinemia in DKD patients. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2168-8184 17 10 2025 Bladder Trigone as a Sensory Hub: A Narrative Review e94951 EN Takuya Sadahira Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuki Maruyama Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yosuke Mitsui Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takanori Sekito Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomofumi Watanabe 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 The bladder trigone is an anatomically and functionally distinct region within the lower urinary tract (LUT), characterized by a dense network of afferent sensory fibers, specialized urothelial interactions, and prominent mechanotransduction mechanisms. Its intricate neuroarchitecture enables precise detection of bladder filling and coordination of micturition, whereas dysregulation of these pathways contributes to lower urinary tract symptoms (LUTS), including urgency, frequency, and bladder pain. Despite its recognized clinical relevance, the structural and functional basis of trigonal sensory signaling - and its role - remain incompletely understood.<br> This review synthesizes current evidence on trigonal afferent organization, integrating data from anatomical mapping, receptor profiling, electrophysiological characterization, and translational research. Seminal anatomical observations are combined with recent advances in mechanotransduction and purinergic, peptidergic, and transient receptor potential (TRP) signaling to provide a comprehensive perspective. The trigone exhibits three principal afferent classes: (1) intraepithelial fibers penetrating umbrella cells, marked by P2X purinoceptor 3 (P2X3), transient receptor potential vanilloid 1 (TRPV1), calcitonin gene-related peptide (CGRP), and substance P (SP); (2) subepithelial plexuses surrounding microvasculature, enriched in vasoactive neuropeptides and exhibiting plastic hypertrophy in overactive bladder (OAB) and interstitial cystitis/bladder pain syndrome (IC/BPS); and (3) encapsulated corpuscular endings at the lamina propria-detrusor junction, expressing PIEZO1/2 and acid-sensing ion channels (ASICs) for rapid adaptation. In trigeminal dorsal root ganglion (DRG) neurons, high expression of PIEZO2, P2RX3, and voltage-gated sodium channel, type 1.8 (Nav1.8) was observed, revealing their role as the foundation for multisensory information processing. Functional assays highlight distinct mechanotransductive and chemosensory pathways, with aging, inflammation, and neurotrophic factors driving afferent plasticity underlying abnormal bladder sensation, such as urgency, frequency, and pain. Early clinical trials of P2X3 antagonists and intravesical TRPV1 inhibitors demonstrate promising symptomatic benefits. Collectively, evidence positions the bladder trigone as a critical sensory hub where neuronal, urothelial, and immune signals converge to regulate bladder sensation. Understanding its molecular and structural specialization may inform the development of region-specific neuromodulatory therapies targeting sensory urgency and afferent-driven bladder dysfunction. No potential conflict of interest relevant to this article was reported.

Royal Society of Chemistry (RSC) Acta Medica Okayama 1477-0520 23 43 2025 Harnessing the reactivity of captodative radicals: photocatalytic α-pyridination of glycyl derivatives through reversible radical coupling 9936 9941 EN Ken Yamazaki Division of Applied Chemistry, Okayama University Shuta Akimoto Division of Applied Chemistry, Okayama University Tomoya Miura Division of Applied Chemistry, Okayama University Captodative radicals that are highly stabilized by the presence of both electron-donating and electron-withdrawing groups exhibit unique reactivity in organic syntheses. These radicals are known to be less reactive towards radical–radical coupling reactions due to the presence of a shielding occupied molecular orbital. Herein we describe a photocatalytic synthetic strategy for the coupling of two different captodative radicals, which are generated from glycyl derivatives and 4-cyanopyridines. An aromatization is incorporated as the driving force after a reversible radical–radical coupling process. This method can be applied to a wide variety of peptides, providing pharmaceutically relevant pyridyl-functionalized products under mild reaction conditions. No potential conflict of interest relevant to this article was reported.

Royal Society of Chemistry (RSC) Acta Medica Okayama 1477-0520 23 27 2025 Fluorescence detection of DNA with a single-base mismatch by a Tm-independent peptide nucleic acid (PNA) twin probe 6557 6563 EN Koki Ishii Department of Applied Chemistry, Kindai University Hajime Shigeto Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) Shohei Yamamura Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) Yoshitane Imai Department of Applied Chemistry, Kindai University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Kindai University There is a need to develop efficient methods for detecting target nucleic acids to enable the rapid diagnosis and early treatment of diseases. We previously demonstrated that a peptide nucleic acid (PNA) twin probe, consisting of two PNAs each containing a fluorescent dye, with pyrene at one end, detects target DNA sequence-specifically through pyrene excimer emission. In this study, to advance the development of this probe system, we further investigated the fluorescence properties of the PNA twin probe P1 and P2, and found that the excimer fluorescence was significantly reduced when a mismatched base in the DNA sequence was present at the site of P1 closest to the pyrene. In other words, this probe was found to detect single-base mismatches without taking into account the thermal stability of the PNA/DNA hybrid. The detection limit of this PNA twin probe for the single-base-mismatched DNA was 2.7 nM. In the future, this probe should lead to a method to detect point mutations in endogenous nucleic acids within cells. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1660-3397 23 5 2025 Novel Anti-MRSA Peptide from Mangrove-Derived Virgibacillus chiguensis FN33 Supported by Genomics and Molecular Dynamics 209 EN Namfa Sermkaew School of Pharmacy, Walailak University Apichart Atipairin School of Pharmacy, Walailak University Phetcharat Boonruamkaew School of Pharmacy, Walailak University Sucheewin Krobthong Center of Excellence in Natural Products Chemistry (CENP), Department of Chemistry, Faculty of Science, Chulalongkorn University Chanat Aonbangkhen Center of Excellence in Natural Products Chemistry (CENP), Department of Chemistry, Faculty of Science, Chulalongkorn University Jumpei Uchiyama Department of Bacteriology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yodying Yingchutrakul National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency Nuttapon Songnaka School of Pharmacy, Walailak University Antimicrobial resistance (AMR) is a global health threat, with methicillin-resistant Staphylococcus aureus (MRSA) being one of the major resistant pathogens. This study reports the isolation of a novel mangrove-derived bacterium, Virgibacillus chiguensis FN33, as identified through genome analysis and the discovery of a new anionic antimicrobial peptide (AMP) exhibiting anti-MRSA activity. The AMP was composed of 23 amino acids, which were elucidated as NH3-Glu-Gly-Gly-Cys-Gly-Val-Asp-Thr-Trp-Gly-Cys-Leu-Thr-Pro-Cys-His-Cys-Asp-Leu-Phe-Cys-Thr-Thr-COOH. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for MRSA were 8 µg/mL and 16 µg/mL, respectively. FN33 AMP induced cell membrane permeabilization, suggesting a membrane-disrupting mechanism. The AMP remained stable at 30–40 °C but lost activity at higher temperatures and following exposure to proteases, surfactants, and extreme pH. All-atom molecular dynamics simulations showed that the AMP adopts a β-sheet structure upon membrane interaction. These findings suggest that Virgibacillus chiguensis FN33 is a promising source of novel antibacterial agents against MRSA, supporting alternative strategies for drug-resistant infections. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2079-6382 13 9 2024 Unveiling a New Antimicrobial Peptide with Efficacy against P. aeruginosa and K. pneumoniae from Mangrove-Derived Paenibacillus thiaminolyticus NNS5-6 and Genomic Analysis 846 EN Namfa Sermkaew School of Pharmacy, Walailak University Apichart Atipairin School of Pharmacy, Walailak University Sucheewin Krobthong Center of Excellence in Natural Products Chemistry (CENP), Department of Chemistry, Faculty of Science, Chulalongkorn University Chanat Aonbangkhen Center of Excellence in Natural Products Chemistry (CENP), Department of Chemistry, Faculty of Science, Chulalongkorn University Yodying Yingchutrakul National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency Jumpei Uchiyama Department of Bacteriology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nuttapon Songnaka School of Pharmacy, Walailak University This study focused on the discovery of the antimicrobial peptide (AMP) derived from mangrove bacteria. The most promising isolate, NNS5-6, showed the closest taxonomic relation to Paenibacillus thiaminolyticus, with the highest similarity of 74.9%. The AMP produced by Paenibacillus thiaminolyticus NNS5-6 exhibited antibacterial activity against various Gram-negative pathogens, especially Pseudomonas aeruginosa and Klebsiella pneumoniae. The peptide sequence consisted of 13 amino acids and was elucidated as Val-Lys-Gly-Asp-Gly-Gly-Pro-Gly-Thr-Val-Tyr-Thr-Met. The AMP mainly exhibited random coil and antiparallel beta-sheet structures. The stability study indicated that this AMP was tolerant of various conditions, including proteolytic enzymes, pH (1.2–14), surfactants, and temperatures up to 40 °C for 12 h. The AMP demonstrated 4 µg/mL of MIC and 4–8 µg/mL of MBC against both pathogens. Time-kill kinetics showed that the AMP acted in a time- and concentration-dependent manner. A cell permeability assay and scanning electron microscopy revealed that the AMP exerted the mode of action by disrupting bacterial membranes. Additionally, nineteen biosynthetic gene clusters of secondary metabolites were identified in the genome. NNS5-6 was susceptible to various commonly used antibiotics supporting the primary safety requirement. The findings of this research could pave the way for new therapeutic approaches in combating antibiotic-resistant pathogens. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 2666-3341 6 S1 2025 Basic biology is not just “for the birds”: how avian studies have informed us about vertebrate reproduction 7 12 EN George E. Bentley Department of Integrative Biology and Helen Wills Neuroscience Institute, University of California at Berkeley Sayaka Aizawa Graduate School of Natural Science and Technology, Okayama University Avian reproductive physiology has been studied for centuries, largely because of the importance of birds as food animals. It is likely that the ubiquity and ease of access to domesticated chickens led to them being used in some of the first experiments on transplantation of endocrine structures―in this case, the testes. Since then, study of seasonal changes in reproductive physiology (photoperiodism) in different orders of bird species has led to advances in the understanding of endocrine regulation of reproductive physiology and behavior. These include mechanisms of adult neuroplasticity, sexual selection, sperm competition, stress physiology, and circadian physiology. Here, we focus mainly on the discovery in birds of a neuropeptide named gonadotropin-inhibitory hormone that mostly has inhibitory effects on reproduction. This hormone has since been shown to exist in all mammals studied to date, including humans (it is known as RFamide-related peptide in mammals). We discuss the history and implications of avian studies on gonadotropin-inhibitory hormone/RFamide-related peptide for human reproductive biology. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1422-0067 26 16 2025 Synergistic Antimicrobial Activity of BrSPR20-P1 Peptide and Silver Nanoparticles Against Pathogenic Bacteria 7832 EN Thanyamai Thongin School of Pharmacy, Walailak University Somchai Sawatdee School of Pharmacy, Walailak University Nuttapon Songnaka School of Pharmacy, Walailak University Jumpei Uchiyama Department of Bacteriology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Theanchai Wiwasuku School of Science, Walailak University Teerapol Srichana Drug Delivery System Excellence Center and Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University Titpawan Nakpheng Drug Delivery System Excellence Center and Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University Apichart Atipairin School of Pharmacy, Walailak University Bacterial infection is a cause of life-threatening diseases. The emergence of antimicrobial-resistant bacteria exacerbates this situation, highlighting the need for the discovery of new antimicrobial agents. Our previous study identified a novel antimicrobial peptide, BrSPR20-P1 (P1), which showed potential activity against MRSA. Additionally, silver nanoparticles (AgNPs) exhibit broad-spectrum antibacterial activity, capable of killing multidrug-resistant bacteria. The combination of antimicrobial agents presents a novel strategy for combating these pathogens. This study aimed to evaluate the antibacterial activity of the combination of P1 and AgNPs. It revealed that the combinations showed synergy. The P1 and AgNP mixture at a concentration of 1 and 8 µg/mL (1:8) doubled the activity against S. aureus and MRSA, while that combination of 64 and 64 µg/mL (64:64) exhibited broad-spectrum activity, expanding to E. coli with a 32-fold increase. These combinations exhibited a bactericidal effect, showing the rapid killing of tested bacteria at 10× MIC, with killing rates during the first 3 h ranging from 4.04 ± 0.01 to 4.31 ± 0.03 h−1. The P1 and AgNP mixtures caused a low risk of antibacterial resistance up to 30 passages. It was demonstrated that the synergistic activity of P1 and AgNPs occurred through the disruption of cell walls and membranes, leakage of intracellular materials, and cell lysis. Additionally, the mixtures appeared to interact with bacterial genomic DNA, as indicated by a gel retardation assay. These activities of the combinations were concentration-dependent. The 1:8 µg/mL mixture caused low hemolysis and cytotoxicity and did not impede the wound healing process. In contrast, although the 64:64 µg/mL mixture showed excellent antibacterial efficacy, it was toxic to erythrocytes and mammalian cells. It implies that dose optimization is required to balance its efficacy and toxicity. Therefore, the P1 and AgNP combinations exhibit synergistic antimicrobial activity and have the potential to resolve bacterial infections. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2059-0105 10 1 2025 Oncolytic virus-mediated p53 activation boosts the antitumor immunity of a p53-transduced dendritic cell vaccine 158 EN Motohiko Yamada Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Tazawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kanto Suemori Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Naohiro Okada Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshinori Kajiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryohei Shoji Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuo Nagai Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroaki Inoue Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Naoyuki Hashimoto Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nobuhiko Kanaya Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Kikuchi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinji Kuroda Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroyuki Michiue Neutron Therapy Research Center, Okayama University Hospital Yasuo Urata Oncolys BioPharma, Inc Shunsuke Kagawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiyoshi Fujiwara Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Dendritic cells (DCs) transduced with replication-deficient, wild-type human p53-expressing adenovirus Ad-p53 (Ad-p53 DCs) induce p53-targeting cytotoxic T lymphocytes (CTLs). However, the antitumor efficacy of Ad-p53 DCs is diminished by weak p53 immunogenicity in tumor cells and poor immune responses. We developed a p53-armed oncolytic adenovirus, OBP-702, to induce tumor-specific p53 expression and antitumor immune response, suggesting a role for OBP-702 in enhancing the antitumor efficacy of Ad-p53 DCs. The combined effect of Ad-p53 DCs and OBP-702 was investigated using murine colon cancer (CC) tumor models. Ad-p53 DCs were obtained by stimulating bone marrow-derived cells with granulocyte-macrophage colony-stimulating factor, interleukin-4, and Ad-p53. Subcutaneous tumor models of CT26 (p53 wild-type) and MC38 (p53 mutant-type) murine CC cell lines were used to evaluate the therapeutic potential of combination therapy in the terms of tumor growth, abscopal effect, antitumor immune response, and presentation of p53 peptides in tumor cells. Combination therapy with Ad-p53 DCs and OBP-702 significantly suppressed the growth of p53-intact CT26 tumors at treated and untreated sites by inducing tumor-infiltration of CD8+ CTLs and CD11c+ DCs. OBP-702-infected tumor cells presented human p53 epitopes in the context of major histocompatibility complex molecules, which were recognized by CTLs induced by Ad-p53 DCs. Combination therapy significantly suppressed the growth of p53-mutant MC38 tumors by activating the antitumor immune response. Our results suggest that OBP-702-mediated presentation of p53 epitopes on tumor cells enhances the antitumor efficacy of Ad-p53 DCs against murine CC tumors by attracting p53-targeting CTLs. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 2772-963X 4 2 2025 Simplified Outcome Prediction in Patients Undergoing Transcatheter Tricuspid Valve Intervention by Survival Tree-Based Modelling 101575 EN Vera Fortmeier Department of General and Interventional Cardiology, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum Mark Lachmann First Department of Medicine, Klinikum rechts der Isar, Technical University of Munich Lukas Stolz DZHK (German Center for Cardiovascular Research), Partner Site Munich Heart Alliance Jennifer von Stein Department of Cardiology, Heart Center, University of Cologne Karl-Philipp Rommel Department of Cardiology, Heart Center Leipzig, University of Leipzig Mohammad Kassar Department of Cardiology, Inselspital Bern, Bern University Hospital Muhammed Gerçek Department of General and Interventional Cardiology, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum Anne R. Schöber Department of Cardiology, Heart Center Leipzig, University of Leipzig Thomas J. Stocker DZHK (German Center for Cardiovascular Research), Partner Site Munich Heart Alliance Hazem Omran Department of General and Interventional Cardiology, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum Michelle Fett Department of General and Interventional Cardiology, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum Jule Tervooren Department of General and Interventional Cardiology, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum Maria I. Körber Department of Cardiology, Heart Center, University of Cologne Amelie Hesse First Department of Medicine, Klinikum rechts der Isar, Technical University of Munich Gerhard Harmsen Department of Physics, University of Johannesburg Kai Peter Friedrichs Department of General and Interventional Cardiology, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum Shinsuke Yuasa Department of Cardiovascular Medicine, Okayama University Tanja K. Rudolph Department of General and Interventional Cardiology, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum Michael Joner DZHK (German Center for Cardiovascular Research), Partner Site Munich Heart Alliance Roman Pfister Department of Cardiology, Heart Center, University of Cologne Stephan Baldus Department of Cardiology, Heart Center, University of Cologne Karl-Ludwig Laugwitz First Department of Medicine, Klinikum rechts der Isar, Technical University of Munich Stephan Windecker Department of Cardiology, Inselspital Bern, Bern University Hospital Fabien Praz Department of Cardiology, Inselspital Bern, Bern University Hospital Philipp Lurz Department of Cardiology, Heart Center Leipzig, University of Leipzig Jörg Hausleiter DZHK (German Center for Cardiovascular Research), Partner Site Munich Heart Alliance Volker Rudolph Department of General and Interventional Cardiology, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum Background Patients with severe tricuspid regurgitation (TR) typically present with heterogeneity in the extent of cardiac dysfunction and extra-cardiac comorbidities, which play a decisive role for survival after transcatheter tricuspid valve intervention (TTVI).<br> Objectives This aim of this study was to create a survival tree-based model to determine the cardiac and extra-cardiac features associated with 2-year survival after TTVI.<br> Methods The study included 918 patients (derivation set, n = 631; validation set, n = 287) undergoing TTVI for severe TR. Supervised machine learning-derived survival tree-based modelling was applied to preprocedural clinical, laboratory, echocardiographic, and hemodynamic data.<br> Results Following univariate regression analysis to pre-select candidate variables for 2-year mortality prediction, a survival tree-based model was constructed using 4 key parameters. Three distinct cluster-related risk categories were identified, which differed significantly in survival after TTVI. Patients from the low-risk category (n = 261) were defined by mean pulmonary artery pressure ≤28 mm Hg and N-terminal pro–B-type natriuretic peptide ≤2,728 pg/mL, and they exhibited a 2-year survival rate of 85.5%. Patients from the high-risk category (n = 190) were defined by mean pulmonary artery pressure >28 mm Hg, right atrial area >32.5 cm2, and estimated glomerular filtration rate ≤51 mL/min, and they showed a significantly worse 2-year survival of only 52.6% (HR for 2-year mortality: 4.3, P < 0.001). Net re-classification improvement analysis demonstrated that this model was comparable to the TRI-Score and outperformed the EuroScore II in identifying high-risk patients. The prognostic value of risk phenotypes was confirmed by external validation.<br> Conclusions This simple survival tree-based model effectively stratifies patients with severe TR into distinct risk categories, demonstrating significant differences in 2-year survival after TTVI. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2041-1723 15 1 2024 A high-protein diet-responsive gut hormone regulates behavioral and metabolic optimization in Drosophila melanogaster 10819 EN Yuto Yoshinari Metabolic Regulation and Genetics, Institute for Molecular and Cellular Regulation, Gunma University Takashi Nishimura Metabolic Regulation and Genetics, Institute for Molecular and Cellular Regulation, Gunma University Taishi Yoshii Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Shu Kondo Department of Biological Science and Technology, Faculty of Advanced Engineering, Tokyo University of Science Hiromu Tanimoto Graduate School of Life Sciences, Tohoku University Tomoe Kobayashi Division of Molecular Genetics, Shigei Medical Research Institute Makoto Matsuyama Division of Molecular Genetics, Shigei Medical Research Institute Ryusuke Niwa Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba Protein is essential for all living organisms; however, excessive protein intake can have adverse effects, such as hyperammonemia. Although mechanisms responding to protein deficiency are well-studied, there is a significant gap in our understanding of how organisms adaptively suppress excessive protein intake. In the present study, utilizing the fruit fly, Drosophila melanogaster, we discover that the peptide hormone CCHamide1 (CCHa1), secreted by enteroendocrine cells in response to a high-protein diet (HPD), is vital for suppressing overconsumption of protein. Gut-derived CCHa1 is received by a small subset of enteric neurons that produce short neuropeptide F, thereby modulating protein-specific satiety. Importantly, impairment of the CCHa1-mediated gut-enteric neuronal axis results in ammonia accumulation and a shortened lifespan under HPD conditions. Collectively, our findings unravel the crosstalk of gut hormone and neuronal pathways that orchestrate physiological responses to prevent and adapt to dietary protein overload. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 2772-4174 13 2025 Photochemical internalization of mRNA using a photosensitizer and nucleic acid carriers 100242 EN Hayaki Maemoto Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ryohei Suzaki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Keiji Itaka Department of Biofunction Research, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University mRNA has great potential for therapeutic applications because it can encode a variety of proteins and antigens, in addition to advantages over DNA in terms of gene expression without genomic integration, nuclear localization, or transcription. However, therapeutic applications of mRNA require safe and effective delivery into target cells. Therefore, we aimed to investigate photochemical internalization (PCI) as a promising strategy for delivering mRNA to target cells. In this strategy, mRNA is taken up into cells by endocytosis, accumulates in endosomes, and is released in a light-dependent manner from the endosomes using an endosome-accumulating photosensitizer, aluminum phthalocyanine disulfonate (AlPcS2a), in combination with nucleic acid carrier molecules. We compared the efficacy of various nucleic acid carriers, including branched polyethyleneimine (bPEI) and poly{N'-[N-(2-aminoethyl)-2-aminoethyl] aspartamide} (PAsp(DET)) under the same conditions for PCI-based mRNA delivery. Our results indicated that bPEI and PAsp(DET) at low N/P ratios exhibited efficient light-enhancement of mRNA expression by PCI with AlPcS2a. Notably, bPEI exhibited the highest light-dependent mRNA delivery among the carriers evaluated (including cationic polymers, cationic peptides, and lipids), whereas PAsp(DET) showed promise for clinical use because of its lower toxicity compared with bPEI. This PCI strategy allows effective cytosolic mRNA delivery at low N/P ratios, thereby reducing cationic carrier molecule-induced cytotoxicity. This method allows spatiotemporal control of protein expression and holds potential for novel light-dependent mRNA therapies. Overall, this study provided valuable insights into optimizing mRNA delivery systems for therapeutic applications. No potential conflict of interest relevant to this article was reported.

Japanese Circulation Society Acta Medica Okayama 1346-9843 89 7 2025 Hemodynamic Changes After Wire Frame Occluders vs. Metal Mesh Devices for Atrial Septal Defect 930 938 EN Mitsutaka Nakashima Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoichi Takaya Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaro Ejiri Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takashi Miki Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Nakayama Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koji Nakagawa Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Teiji Akagi Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazufumi Nakamura Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinsuke Yuasa Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: Transcatheter atrial septal defect (ASD) closure is the first treatment option for secundum ASD, but parameters for optimal device selection have not been established. We compared outcomes between occluders with a wire frame and metal mesh devices.<br> Methods and Results: This study included secundum ASD patients implanted with a wire frame occluder (GORE®CARDIOFORM ASD occluder [GCA]; W.L. Gore & Associates) or metal mesh devices (Amplatzer septal occluder device [Abbott] and Occlutech Figulla Flex II device [Occlutech]). The presence of residual shunt and B-type natriuretic peptide (BNP) levels after implantation were compared. Of the 970 patients with either GCA (n=48) or a metal mesh device (n=922; control), 42 patients from each group were analyzed after propensity score matching. The prevalence of residual shunt was significantly lower in the GCA group 1 day and 1 month after implantation (P<0.001 and P=0.017, respectively), whereas there was no significant difference between the 2 groups 6 months later (P=0.088). BNP levels at 1 month were significantly higher in the GCA group (ratio of change 1.36; 95% confidence interval [CI] 1.01–1.83), but did not differ significantly between the 2 groups at 6 months (ratio of change 1.04; 95% CI 0.65–1.65).<br> Conclusions: Patients implanted with a wire frame occluder had a lower prevalence of residual shunt and a greater increase in BNP levels in the early period after implantation. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2025 Preventive effects of vasohibin-2-targeting peptide vaccine for diabetic nephropathy EN Yuri NAKASHIMA Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 17 9 2025 Impacts of Dental Follicle Cells and Periodontal Ligament Cells on the Bone Invasion of Well-Differentiated Oral Squamous Cell Carcinoma 1559 EN Anqi Chang Department of Oral Pathology and Medicine, Okayama University Kiyofumi Takabatake Department of Oral Pathology and Medicine, Okayama University Tianyan Piao Department of Oral Pathology and Medicine, Okayama University Takuma Arashima Department of Oral Pathology and Medicine, Okayama University Hotaka Kawai Department of Oral Pathology and Medicine, Okayama University Htoo Shwe Eain Department of Oral Pathology and Medicine, Okayama University Yamin Soe Department of Oral Pathology and Medicine, Okayama University Zin Zin Min Department of Oral Pathology and Medicine, Okayama University Keisuke Nakano Department of Oral Pathology and Medicine, Okayama University Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Okayama University Background: Oral squamous cell carcinoma (OSCC) frequently invades the jawbone, leading to diagnostic and therapeutic challenges. While tumor-bone interactions have been studied, the specific roles of dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) in OSCC-associated bone resorption remain unclear. This study aimed to compare the effects of DFCs and PDLCs on OSCC-induced bone invasion and elucidate the underlying mechanisms. Methods: Primary human DFCs and PDLCs were isolated from extracted third molars and characterized by Giemsa and immunofluorescence staining. An in vitro co-culture system and an in vivo xenograft mouse model were established using the HSC-2 OSCC cell line. Tumor invasion and osteoclast activation were assessed by hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase (TRAP) staining. Immunohistochemical analysis was performed to evaluate the expression of receptor activator of NF-kappa B ligand (RANKL) and parathyroid hormone-related peptide (PTHrP). Results: DFCs significantly enhanced OSCC-induced bone resorption by promoting osteoclastogenesis and upregulating RANKL and PTHrP expression. In contrast, PDLCs suppressed RANKL expression and partially modulated PTHrP levels, thereby reducing osteoclast activity. Conclusions: DFCs and PDLCs exert opposite regulatory effects on OSCC-associated bone destruction. These findings underscore the importance of stromal heterogeneity and highlight the therapeutic potential of targeting specific stromal-tumor interactions to mitigate bone-invasive OSCC. No potential conflict of interest relevant to this article was reported.

American Chemical Society Acta Medica Okayama 2470-1343 2025 High-Resolution HPLC for Separating Peptide-Oligonucleotide Conjugates EN Miyako Naganuma Division of Organic Chemistry, National Institute of Health Sciences Genichiro Tsuji Division of Organic Chemistry, National Institute of Health Sciences Misato Amiya YMC CO., LTD. Reira Hirai YMC CO., LTD. Yuki Higuchi YMC CO., LTD. Naoko Hata YMC CO., LTD. Saoko Nozawa YMC CO., LTD. Daishi Watanabe Division of Organic Chemistry, National Institute of Health Sciences Taeko Nakajima YMC CO., LTD. Yosuke Demizu Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Division of Pharmaceutical Science, Okayama University Peptide-oligonucleotide conjugates (POCs) are chimeric molecules that combine the specificity of oligonucleotides with the functionality of peptides, improving the delivery and therapeutic potential of nucleic acid-based drugs. However, the analysis of POCs, particularly those containing arginine-rich sequences, poses major challenges because of aggregation caused by electrostatic interactions. In this study, we developed an optimized high-performance liquid chromatography (HPLC) method for analyzing POCs. Using a conjugate of DNA and nona-arginine as a model compound, we systematically investigated the effects of various analytical parameters, including column type, column temperature, mobile-phase composition, and pH. A column packed with C18 resin with wide pores combined with butylammonium acetate as the ion-pairing reagent and an optimal column temperature of 80 degrees C provided superior peak resolution and sensitivity. The optimized conditions gave clear separation of POCs from unlinked oligonucleotides and enabled the detection of nucleic acid fragments lacking an alkyne moiety as a linkage part, which is critical for quality control. Our HPLC method is robust and reproducible and substantially reduces the complexity, time, and cost associated with the POC analysis. The method may improve the efficiency of quality control in the production of POCs, thereby supporting their development as promising therapeutic agents for clinical applications. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2041-1723 16 1 2025 A mini-hairpin shaped nascent peptide blocks translation termination by a distinct mechanism 2323 EN Yushin Ando Department of Biological Sciences, Graduate School of Science, The University of Tokyo Akinao Kobo School of Life Science and Technology, Institute of Science Tokyo Tatsuya Niwa School of Life Science and Technology, Institute of Science Tokyo Ayako Yamakawa School of Life Science and Technology, Institute of Science Tokyo Suzuna Konoma School of Life Science and Technology, Institute of Science Tokyo Yuki Kobayashi School of Life Science and Technology, Institute of Science Tokyo Osamu Nureki Department of Biological Sciences, Graduate School of Science, The University of Tokyo Hideki Taguchi School of Life Science and Technology, Institute of Science Tokyo Yuzuru Itoh Department of Biological Sciences, Graduate School of Science, The University of Tokyo Yuhei Chadani Faculty of Environmental, Life, Natural Science and Technology, Okayama University Protein synthesis by ribosomes produces functional proteins but also serves diverse regulatory functions, which depend on the coding amino acid sequences. Certain nascent peptides interact with the ribosome exit tunnel to arrest translation and modulate themselves or the expression of downstream genes. However, a comprehensive understanding of the mechanisms of such ribosome stalling and its regulation remains elusive. In this study, we systematically screen for unidentified ribosome arrest peptides through phenotypic evaluation, proteomics, and mass spectrometry analyses, leading to the discovery of the arrest peptides PepNL and NanCL in E. coli. Our cryo-EM study on PepNL reveals a distinct arrest mechanism, in which the N-terminus of PepNL folds back towards the tunnel entrance to prevent the catalytic GGQ motif of the release factor from accessing the peptidyl transferase center, causing translation arrest at the UGA stop codon. Furthermore, unlike sensory arrest peptides that require an arrest inducer, PepNL uses tryptophan as an arrest inhibitor, where Trp-tRNATrp reads through the stop codon. Our findings illuminate the mechanism and regulatory framework of nascent peptide-induced translation arrest, paving the way for exploring regulatory nascent peptides. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0026-265X 210 2025 A microfluidic paper-based analytical device that uses gelatin film to assay protease activity via time readout 112952 EN Jianchao Ren Department of Chemistry, Okayama University Kaewta Danchana Department of Chemistry, Okayama University Takashi Kaneta Department of Chemistry, Okayama University Food processing, detergents, and pharmaceuticals frequently employ proteases, which are enzymes that break the chemical bonds of both proteins and peptides. In this work, we developed a microfluidic paper-based analytical device (µPAD) for protease activity assays via time readout. To accomplish this, we folded the µPAD to form layers, then inserted a water-insoluble gelatin film between the layers of paper to form the device. Lamination helps to maintain the gelatin film between the introduction zone, which is the upper layer, and the detection channel, which is the lower layer. Proteases decompose the gelatin film when it enters the introduction zone, which then allows it to flow into the detection channel. The protease activity in the sample solution determines the time required to dissolve the gelatin film, which leads to a linear relationship between the logarithm of the protease concentration and the time required to flow the solution a specific distance on the detection channel. The µPAD was used to measure proteases in concentrations that ranged from 0.25 to 1 mg L−1 for bromelain, 2.5 to 10 mg L−1 for papain, and 1 to 8 mg L−1 for trypsin. The limits of quantification for bromelain, papain, and trypsin were 0.41, 2.7, and 9.2 mg mL−1, respectively. The relative standard deviations for bromelain were smaller than 2 % for concentrations ranging from 0.5 to 1.0 mg L−1. We compared the µPAD to a commercially available protease activity assay kit, which relies on quenching fluorescein isothiocyanate-labeled casein. Both methods demonstrated the same order of activity: bromelain > papain > trypsin. The proposed device allowed the assay of bromelain in both pineapple pulp and juice, which were stored at room temperature. When first using the proposed device, the bromelain in the pulp gradually lost its activity, while the activity of the bromelain in the juice showed no significant change for five days. The µPAD requires no analytical instruments for quality control and monitoring of the protease activity in food. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2077-0383 14 3 2025 Interrelationships Between Plasma Levels of Brain Natriuretic Peptide and Prolonged Symptoms Due to Long COVID 817 EN Yohei Masuda Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuki Otsuka Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuki Tokumasu Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroyuki Honda Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasue Sakurada Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yui Matsuda Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuhiro Nakano Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryosuke Takase Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Daisuke Omura Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toru Hasegawa Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keigo Ueda Department of General Medicine, 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 Objectives: Evidence for the usefulness of biomarkers that aid in diagnosis, assessment of severity, and prediction of prognosis in patients with long COVID is limited. The aim of this study was to clarify the characteristics of brain natriuretic peptide (BNP) in long COVID. Methods: We conducted a retrospective observational study of patients who visited the COVID-19 aftercare outpatient clinic at Okayama University Hospital from February 2021 to April 2024. Results: A total of 428 patients were enrolled in this study, and the patients were divided into a group with normal BNP (n = 314, <= 18.4 pg/mL) and a group with increased BNP (n = 114, >18.4 pg/mL). The long COVID group with increased BNP had a higher proportion of females (44.3% vs. 73.7%, p < 0.01) and an older median age (38 vs. 51 years, p < 0.01). Fatigue and brain fog were commonly manifested in both groups, while dyspnea was a more frequent complaint in the group with increased BNP. Various symptoms including fatigue, palpitations, and taste and/or olfactory disorders were associated with elevated BNP (23 to 24 pg/mL). Memory impairment was also linked to higher BNP (OR: 2.36, p = 0.05). In long COVID patients, plasma BNP elevation appears to be more pronounced in females and is often related to cardiogenic factors, in which inflammatory responses are also involved. Conclusions: Plasma BNP measurement may be useful for evaluating the severity of long COVID, especially in female patients and those with respiratory symptoms and/or memory impairment. No potential conflict of interest relevant to this article was reported.

American Physiological Society Acta Medica Okayama 1931-857X 326 6 2024 Preventive effects of vasohibin-2-targeting peptide vaccine for diabetic nephropathy F1054 F1065 EN Yuri Nakashima Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Katsuyuki Tanabe Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Tomoyo Mifune Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Takato Nakadoi Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hiroki Hayashi Department of Health Development and Medicine, Osaka University Graduate School of Medicine Hironori Nakagami Department of Health Development and Medicine, Osaka University Graduate School of Medicine Yasufumi Sato New Industry Creation Hatchery Center, Tohoku University Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Diabetic nephropathy remains the leading cause of end-stage kidney disease in many countries, and additional therapeutic targets are needed to prevent its development and progression. Some angiogenic factors are involved in the pathogenesis of diabetic nephropathy. Vasohibin-2 (VASH2) is a novel proangiogenic factor, and our previous study showed that glomerular damage is inhibited in diabetic Vash2 homozygous knockout mice. Therefore, we established a VASH2-targeting peptide vaccine as a tool for anti-VASH2 therapy in diabetic nephropathy. In this study, the preventive effects of the VASH2-targeting peptide vaccine against glomerular injury were examined in a streptozotocin (STZ)-induced diabetic mouse model. The mice were subcutaneously injected with the vaccine at two doses 2 wk apart and then intraperitoneally injected with 50 mg/kg STZ for 5 consecutive days. Glomerular injury was evaluated 20 wk after the first vaccination. Treatment with the VASH2-targeting peptide vaccine successfully induced circulating anti-VASH2 antibody without inflammation in major organs. Although the vaccination did not affect blood glucose levels, it significantly prevented hyperglycemia-induced increases in urinary albumin excretion and glomerular volume. The vaccination did not affect increased VASH2 expression but significantly inhibited renal angiopoietin-2 (Angpt2) expression in the diabetic mice. Furthermore, it significantly prevented glomerular macrophage infiltration. The preventive effects of vaccination on glomerular injury were also confirmed in db/db mice. Taken together, the results of this study suggest that the VASH2-targeting peptide vaccine may prevent diabetic glomerular injury in mice by inhibiting Angpt2-mediated microinflammation. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 1664-2392 15 2024 HOMA-beta independently predicts survival in patients with advanced cancer on treatment with immune checkpoint inhibitors 1439705 EN Mayu Watanabe Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jun Eguchi Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Atsushi Takamoto Department of Urology, Fukuyama City Hospital Hiromitsu Kanzaki Department of Internal Medicine, Tsuyama Chuo Hospital Yohei Noda Department of Urology, Fukuyama City Hospital Syunsuke Kagawa Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: Although immune checkpoint inhibitors (ICIs) are effective cancer drugs, ICI-induced diabetes is a rare but a life-threatening adverse event for patients. The deleterious action of ICI on pancreatic beta-cell function is a concern. However, the influence of ICI on insulin synthesis and secretion in patients with cancer without diabetes remains unknown. <br> Methods: This study included 87 patients diagnosed with advanced cancer. Glucose metabolism markers (HbA1c, HOMA-IR) and indicators of insulin secretory capacity (HOMA-beta, C-peptide) were prospectively evaluated in patients with ICI-treated cancers to determine their association with cancer prognosis. <br> Results: Patients with overall survival (OS) >= 7 months had substantially higher HOMA-beta levels at baseline (p=0.008) and 1 month after ICI administration (p=0.006) compared to those with OS <7 months. The median OS was significantly longer in patients with HOMA-beta >= 64.24 (13 months, 95%CI: 5.849-20.151, 37 events) than in those with HOMA-beta < 64.24 (5 months, 95%CI: 3.280-6.720, 50 events) (p=0.013). Further, the median progression-free survival (PFS) was significantly longer in patients with HOMA-beta >= 66.43 (4 months, 95%CI: 3.073-4.927, 33 events) than in those with HOMA-beta < 66.43 (2 months, 95%CI: 1.410-2.590, 54 events) (p=0.025). Additionally, multivariable logistic regression analysis revealed that a HOMA-beta value >= 64.24 independently predicted longer OS in ICI-treated patients. <br> Conclusions: Pre-ICI HOMA-beta level is linked to longer OS in ICI-treated patients. This connection is significant and shows that insulin secretory capacity may predict ICI efficacy. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2296-2565 12 2024 Efficacy of extracting and preventively intervening late-stage older adults who are at high risk for spending high medical costs by using the health check-up system in Japan: a pilot study 1434800 EN Kana Kazawa Faculty of Health Sciences, Okayama University Madoka Kawai Division of Nursing Science, Graduate School of Biomedical and Health Sciences, Hiroshima University Michiko Moriyama Division of Nursing Science, Graduate School of Biomedical and Health Sciences, Hiroshima University Objectives: In Japan, the seven diseases (femur fracture, cerebral infarction, chronic renal failure, heart failure, dementia, pneumonia, and chronic obstructive pulmonary disease) are the top causes of inpatient medical costs among the late-stage older adults aged 75 years and over. This pilot study was conducted with the following two objectives; (1) to examine the proportion of risks of onset and severity of seven diseases among the late-stage older adults, and (2) to examine the efficacy of interventions focusing on the prevention of unplanned hospitalization. <br> Methods: Participants were 45,233 older adults aged 75 and over living in Kure City, Japan. In addition to the government-mandated health checkup items, the Intervention group underwent additional risk screening tests included questionnaires, physical examinations, blood tests, and educational guidance by nurses. The efficacy of the intervention was examined whether there were differences in the number of hospitalizations, the use of emergency and critical care, and the incidence of hemodialysis induction between the Intervention and control groups (Usual Health Checkup group and No Health Checkup group) for the 2 years. <br> Results: There were 485 participants in the Intervention group, 1,067 in the Usual Health Checkup group, and 43,712 in the No Health Checkup group. As the risks of seven diseases in the Intervention group, the largest proportion of deviations occurred for systolic blood pressure (63.3%), estimated salt intake (60.3%), and low-density lipoprotein cholesterol (51.5%). Estimated glomerular filtration rate deviated in 41.0%, N-terminal pro b-type natriuretic peptide in 37.9%. 7.5% scored <2 points on the Mini-Cog (c), and 9.1% performed the Timed Up and Go test in >12 s. The incidence of hospitalization due to any of the seven diseases was significantly higher in the No Health Checkup group (p < 0.001). There were no differences among the three groups in the use of emergency and critical care or the introduction of hemodialysis. <br> Conclusion: This study revealed that additional health checkup tests and intervention methods could be prevented hospitalization among the adults of 75 years and older. It is necessary to make health checkups and follow-ups more accessible those are already available within the existing health system in Japan. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2024 Development of Peptide Nanomicelle for siRNA Delivery into Cancer Cells EN TAUFIK FATWA NUR HAKIM Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2024 Application of novel tumor-homing peptide-modified magnetic nanoparticles for magnetic hyperthermia and tumor cell detection EN SHENGLI ZHOU Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2024 Comparative research on antibacterial peptides, bacteriocins, produced by two strains of lactic acid bacteria, Leuconostoc mesenteroides subsp. mesenteroides 406 and 213M0, isolated from Mongolian traditional fermented milk, airag EN HASIQIMUGE Graduate School of Environmental and Life Science, Okayama University No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1083-351X 300 6 2024 Nonspecific N-terminal tetrapeptide insertions disrupt the translation arrest induced by ribosome-arresting peptide sequences 107360 EN Akinao Kobo School of Life Science and Technology, Tokyo Institute of Technology Hideki Taguchi School of Life Science and Technology, Tokyo Institute of Technology Yuhei Chadani Faculty of Environmental, Life, Natural Science and Technology, Okayama University The nascent polypeptide chains passing through the ribosome tunnel not only serve as an intermediate of protein synthesis but also, in some cases, act as dynamic genetic information, controlling translation through interaction with the ribosome. One notable example is Escherichia coli SecM, in which translation of the ribosome arresting peptide (RAP) sequence in SecM leads to robust elongation arrest. Translation regulations, including the SecM-induced translation arrest, play regulatory roles such as gene expression control. Recent investigations have indicated that the insertion of a peptide sequence, SKIK (or MSKIK), into the adjacent N-terminus of the RAP sequence of SecM behaves as an "arrest canceler". As the study did not provide a direct assessment of the strength of translation arrest, we conducted detailed biochemical analyses. The results revealed that the effect of SKIK insertion on weakening SecM-induced translation arrest was not specific to the SKIK sequence, that is, other tetrapeptide sequences inserted just before the RAP sequence also attenuated the arrest. Our data suggest that SKIK or other tetrapeptide insertions disrupt the context of the RAP sequence rather than canceling or preventing the translation arrest. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 78 5 2024 Effect of Radon Inhalation on Murine Brain Proteins: Investigation Using Proteomic and Multivariate Analyses 387 399 EN Shota Naoe Graduate School of Health Sciences, Okayama University Ayumi Tanaka Graduate School of Health Sciences, Okayama University Norie Kanzaki Ningyo-toge Environmental Engineering Center, Japan Atomic Energy Agency Reiju Takenaka Graduate School of Health Sciences, Okayama University Akihiro Sakoda Ningyo-toge Environmental Engineering Center, Japan Atomic Energy Agency Takaaki Miyaji Advanced Science Research Center, Okayama University Kiyonori Yamaoka Faculty of Health Sciences, Okayama University Takahiro Kataoka Faculty of Health Sciences, Okayama University Original Article /10.18926/AMO/67663 Radon is a known risk factor for lung cancer; however, it can be used beneficially, such as in radon therapy. We have previously reported the enhancement of antioxidant effects associated with trace amounts of oxidative stress as one of the positive biological effects of radon inhalation. However, the biological effects of radon inhalation are incompletely understood, and more detailed and comprehensive studies are required. Although several studies have used proteomics to investigate the effects of radon inhalation on body proteins, none has focused on brain proteins. In this study, we evaluated the expression status of proteins in murine brains using proteomic and multivariate analyses to identify those whose expressions changed following two days of radon inhalation at a concentration of 1,500 Bq/m3. We found associations of radon inhalation with the expressions of seven proteins related to neurotransmission and heat shock. These proteins may be proposed as biomarkers indicative of radon inhalation. Although further studies are required to obtain the detailed biological significance of these protein alterations, this study contributes to the elucidation of the biological effects of radon inhalation as a low-dose radiation. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2076-2607 12 9 2024 A Novel C-Terminal Truncated Bacteriocin Found by Comparison between Leuconostoc mesenteroides 406 and 213M0 Isolated from Mongolian Traditional Fermented Milk, Airag 1781 EN Chihiro Hasiqimuge Graduate School of Environmental and Life Science, Okayama University Chihiro Hano Graduate School of Environmental and Life Science, Okayama University Kensuke Arakawa Graduate School of Environmental and Life Science, Okayama University Saki Yoshida Graduate School of Environmental and Life Science, Okayama University Junliang Zhao Graduate School of Environmental and Life Science, Okayama University Hidehiro Toh Advanced Genomics Center, National Institute of Genetics Hidetoshi Morita Graduate School of Environmental and Life Science, Okayama University Taku Miyamoto Graduate School of Environmental and Life Science, Okayama University Bacteriocins produced by lactic acid bacteria are known to be useful tools for food biopreservation and fermentation control. Leuconostoc mesenteroides subsp. mesenteroides 406 and 213M0 isolated from different samples of Mongolian traditional fermented milk, airag, had been reported to produce listericidal bacteriocin-like inhibitory substances with similar but slightly different properties. In this study, the antibacterial properties and the related gene sequences of both strains were compared, and then their bacteriocins were purified and identified. Strain 406 was superior to strain 213M0 in cell growth and antibacterial activity against many strains. However, the activity of 213M0 was stronger than that of 406 against a few strains. DNA sequencing revealed two and three plasmids in 406 and 213M0, respectively, and each one of them harbored an almost identical mesentericin Y105-B105 gene cluster. Removal of these plasmids resulted in a complete loss of activity, indicating that the antibacterial activity of both strains was generated by bacteriocins encoded on the plasmids. Mesentericins Y105 and B105 were purified from both cultures, and another novel bacteriocin, named mesentericin M, was identified from the 213M0 culture only. Its structural gene was coded on a 213M0 plasmid and, surprisingly, its C-terminal three amino acid residues were post-translationally cleaved. To our knowledge, this is the first report of a C-terminal truncated bacteriocin. In conclusion, the novel bacteriocin should be mainly responsible for the difference in antibacterial properties between the two strains. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 1046-5928 149 2018 Functional analysis of N-terminal propeptide in the precursor of Vibrio vulnificus metalloprotease by using cell-free translational system 13 16 EN Tomoka Kawase Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Fumi Miura Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Anusuya Debnath Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kinuyo Imakura Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shin-ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Vibrio vulnificus is a human pathogen causing fatal septicemia with edematous and hemorrhagic skin damage. Among multiple virulence factors, an extracellular metalloprotease termed as V. vulnificus protease (VVP) is known to play a crucial role in eliciting the skin damage. The mature VVP (413 aa) is composed of two domains, the N-terminal core domain with proteolytic activity and the C-terminal domain mediates efficient attachment to protein substrates. However, VVP is produced as an inactive precursor (609 aa) with a signal peptide (24 aa) and propeptide (172 aa). In order to clarify the function of propeptide, a series of DNA fragments encoding the VVP precursor and its various domains were designed and the proteins were expressed in vitro by using cell-free translational system. The results indicated that the propeptide might function as an intramolecular chaperon to promote the proper folding of both N-terminal and C-terminal domains. The obtained results also suggest that the propeptide, itself was unstable and thus digested easily by the enzymes present in cell lysate used for cell-free system. Additionally, the C-terminal domain in VVP found to inhibit the folding of the N-terminal domain in absence of propeptide. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2077-0383 13 15 2024 Impact of Serum Indoxyl Sulfate on One-Year Adverse Events in Chronic Kidney Disease Patients with Heart Failure 4384 EN Keiichiro Iwasaki Department of Cardiovascular Medicine, Okayama University Institute of Academic and Research, Medicine, Dentistry and Pharmaceutical Sciences Toru Miyoshi Department of Cardiovascular Medicine, Okayama University Institute of Academic and Research, Medicine, Dentistry and Pharmaceutical Sciences Chikara Urabe Department of Cardiovascular Medicine, Okayama University Institute of Academic and Research, Medicine, Dentistry and Pharmaceutical Sciences Satoru Sakuragi Department of Cardiovascular Medicine, Iwakuni Clinical Center Yusuke Kawai Department of Cardiovascular Medicine, Okayama City Hospital Soichiro Fuke Department of Cardiovascular Medicine, Japanese Red Cross Okayama Hospital Masayuki Doi Department of Cardiology, Kagawa Prefectural Central Hospital Atsushi Takaishi Department of Cardiology, Mitoyo General Hospital Takefumi Oka Department of Cardiology, Tsuyama Chuo Hospital Naoto Tokunaga Department of Cardiology, Ibara City Hospital Hiroshi Ito Department of General Internal Medicine 3, Kawasaki Medical School Background/Objectives: Indoxyl sulfate, a uremic toxin, is associated with mortality and cardiovascular events in patients with chronic kidney disease (CKD). This study aimed to evaluate the prognostic implications of serum indoxyl sulfate levels in patients with heart failure and CKD. Methods and Results: This was a prospective multicenter observational study. Overall, 300 patients with chronic heart failure with a previous history of hospitalization and an estimated glomerular filtration rate (eGFR) of 45 mL/min/1.73 m2 or less (CKD stage G3b to G5) without dialysis were analyzed. The primary outcome assessed in a time-to-event analysis from the measurement of indoxyl sulfate was a composite of all-cause death, hospitalization for heart failure, nonfatal myocardial infarction, and nonfatal stroke. Clinical events were followed-up to one year after indoxyl sulfate measurement. The median patient age was 75 years, and 57% of the patients were men. We divided the cohort into low and high indoxyl sulfate categories according to a median value of 9.63 mg/mL. The primary outcome occurred in 27 of 150 patients (18.0%) in the low indoxyl sulfate group and 27 of 150 patients (18.0%) in the high indoxyl sulfate group (hazard ratio, 1.00; 95% confidence interval, 0.58 to 1.70, p = 0.99). In the post hoc exploratory analyses, the results were consistent across age, sex, body mass index, left ventricular ejection fraction, eGFR, and N-terminal pro b-type natriuretic peptide. Conclusions: Among heart failure patients with CKD stages G3b to 5G, serum indoxyl sulfate concentrations were not significantly associated with the subsequent occurrence of cardiovascular events. No potential conflict of interest relevant to this article was reported.

IOS Press Acta Medica Okayama 1387-2877 100 1 2024 A Novel Peptidome Technology for the Diagnosis of Mild Cognitive Impairment and Alzheimer’s Disease by Selected Reaction Monitoring 219 228 EN Yusuke Fukui Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Koh Tadokoro Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Minaki Hamada Protosera, Inc. Kyoichi Asada Protosera, Inc. Lyang-Ja Lee Protosera, Inc. Hidehisa Tachiki Protosera, Inc. Ryuta Morihara Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Koji Abe Department of Neurology, National Center of Neurology and Psychiatry Toru Yamashita Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Background:With the aging of populations worldwide, Alzheimer’s disease (AD) has become a concern due to its high prevalence and the continued lack of established treatments. Early diagnosis is required as a preventive intervention to modify the disease’s progression. In our previous study, we performed peptidomic analysis of serum samples obtained from AD patients and age-matched healthy subjects to seek peptide biomarker candidates for AD by using BLOTCHIP-MS analysis, and identified four peptides as AD biomarker candidates. <br> Objective:The objective was to validate the serum biomarker peptides to distinguish mild cognitive impairment (MCI) and AD in comparison to cognitively healthy controls using a new peptidome technology, the Dementia Risk Test. <br> Methods:We enrolled 195 subjects with normal cognitive function (NC; n = 70), MCI (n = 55), and AD (n = 70), The concentrations of cognitive impairment marker peptides (Fibrinogen α chain (FAC), Fibrinogen β chain (FBC), Plasma protease C1 inhibitor (PPC1I), α2-HS-glycoprotein (AHSG)) were quantified by using a selected reaction monitoring assay based on liquid chromatography-MS/MS. <br> Results:The present study confirmed that three peptides, FAC, FBC, and PPC1I, were significantly upregulated during the onset of AD. This three-peptide set was both highly sensitive in determining AD (sensitivity: 85.7%, specificity: 95.7%, AUC: 0.900) and useful in distinguishing MCI (sensitivity: 61.8%, specificity: 98.6%, AUC: 0.824) from NC. <br> Conclusions:In this validation study, we confirmed the high diagnostic potential of the three peptides identified in our previous study as candidate serum biomarkers for AD. The Dementia Risk Test may be a powerful tool for detecting AD-related pathological changes. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1420-3049 29 11 2024 In Vitro Study of Tumor-Homing Peptide-Modified Magnetic Nanoparticles for Magnetic Hyperthermia 2632 EN Shengli Zhou Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kaname Tsutsumiuchi College of Bioscience and Biotechnology, Chubu University Ritsuko Imai College of Bioscience and Biotechnology, Chubu University Yukiko Miki College of Bioscience and Biotechnology, Chubu University Anna Kondo College of Bioscience and Biotechnology, Chubu University Hiroshi Nakagawa College of Bioscience and Biotechnology, Chubu University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cancer cells have higher heat sensitivity compared to normal cells; therefore, hyperthermia is a promising approach for cancer therapy because of its ability to selectively kill cancer cells by heating them. However, the specific and rapid heating of tumor tissues remains challenging. This study investigated the potential of magnetic nanoparticles (MNPs) modified with tumor-homing peptides (THPs), specifically PL1 and PL3, for tumor-specific magnetic hyperthermia therapy. The synthesis of THP-modified MNPs involved the attachment of PL1 and PL3 peptides to the surface of the MNPs, which facilitated enhanced tumor cell binding and internalization. Cell specificity studies revealed an increased uptake of PL1- and PL3-MNPs by tumor cells compared to unmodified MNPs, indicating their potential for targeted delivery. In vitro hyperthermia experiments demonstrated the efficacy of PL3-MNPs in inducing tumor cell death when exposed to an alternating magnetic field (AMF). Even without exposure to an AMF, an additional ferroptotic pathway was suggested to be mediated by the nanoparticles. Thus, this study suggests that THP-modified MNPs, particularly PL3-MNPs, hold promise as a targeted approach for tumor-specific magnetic hyperthermia therapy. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 78 3 2024 Effect of Humidified High-Flow Nasal Cannula Oxygen Therapy with a Pulmonary Infection Control Window as a Ventilation Switching Indication in Combination with Atomizing Inhalation of Terbutaline on the Lung Function of Patients with Acute Exacerbation of COPD 271 279 EN Mengjiao Ye Department of Respiratory and Critical Care Medicine, Tiantai Hospital of Traditional Chinese Medicine Renwei Zhang Department of Respiratory and Critical Care Medicine, Tiantai Hospital of Traditional Chinese Medicine Original Article /10.18926/AMO/67202 We investigated how humidified high-flow nasal cannula oxygen therapy (HFNC) with a pulmonary infection control (PIC) window as a ventilation switching indication in combination with atomizing inhalation of terbutaline affects the lung function of patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). We examined 140 hospitalized AECOPD patients randomized to control and observation groups. Conventional supportive therapy and invasive mechanical ventilation with tracheal intubation were conducted in both groups, with a PIC window as the indication for ventilation switching. Noninvasive positive pressure ventilation (NIPPV) plus atomizing inhalation of terbutaline was used in the control group. In the observation group, HFNC combined with atomizing inhalation of terbutaline was used. Compared to the control group, after 48-hr treatment and treatment completion, the observation group had significantly increased levels of lung function indicators (maximal voluntary ventilation [MVV] plus forced vital capacity [FVC], p<0.05) and oxygen metabolism indicators (arterial oxygen partial pressure [PaO2], arterial oxygen content [CaO2], and oxygenation index, p<0.05). The comparison of the groups revealed that the levels of airway remodeling indicators (matrix metalloproteinase-2 [MMP-2], tissue inhibitor of metalloproteinase 2 [TIMP-2] plus MMP-9) and inflammatory indicators (interferon gamma [IFN-γ] together with interleukin-17 [IL-17], IL-10 and IL-4) were significantly lower after 48 h of treatment as well as after treatment completion (both p<0.05). These results demonstrate that HFNC with a PIC window as the indication for ventilation switching combined with atomizing inhalation of terbutaline can relieve the disorder of oxygen metabolism and correct airway hyper-reactivity. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2227-9717 12 4 2024 Pyrene-Modified Cyclic Peptides Detect Cu2+ Ions by Fluorescence in Water 746 EN Yuhi Maekawa Department of Applied Chemistry, Faculty of Science and Engineering, Kindai University Sora Sakura Department of Applied Chemistry, Faculty of Science and Engineering, Kindai University Yuji Furutani Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Rento Fujihara Department of Applied Chemistry, Faculty of Science and Engineering, Kindai University Hisashi Sugime Department of Applied Chemistry, Faculty of Science and Engineering, Kindai University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Faculty of Science and Engineering, Kindai University The detection of metal ions is an option for maintaining water quality and diagnosing metal ion-related diseases. In this study, we successfully detected metal ions using fluorescent peptides in water. First, we prepared seven linear (L1-L7) and seven cyclic (C1-C7) peptides containing two pyrenyl (Pyr) units and assessed the response to various metal ions by fluorescence. The results indicated that C1, which contains a hexameric cyclic peptide moiety consisting of Pyr and Gly units, did not show a fluorescent response to metal ions, while the linear L1 corresponding to C1 showed a response to Cu2+, but its selectivity was found to be poor through a competition assay for each metal ion. We then assessed C2-C7 and L2-L7, in which Gly was replaced by His units at various positions in the same manner. The results showed that C2-C7 responded to Cu2+ in a manner dependent on the His position. Additionally, superior selectivity was observed in C7 through a competition assay. These results demonstrate that the structural restriction of peptides and the sequence affect the selective detection of Cu2+ and reveal that peptides with an appropriate structure can accomplish the fluorescent detection of Cu2+ specifically. No potential conflict of interest relevant to this article was reported.

Oxford University Press Acta Medica Okayama 0305-1048 52 10 2024 The ABCF proteins in Escherichia coli individually cope with 'hard-to-translate' nascent peptide sequences 5825 5840 EN Yuhei Chadani Faculty of Environmental, Life, Natural Science and Technology, Okayama University Shun Yamanouchi Department of Biological Sciences, Graduate School of Science, the University of Tokyo Eri Uemura Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology Kohei Yamasaki Faculty of Science, Okayama University Tatsuya Niwa Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology Toma Ikeda School of Life Science and Technology, Tokyo Institute of Technology Miku Kurihara School of Life Science and Technology, Tokyo Institute of Technology Wataru Iwasaki Department of Biological Sciences, Graduate School of Science, the University of Tokyo Hideki Taguchi Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology Organisms possess a wide variety of proteins with diverse amino acid sequences, and their synthesis relies on the ribosome. Empirical observations have led to the misconception that ribosomes are robust protein factories, but in reality, they have several weaknesses. For instance, ribosomes stall during the translation of the proline-rich sequences, but the elongation factor EF-P assists in synthesizing proteins containing the poly-proline sequences. Thus, living organisms have evolved to expand the translation capability of ribosomes through the acquisition of translation elongation factors. In this study, we have revealed that Escherichia coli ATP-Binding Cassette family-F (ABCF) proteins, YheS, YbiT, EttA and Uup, individually cope with various problematic nascent peptide sequences within the exit tunnel. The correspondence between noncanonical translations and ABCFs was YheS for the translational arrest by nascent SecM, YbiT for poly-basic sequence-dependent stalling and poly-acidic sequence-dependent intrinsic ribosome destabilization (IRD), EttA for IRD at the early stage of elongation, and Uup for poly-proline-dependent stalling. Our results suggest that ATP hydrolysis-coupled structural rearrangement and the interdomain linker sequence are pivotal for handling 'hard-to-translate' nascent peptides. Our study highlights a new aspect of ABCF proteins to reduce the potential risks that are encoded within the nascent peptide sequences. Graphical Abstract No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 78 2 2024 The Roles of Neuropeptide Y in Respiratory Disease Pathogenesis via the Airway Immune Response 95 106 EN Junko Itano Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Katsuyuki Kiura Department of Allergy and Respiratory Medicine, Okayama University Hospital Yoshinobu Maeda Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nobuaki Miyahara Department of Allergy and Respiratory Medicine, Okayama University Hospital Review /10.18926/AMO/66912 The lungs are very complex organs, and the respiratory system performs the dual roles of repairing tissue while protecting against infection from various environmental stimuli. Persistent external irritation disrupts the immune responses of tissues and cells in the respiratory system, ultimately leading to respiratory disease. Neuropeptide Y (NPY) is a 36-amino-acid polypeptide and a neurotransmitter that regulates homeostasis. The NPY receptor is a seven-transmembrane-domain G-protein-coupled receptor with six subtypes (Y1, Y2, Y3, Y4, Y5, and Y6). Of these receptors, Y1, Y2, Y4, and Y5 are functional in humans, and Y1 plays important roles in the immune responses of many organs, including the respiratory system. NPY and the Y1 receptor have critical roles in the pathogenesis of asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis. The effects of NPY on the airway immune response and pathogenesis differ among respiratory diseases. This review focuses on the involvement of NPY in the airway immune response and pathogenesis of various respiratory diseases. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 2189-7948 25 3 2024 Idiopathic ventricular tachycardia detected after coronavirus disease 2019 164 165 EN Koichiro Yamamoto Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koji Nakagawa Department of Cardiovascular Medicine, Okayama University Hospital Fumio Otsuka Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences We present a 23-year-old woman with depression and long COVID in whom a diagnosis of idiopathic ventricular tachycardia (VT) was made. Although the relationship between idiopathic VT and long COVID remains unknown, this is the first report of idiopathic VT detected in a patient with long COVID. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 1574-6968 371 2024 Knockout of adenylosuccinate synthase purA increases susceptibility to colistin in Escherichia coli fnae007 EN Tomonori Kano Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kazuya Ishikawa Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kazuyuki Furuta Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Chikara Kaito Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Colistin is a cationic cyclic antimicrobial peptide used as a last resort against multidrug-resistant gram-negative bacteria. To understand the factors involved in colistin susceptibility, we screened colistin-sensitive mutants from an E. coli gene-knockout library (Keio collection). The knockout of purA, whose product catalyzes the synthesis of adenylosuccinate from IMP in the de novo purine synthesis pathway, resulted in increased sensitivity to colistin. Adenylosuccinate is subsequently converted to AMP, which is phosphorylated to produce ADP, a substrate for ATP synthesis. The amount of ATP was lower in the purA-knockout mutant than that in the wild-type strain. ATP synthesis is coupled with proton transfer, and it contributes to the membrane potential. Using the membrane potential probe, 3,3′-diethyloxacarbocyanine iodide [DiOC2(3)], we found that the membrane was hyperpolarized in the purA-knockout mutant compared to that in the wild-type strain. Treatment with the proton uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), abolished the hyperpolarization and colistin sensitivity in the mutant. The purA-knockout mutant exhibited increased sensitivity to aminoglycosides, kanamycin, and gentamicin; their uptake requires a membrane potential. Therefore, the knockout of purA, an adenylosuccinate synthase, decreases ATP synthesis concurrently with membrane hyperpolarization, resulting in increased sensitivity to colistin. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1661-6596 25 3 2024 Mutual Effects of Orexin and Bone Morphogenetic Proteins on Catecholamine Regulation Using Adrenomedullary Cells 1585 EN Yoshiaki Soejima Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Iwata Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koichiro Yamamoto Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Atsuhito Suyama Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuhiro Nakano Department of General Medicine, 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 Orexins are neuronal peptides that play a prominent role in sleep behavior and feeding behavior in the central nervous system, though their receptors also exist in peripheral organs, including the adrenal gland. In this study, the effects of orexins on catecholamine synthesis in the rat adrenomedullary cell line PC12 were investigated by focusing on their interaction with the adrenomedullary bone morphogenetic protein (BMP)-4. Orexin A treatment reduced the mRNA levels of key enzymes for catecholamine synthesis, including tyrosine hydroxylase (Th), 3,4-dihydroxyphenylalanie decarboxylase (Ddc) and dopamine beta-hydroxylase (Dbh), in a concentration-dependent manner. On the other hand, treatment with BMP-4 suppressed the expression of Th and Ddc but enhanced that of Dbh with or without co-treatment with orexin A. Of note, orexin A augmented BMP-receptor signaling detected by the phosphorylation of Smad1/5/9 through the suppression of inhibitory Smad6/7 and the upregulation of BMP type-II receptor (BMPRII). Furthermore, treatment with BMP-4 upregulated the mRNA levels of OX1R in PC12 cells. Collectively, the results indicate that orexin and BMP-4 suppress adrenomedullary catecholamine synthesis by mutually upregulating the pathway of each other in adrenomedullary cells. No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 1549-9596 64 2 2023 pSPICA Force Field Extended for Proteins and Peptides 532 542 EN Yusuke Miyazaki Research Institute for Interdisciplinary Science, Okayama University Wataru Shinoda Research Institute for Interdisciplinary Science, Okayama University Many coarse-grained (CG) molecular dynamics (MD) studies have been performed to investigate biological processes involving proteins and lipids. CG force fields (FFs) in these MD studies often use implicit or nonpolar water models to reduce computational costs. CG-MD using water models cannot properly describe electrostatic screening effects owing to the hydration of ionic segments and thus cannot appropriately describe molecular events involving water channels and pores through lipid membranes. To overcome this issue, we developed a protein model in the pSPICA FF, in which a polar CG water model showing the proper dielectric response was adopted. The developed CG model greatly improved the transfer free energy profiles of charged side chain analogues across the lipid membrane. Application studies on melittin-induced membrane pores and mechanosensitive channels in lipid membranes demonstrated that CG-MDs using the pSPICA FF correctly reproduced the structure and stability of the pores and channels. Furthermore, the adsorption behavior of the highly charged nona-arginine peptides on lipid membranes changed with salt concentration, indicating the pSPICA FF is also useful for simulating protein adsorption on membrane surfaces. No potential conflict of interest relevant to this article was reported.

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

Frontiers Media Acta Medica Okayama 1664-3224 14 2023 “Input/output cytokines” in epidermal keratinocytes and the involvement in inflammatory skin diseases 1239598 EN Shin Morizane Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomoyuki Mukai Department of Immunology and Molecular Genetics, Kawasaki Medical School Ko Sunagawa Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kota Tachibana Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshio Kawakami 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 Considering the role of epidermal keratinocytes, they occupy more than 90% of the epidermis, form a physical barrier, and also function as innate immune barrier. For example, epidermal keratinocytes are capable of recognizing various cytokines and pathogen-associated molecular pattern, and producing a wide variety of inflammatory cytokines, chemokines, and antimicrobial peptides. Previous basic studies have shown that the immune response of epidermal keratinocytes has a significant impact on inflammatory skin diseases. The purpose of this review is to provide foundation of knowledge on the cytokines which are recognized or produced by epidermal keratinocytes. Since a number of biologics for skin diseases have appeared, it is necessary to fully understand the relationship between epidermal keratinocytes and the cytokines. In this review, the cytokines recognized by epidermal keratinocytes are specifically introduced as "input cytokines", and the produced cytokines as "output cytokines". Furthermore, we also refer to the existence of biologics against those input and output cytokines, and the target skin diseases. These use results demonstrate how important targeted cytokines are in real skin diseases, and enhance our understanding of the cytokines. No potential conflict of interest relevant to this article was reported.

Spandidos Publications Acta Medica Okayama 1792-1074 24 5 2022 Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line‑derived xenograft model 382 EN Qiusheng Shan Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kiyofumi Takabatake Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Haruka Omori Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hotaka Kawai Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences May Wathone Oo Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shintaro Sukegawa Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masae Fujii Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasunori Inada Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Sho Sano Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keisuke Nakano Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The cancer stroma regulates bone invasion in oral squamous cell carcinoma (OSCC). However, data on normal stroma are limited. In the present study, the effects of gingival and periodontal ligament tissue‑derived stromal cells (G‑SCs and P‑SCs, respectively) and human dermal fibroblasts (HDFs) on bone resorption and osteoclast activation were assessed using hematoxylin and eosin and tartrate‑resistant acid phosphatase staining in a cell line‑derived xenograft model. The results demonstrated that G‑SCs promoted bone invasion and osteoclast activation and inhibited osteoclast proliferation following crosstalk with the human OSCC HSC‑3 cell line, whereas P‑SCs inhibited bone resorption and promoted osteoclast proliferation in vitro but had a minimal effect on osteoclast activation both in vitro and in vivo following crosstalk with HSC‑3 cells. Furthermore, the effects of G‑SCs, P‑SCs and HDFs on protein expression levels of matrix metalloproteinase (MMP)‑9, membrane type 1 MMP (MT1‑MMP), Snail, parathyroid hormone‑related peptide (PTHrP) and receptor activator of NF‑κB ligand (RANKL) in HSC‑3 cells in OSCC bone invasion regions were assessed using immunohistochemistry. The results demonstrated that G‑SCs had a more prominent effect on the expression of MMP‑9, MT1‑MMP, Snail, PTHrP, and RANKL, whereas P‑SCs only promoted RANKL and PTHrP expression and exerted a minimal effect on MMP‑9, MT1‑MMP and Snail expression. The potential genes underlying the differential effects of G‑SCs and P‑SCs on bone invasion in OSCC were evaluated using a microarray, which indicated that cyclin‑dependent kinase 1, insulin, aurora kinase A, cyclin B1 and DNA topoisomerase II alpha underlaid these differential effects. Therefore, these results demonstrated that G‑SCs promoted bone invasion in OSCC by activating osteoclasts on the bone surface, whereas P‑SCs exerted an inhibitory effect. These findings could indicate a potential regulatory mechanism for bone invasion in OSCC. No potential conflict of interest relevant to this article was reported.

Spandidos Publications Acta Medica Okayama 1021-335X 47 4 2022 Significance of cancer stroma for bone destruction in oral squamous cell carcinoma using different cancer stroma subtypes 81 EN Qiusheng Shan Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kiyofumi Takabatake Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hotaka Kawai Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences May Wathone Oo Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasunori Inada Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shintaro Sukegawa Department of Oral and Maxillofacial Surgery, Kagawa Prefectural Central Hospital Shigeko Fushimi Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keisuke Nakano Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Stromal cells in the tumor microenvironment (TME) can regulate the progression of numerous types of cancer; however, the bone invasion of oral squamous cell carcinoma (OSCC) has been poorly investigated. In the present study, the effect of verrucous SCC‑associated stromal cells (VSCC‑SCs), SCC‑associated stromal cells (SCC‑SCs) and human dermal fibroblasts on bone resorption and the activation of HSC‑3 osteoclasts in vivo were examined by hematoxylin and eosin, AE1/3 (pan‑cytokeratin) and tartrate‑resistant acid phosphatase staining. In addition, the expression levels of matrix metalloproteinase (MMP)9, membrane‑type 1 MMP (MT1‑MMP), Snail, receptor activator of NF‑κB ligand (RANKL) and parathyroid hormone‑related peptide (PTHrP) in the bone invasion regions of HSC‑3 cells were examined by immunohistochemistry. The results suggested that both SCC‑SCs and VSCC‑SCs promoted bone resorption, the activation of osteoclasts, and the expression levels of MMP9, MT1‑MMP, Snail, RANKL and PTHrP. However, SCC‑SCs had a more prominent effect compared with VSCC‑SCs. Finally, microarray data were used to predict potential genes underlying the differential effects of VSCC‑SCs and SCC‑SCs on bone invasion in OSCC. The results revealed that IL1B, ICAM1, FOS, CXCL12, INS and NGF may underlie these differential effects. In conclusion, both VSCC‑SCs and SCC‑SCs may promote bone invasion in OSCC by enhancing the expression levels of RANKL in cancer and stromal cells mediated by PTHrP; however, SCC‑SCs had a more prominent effect. These findings may represent a potential regulatory mechanism underlying the bone invasion of OSCC. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2005 Conformationally-restricted peptide nucleic acids with pyrrolidine rings in the main chains EN Mizuki Kitamatsu No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0168-1702 334 2023 Exploration of the yadokari/yadonushi nature of YkV3 and RnMBV3 in the original host and a model filamentous fungus 199155 EN Yukiyo Sato Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Sakae Hisano Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University Nobuhiro Suzuki Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University The yadokari/yadonushi nature is a recently discovered virus lifestyle; “yadokari” refers to the ability of capsidless positive-sense (+) RNA viruses (yadokariviruses) to utilize the capsids of phylogenetically distant double-stranded RNA (dsRNA) viruses possibly as the replication site, while “yadonushi” refers to the ability of dsRNA viruses to provide capsids to yadokariviruses. This virus–virus interaction, however, has been only studied with limited pathosystems. Here, we established a new study model with a capsidless (+)RNA yadokarivirus YkV3 (family Yadokariviridae) and its capsid donor RnMBV3 (family Megabirnaviridae) in the original host fungus Rosellinia necatrix and a model filamentous fungal host Cryphonectria parasitica. YkV3 has a simple genome structure with one open reading frame of 4305 nucleotides encoding a single polyprotein with an RNA-dependent RNA polymerase and a 2A-like self-cleavage peptide domain. Reverse genetics of YkV3 in R. necatrix showed that YkV3 tolerates a nucleotide substitution in the extreme 5′-terminus. The insertion of two termination codons immediately downstream of the 2A-like cleavage site abolished YkV3 viability, suggesting the importance of the C-terminal portion of the polyprotein of unknown function. Transfection of RnMBV3 and YkV3 into an RNA silencing-deficient mutant Δdcl2 of C. parasitica showed the replication competency of both viruses. Comparison between the wild-type and Δdcl2 strains of C. parasitica in virus accumulation suggested that RnMBV3 and YkV3 are susceptible to RNA silencing in C. parasitica. Taken together, we have established a platform to further explore the yadokari/yadonushi nature using genetically manipulable host fungal and virus strains. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 77 4 2023 Association of Tumor Necrosis Factor-Alpha with Psychopathology in Patients with Schizophrenia 395 405 EN Marko Pavlovic University Hospital Center Mostar, University of Mostar Dragan Babic University Hospital Center Mostar, University of Mostar Pejana Rastovic University Hospital Center Mostar, University of Mostar Jurica Arapovic University Hospital Center Mostar, University of Mostar Marko Martinac Health Care Center Mostar, University of Mostar Sanja Jakovac University Hospital Center Mostar, University of Mostar Romana Barbaric University Hospital Center Mostar, University of Mostar Original Article /10.18926/AMO/65750 We investigated the relationship between serum tumor necrosis factor-alpha (TNF-α) levels and psychopathological symptoms, clinical and socio-demographic characteristics and antipsychotic therapy in individuals with schizophrenia. TNF-α levels were measured in 90 patients with schizophrenia and 90 healthy controls matched by age, gender, smoking status, and body mass index. The Positive and Negative Syndrome Scale (PANSS) was used to assess the severity of psychopathology in patients. No significant differences in TNF-α levels were detected between the patients and controls (p=0.736). TNF-α levels were not correlated with total, positive, negative, general, or composite PANSS scores (all p>0.05). A significant negative correlation was observed between TNF-α levels and the PANSS cognitive factor (ρ=−0.222, p=0.035). A hierarchical regression analysis identified the cognitive factor as a significant predictor of the TNF-α level (beta=−0.258, t=−2.257, p=0.027). There were no significant differences in TNF-α levels among patients treated with different types of antipsychotics (p=0.596). TNF-α levels correlated positively with the age of onset (ρ=0.233, p=0.027) and negatively with illness duration (ρ=−0.247, p=0.019) and antipsychotic treatment duration (ρ=−0.256, p=0.015). These results indicate that TNF-α may be involved in cognitive impairment in schizophrenia, and would be a potential clinical-state marker in schizophrenia. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 77 4 2023 Changes in TRPV1 Receptor, CGRP, and BDNF Expression in Rat Dorsal Root Ganglion with Resiniferatoxin-Induced Neuropathic Pain: Modulation by Pulsed Radiofrequency Applied to the Sciatic Nerve 359 364 EN Tomohiro Koshida Department of Anesthesiology and Pain Clinic, Faculty of Medicine, University of Miyazaki Toyoaki Maruta Department of Anesthesiology and Pain Clinic, Faculty of Medicine, University of Miyazaki Nobuhiko Tanaka Tanaka homecare clinic Kotaro Hidaka Department of Anesthesiology and Pain Clinic, Faculty of Medicine, University of Miyazaki Mio Kurogi Department of Anesthesiology and Pain Clinic, Faculty of Medicine, University of Miyazaki Takayuki Nemoto Department of Pharmacology, Faculty of Medicine, Fukuoka University Toshihiko Yanagita Department of Clinical Pharmacology, School of Nursing, Faculty of Medicine, University of Miyazaki Ryu Takeya Department of Pharmacology, Faculty of Medicine, University of Miyazaki Isao Tsuneyoshi Department of Anesthesiology and Pain Clinic, Faculty of Medicine, University of Miyazaki Original Article /10.18926/AMO/65741 Pulsed radiofrequency (PRF) is a safe method of treating neuropathic pain by generating intermittent electric fields at the needle tip. Resiniferatoxin (RTX) is an ultrapotent agonist of transient receptor potential vanilloid subtype-1 (TRPV1) receptors. We investigated the mechanism of PRF using a rat model of RTX-induced neuropathic pain. After administering RTX intraperitoneally, PRF was applied to the right sciatic nerve. We observed the changes in TRPV1, calcitonin gene-related peptide (CGRP), and brain-derived neurotrophic factor (BDNF) in the dorsal root ganglia by western blotting. Expressions of TRPV1 and CGRP were significantly lower in the contralateral (RTX-treated, PRF-untreated) tissue than in control rats (p<0.0001 and p<0.0001, respectively) and the ipsilateral tissues (p<0.0001 and p<0.0001, respectively). BDNF levels were significantly higher in the contralateral tissues than in the control rats (p<0.0001) and the ipsilateral tissues (p<0.0001). These results suggest that, while TRPV1 and CGRP are decreased by RTX-induced neuronal damage, increased BDNF levels result in pain development. PRF may promote recovery from neuronal damage with concomitant restoration of TRPV1 and CGRP, and exert its analgesic effect by reversing BDNF increase. Further research is required to understand the role of TRPV1 and CGRP restoration in improving mechanical allodynia. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0953-8194 35 9 2023 Neuropeptidergic control circuits in the spinal cord for male sexual behaviour: Oxytocin–gastrin‐releasing peptide systems e13324 EN Takumi Oti Ushimado Marine Institute (UMI), Faculty of Environmental, Life, Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Faculty of Environmental, Life, Natural Science and Technology, Okayama University The neuropeptidergic mechanisms controlling socio-sexual behaviours consist of complex neuronal circuitry systems in widely distributed areas of the brain and spinal cord. At the organismal level, it is now becoming clear that “hormonal regulations” play an important role, in addition to the activation of neuronal circuits. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the “spinal ejaculation generator (SEG).” Oxytocin, long known as a neurohypophyseal hormone, is now known to be involved in the regulation of socio-sexual behaviors in mammals, ranging from social bonding to empathy. However, the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system remains unclear. Oxytocin is known to be synthesised mainly in hypothalamic neurons and released from the posterior pituitary into the circulation. Oxytocin is also released from the dendrites of the neurons into the hypothalamus where they have important roles in social behaviours via non-synaptic volume transmission. Because the most familiar functions of oxytocin are to regulate female reproductive functions including parturition, milk ejection, and maternal behaviour, oxytocin is often thought of as a “feminine” hormone. However, there is evidence that a group of parvocellular oxytocin neurons project to the lower spinal cord and control male sexual function in rats. In this report, we review the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system and effects of these neuropeptides on male sexual behaviour. Furthermore, we discuss the finding of a recently identified, localised “volume transmission” role of oxytocin in the spinal cord. Findings from our studies suggest that the newly discovered “oxytocin-mediated spinal control of male sexual function” may be useful in the treatment of erectile and ejaculatory dysfunction. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1661-6596 24 14 2023 Development of Hydrophobic Cell-Penetrating Stapled Peptides as Drug Carriers 11768 EN Keisuke Tsuchiya Division of Organic Chemistry, National Institute of Health Sciences Kanako Horikoshi Division of Organic Chemistry, National Institute of Health Sciences Minami Fujita Division of Organic Chemistry, National Institute of Health Sciences Motoharu Hirano Division of Organic Chemistry, National Institute of Health Sciences Maho Miyamoto Division of Organic Chemistry, National Institute of Health Sciences Hidetomo Yokoo Division of Organic Chemistry, National Institute of Health Sciences Yosuke Demizu Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of a variety of cargo molecules, including small molecules, peptides, nucleic acids, and proteins. Many cationic and amphiphilic CPPs have been developed; however, there have been few reports regarding hydrophobic CPPs. Herein, we have developed stapled hydrophobic CPPs based on the hydrophobic CPP, TP10, by introducing an aliphatic carbon side chain on the hydrophobic face of TP10. This side chain maintained the hydrophobicity of TP10 and enhanced the helicity and cell penetrating efficiency. We evaluated the preferred secondary structures, and the ability to deliver 5(6)-carboxyfluorescein (CF) as a model small molecule and plasmid DNA (pDNA) as a model nucleotide. The stapled peptide F-3 with CF, in which the stapling structure was introduced at Gly residues, formed a stable & alpha;-helical structure and the highest cell-membrane permeability via an endocytosis process. Meanwhile, peptide F-4 demonstrated remarkable stability when forming a complex with pDNA, making it the optimal choice for the efficient intracellular delivery of pDNA. The results showed that stapled hydrophobic CPPs were able to deliver small molecules and pDNA into cells, and that different stapling positions in hydrophobic CPPs can control the efficiency of the cargo delivery. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6643 15 13 2023 Adrenomedullin Enhances Mouse Gustatory Nerve Responses to Sugars via T1R-Independent Sweet Taste Pathway 2941 EN Shusuke Iwata Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University Ryusuke Yoshida Department of Oral Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shingo Takai Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University Keisuke Sanematsu Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University Noriatsu Shigemura Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University Yuzo Ninomiya Department of Oral Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University On the tongue, the T1R-independent pathway (comprising glucose transporters, including sodium-glucose cotransporter (SGLT1) and the K-ATP channel) detects only sugars, whereas the T1R-dependent (T1R2/T1R3) pathway can broadly sense various sweeteners. Cephalic-phase insulin release, a rapid release of insulin induced by sensory signals in the head after food-related stimuli, reportedly depends on the T1R-independent pathway, and the competitive sweet taste modulators leptin and endocannabinoids may function on these two different sweet taste pathways independently, suggesting independent roles of two oral sugar-detecting pathways in food intake. Here, we examined the effect of adrenomedullin (ADM), a multifunctional regulatory peptide, on sugar sensing in mice since it affects the expression of SGLT1 in rat enterocytes. We found that ADM receptor components were expressed in T1R3-positive taste cells. Analyses of chorda tympani (CT) nerve responses revealed that ADM enhanced responses to sugars but not to artificial sweeteners and other tastants. Moreover, ADM increased the apical uptake of a fluorescent D-glucose derivative into taste cells and SGLT1 mRNA expression in taste buds. These results suggest that the T1R-independent sweet taste pathway in mouse taste cells is a peripheral target of ADM, and the specific enhancement of gustatory nerve responses to sugars by ADM may contribute to caloric sensing and food intake. No potential conflict of interest relevant to this article was reported.

Wiley Periodicals, INC Acta Medica Okayama 2055-5822 2023 Prevalence of transthyretin amyloidosis among heart failure patients with preserved ejection fraction in Japan EN Takanori Naito Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazufumi Nakamura Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yukio Abe Department of Cardiology, Osaka City General Hospital Hiroyuki Watanabe Department of Cardiovascular Medicine, Tokyo Bay Urayasu Ichikawa Medical Center Satoru Sakuragi Department of Cardiovascular Medicine, National Hospital Organization Iwakuni Clinical Center Yusuke Katayama Department of Cardiovascular Medicine, National Hospital Organization Iwakuni Clinical Center Hajime Kihara Department of Internal Medicine, Kihara Cardiovascular Clinic Atsutaka Okizaki Department of Radiology, Asahikawa Medical University Yusuke Kawai Department of Cardiovascular Medicine, Okayama City Hospital Masaki Yoshikawa Department of Cardiovascular Medicine, Fukuyama City Hospital Atsushi Takaishi Department of Cardiology, Mitoyo General Hospital Hideki Fujio Department of Cardiovascular Medicine, Japanese Red Cross Society Himeji Hospital Hiroaki Otsuka Department of Cardiovascular Medicine, National Hospital Organization Iwakuni Clinical Center Soichiro Ogura Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Ito Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences ATTR HFpEF Registry Investigators Aims Heart failure with preserved ejection fraction (HFpEF), which is caused by wide various conditions, has become a major public health problem. Transthyretin amyloid cardiomyopathy (ATTR-CM), which is thought to be an underdiagnosed disease, can cause HFpEF. Non-invasive diagnosis using 99mTechnetium (Tc)-pyrophosphate (PYP) scintigraphy enables accurate diagnosis of ATTR-CM. The aim of this study was to clarify the prevalence and characteristics of ATTR-CM among Japanese patients with HFpEF.<br> Methods and results This study was a multicentre, prospective, observational study conducted in Japan. We enrolled 373 patients with HFpEF [left ventricular (LV) ejection fraction ≥50%] aged ≥65 years who were admitted to the department of cardiology from September 2018 to January 2022. A 99mTc-PYP scintigraphy scan was performed during admission in all eligible patients. Cardiac 99mTc-PYP retention was graded according to a previously reported visual scale ranging from 0 to 3 points. The scan was considered positive when it revealed moderate-to-severe 99mTc-PYP uptake (Grade 2–3) in both ventricles. Patients were divided into ATTR-CM and non-ATTR-CM patients according to positive (Grade 2–3) or negative (Grade 0–1) 99mTc-PYP scintigraphy, respectively. Medical history, blood tests, electrocardiogram, echocardiography, and magnetic resonance imaging in the two groups of patients were compared. Among the 373 patients with HFpEF, 53 patients (14.2%; 95% confidence interval: 10.7–17.7) showed positive uptake on 99mTc-PYP scintigraphy. An endomyocardial biopsy was performed in 32 patients and confirmed amyloidosis in all cases. There were no significant differences between the two groups in age, severity of heart failure as assessed by the New York Heart Association (NYHA) functional classification, renal function values, left ventricular ejection fraction, and tricuspid regurgitant pressure gradient (ATTR-CM, n = 53 vs. non-ATTR-CM, n = 320). Patients in the ATTR-CM group had a higher N-terminal pro-brain natriuretic peptide level [2314 (1081–3398) vs. 900 (415–1828), P < 0.001], higher sensitive troponin T level (0.074 ± 0.049 vs. 0.035 ± 0.038, P < 0.001), and higher mean LV maximal wall thickness [12.5 (11–14) vs. 10.5 (9.5–11.5), P < 0.001].<br> Conclusions ATTR-CM is an underdiagnosed disease with a significant prevalence in Japanese patients with HFpEF. This study showed that results of examinations for ATTR-CM patients appear to be worse than those for non-ATTR-CM patients, but clinical severities of heart failure as assessed by the NYHA functional classification are similar in ATTR-CM and non-ATTR-CM patients, and the clinical overlap between ATTR-CM and non-ATTR-CM is high. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 12 3 2023 The Pursuit of the "Inside" of the Amyloid Hypothesis-Is C99 a Promising Therapeutic Target for Alzheimer's Disease? 454 EN Nobumasa Takasugi Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masato Komai Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nanaka Kaneshiro Division of Biomedical Sciences, School of Medicine, University of California Atsuya Ikeda Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuji Kamikubo Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine Takashi Uehara Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Aducanumab, co-developed by Eisai (Japan) and Biogen (U.S.), has received Food and Drug Administration approval for treating Alzheimer's disease (AD). In addition, its successor antibody, lecanemab, has been approved. These antibodies target the aggregated form of the small peptide, amyloid-beta (A beta), which accumulates in the patient brain. The "amyloid hypothesis " based therapy that places the aggregation and toxicity of A beta at the center of the etiology is about to be realized. However, the effects of immunotherapy are still limited, suggesting the need to reconsider this hypothesis. A beta is produced from a type-I transmembrane protein, A beta precursor protein (APP). One of the APP metabolites, the 99-amino acids C-terminal fragment (C99, also called beta CTF), is a direct precursor of A beta and accumulates in the AD patient's brain to demonstrate toxicity independent of A beta. Conventional drug discovery strategies have focused on A beta toxicity on the "outside " of the neuron, but C99 accumulation might explain the toxicity on the "inside " of the neuron, which was overlooked in the hypothesis. Furthermore, the common region of C99 and A beta is a promising target for multifunctional AD drugs. This review aimed to outline the nature, metabolism, and impact of C99 on AD pathogenesis and discuss whether it could be a therapeutic target complementing the amyloid hypothesis. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 2186-7755 112 2023 鳥類におけるアルギニンバソトシン神経の性差 19 22 EN Noboru Saito Course of Applied Animal Science Arginine vasotocin (AVT) is one of neurohypophysial peptides in birds and is well known both as an antidiuretic and oxytocic hormone. AVT is produced in the hypothalamus and mainly synthesized in magnocellular neurons, supraoptic nucleus (SON) and paraventricular nucleus (PVN). Recent, immunocytochemistry and in situ hybridization results indicate that AVT is also synthesized in parvocellular neurons corresponding to mammalian stria terminalis (nST). The AVT neurons in nST in the male are more numerous than those in the female. Therefore, these data suggest that there is sexual dimorphism in the distribution of AVT neurons. Several experiments involving electrical lesion, testosterone implantation, and the detection of the immediately-early gene expression in birds expressing copulatory behavior suggested that nST and nucleus praeopticus medialis (POM) were related to the reproductive behavior. When male embryo was treated with estradiol, the distribution of AVT neurons assumes the same distribution as found in the female, and reproductive behavior was abolished, suggesting that AVT in the brain has an important role in reproductive behavior. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0031-9384 261 2023 Oxytocin increased intragastric pressure in the forestomach of rats via the dorsal vagal complex 114087 EN Motoi Kobashi Department of Oral Physiology, Faculty of Medicine, Dentistry and Pharmaceutical Science, Okayama University Yuichi Shimatani Department of Medical Engineering, Faculty of Engineering, Tokyo City University Masako Fujita Department of Oral Physiology, Faculty of Medicine, Dentistry and Pharmaceutical Science, Okayama University We previously reported that appetite enhancing peptides facilitated phasic contractions of the distal stomach and relaxed the forestomach via the dorsal vagal complex (DVC). The present study investigated the effects of anorectic substances on gastric reservoir function. The effects of oxytocin on the motility of the forestomach were examined in rats anesthetized with urethane chloralose. Gastric motor responses were measured using an intragastric balloon. The fourth ventricular administration of oxytocin (0.1 1.0 nmol) increased intragastric pressure (IGP) in the forestomach in a dose dependent manner. Conversely, the administration of oxytocin (0.3 nmol) suppressed phasic contractions of the distal stomach. These responses were opposite to those of appetite enhancing peptides in previous studies. The oxytocin response in the forestomach was not observed after bilateral cervical vagotomy. The effects of oxytocin on forestomach motility were examined in animals that underwent ablation of the area postrema (AP) to clarify its involvement. Although the magnitude of the response to the fourth ventricular administration of oxytocin decreased, a significant response was still observed. A microinjection of oxytocin (3 pmol) into the AP, the left medial nucleus of the nucleus tractus solitarius (mNTS), the left commissural part of the NTS, or the left dorsal motor nucleus of the vagus was performed. The oxytocin injection into the AP and/or mNTS induced a rapid and large increase in IGP in the forestomach. Prior injection of L-368,899, an oxytocin receptor antagonist, into both the AP and mNTS attenuated the oxytocin response of the forestomach induced by fourth ventricular administration of oxytocin. These results indicate that oxytocin acts on the AP and/or mNTS to increase IGP in the forestomach via vagal preganglionic neurons. No potential conflict of interest relevant to this article was reported.

American Chemical Society Acta Medica Okayama 2470-1343 7 50 2022 Oligoarginine-Conjugated Peptide Foldamers Inhibiting Vitamin D Receptor-Mediated Transcription 46573 46582 EN Mami Takyo National Institute of Health Sciences Yumi Sato National Institute of Health Sciences Naoya Hirata National Institute of Health Sciences Keisuke Tsuchiya National Institute of Health Sciences Hiroaki Ishida Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University Takashi Kurohara National Institute of Health Sciences Yuta Yanase National Institute of Health Sciences Takahito Ito National Institute of Health Sciences Yasunari Kanda National Institute of Health Sciences Keiko Yamamoto Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University Takashi Misawa National Institute of Health Sciences Yosuke Demizu National Institute of Health Sciences The vitamin D receptor (VDR) is a nuclear receptor, which is involved in several physiological processes, including differentiation and bone homeostasis. The VDR is a promising target for the development of drugs against cancer and bone-related diseases. To date, several VDR antagonists, which bind to the ligand binding domain of the VDR and compete with the endogenous agonist 1 alpha,25(OH)D3, have been reported. However, these ligands contain a secosteroidal skeleton, which is chemically unstable and complicated to synthesize. A few VDR antagonists with a nonsecosteroidal skeleton have been reported. Alternative inhibitors against VDR transactivation that act via different mechanisms are desirable. Here, we developed peptide-based VDR inhibitors capable of disrupting the VDR-coactivator interaction. It was reported that helical SRC2-3 peptides strongly bound to the VDR and competed with the coactivator in vitro. Therefore, we designed and synthesized a series of SRC2-3 derivatives by the introduction of nonproteinogenic amino acids, such as beta-amino acids, and by side-chain stapling to stabilize helical structures and provide resistance against digestive enzymes. In addition, conjugation with a cell-penetrating peptide increased the cell membrane permeability and was a promising strategy for intracellular VDR inhibition. The nona-arginine-conjugated peptides 24 with side-chain stapling and 25 with cyclic beta-amino acids showed strong intracellular VDR inhibitory activity, resulting in suppression of the target gene expression and inhibition of the cell differentiation of HL-60 cells. Herein, the peptide design, structure-activity relationship (SAR) study, and biological evaluation of the peptides are described. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 0022-0957 74 3 2022 The secreted immune response peptide 1 functions as a phytocytokine in rice immunity 1059 1073 EN Pingyu Wang Key Laboratory of Plant Hormones and Development Regulation of Chongqing, School of Life Sciences, Chongqing University Huimin Jia Shanghai Center for Plant Stress Biology and Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Ting Guo Shanghai Center for Plant Stress Biology and Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Yuanyuan Zhang Shanghai Center for Plant Stress Biology and Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Wanqing Wang Institute of Plant Science and Resources, Okayama University Hideki Nishimura Institute of Plant Science and Resources, Okayama University Zhengguo Li Key Laboratory of Plant Hormones and Development Regulation of Chongqing, School of Life Sciences, Chongqing University Yoji Kawano Shanghai Center for Plant Stress Biology and Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Small signalling peptides play important roles in various plant processes, but information regarding their involvement in plant immunity is limited. We previously identified a novel small secreted protein in rice, called immune response peptide 1 (IRP1). Here, we studied the function of IRP1 in rice immunity. Rice plants overexpressing IRP1 enhanced resistance to the virulent rice blast fungus. Application of synthetic IRP1 to rice suspension cells triggered the expression of IRP1 itself and the defence gene phenylalanine ammonia-lyase 1 (PAL1). RNA-seq results revealed that 84% of genes up-regulated by IRP1, including 13 OsWRKY transcription factors, were also induced by a microbe-associated molecular pattern (MAMP), chitin, indicating that IRP1 and chitin share a similar signalling pathway. Co-treatment with chitin and IRP1 elevated the expression level of PAL1 and OsWRKYs in an additive manner. The increased chitin concentration arrested the induction of IRP1 and PAL1 expression by IRP1, but did not affect IRP1-triggered mitogen-activated protein kinases (MAPKs) activation. Collectively, our findings indicate that IRP1 functions as a phytocytokine in rice immunity regulating MAPKs and OsWRKYs that can amplify chitin and other signalling pathways, and provide new insights into how MAMPs and phytocytokines cooperatively regulate rice immunity. No potential conflict of interest relevant to this article was reported.

The Royal Society Acta Medica Okayama 0962-8452 289 1985 2022 Footedness for scratching itchy eyes in rodents 20221126 EN Yukitoshi Katayama Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi Ayane Miura Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi Keiko Takanami Mouse Genomics Resources Laboratory, National Institute of Genetics, Yata, Mishima Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi The neural bases of itchy eye transmission remain unclear compared with those involved in body itch. Here, we show in rodents that the gastrin-releasing peptide receptor (GRPR) of the trigeminal sensory system is involved in the transmission of itchy eyes. Interestingly, we further demonstrate a difference in scratching behaviour between the left and right hindfeet in rodents; histamine instillation into the conjunctival sac of both eyes revealed right-foot biased laterality in the scratching movements. Unilateral histamine instillation specifically induced neural activation in the ipsilateral sensory pathway, with no significant difference between the activations following left- and right-eye instillations. Thus, the behavioural laterality is presumably due to right-foot preference in rodents. Genetically modified rats with specific depletion of Grpr-expressing neurons in the trigeminal sensory nucleus caudalis of the medulla oblongata exhibited fewer and shorter histamine-induced scratching movements than controls and eliminated the footedness. These results taken together indicate that the Grp-expressing neurons are required for the transmission of itch sensation from the eyes, but that foot preference is generated centrally. These findings could open up a new field of research on the mechanisms of the laterality in vertebrates and also offer new potential therapeutic approaches to refractory pruritic eye disorders. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 76 6 2022 The Impact of Tofogliflozin on Physiological and Hormonal Function, Serum Electrolytes, and Cardiac Diastolic Function in Elderly Japanese Patients with Type 2 Diabetes Mellitus 705 713 EN Toshihiro Higashikawa Kanazawa Medical University Himi Municipal Hospital Tomohiko Ito Kanazawa Medical University Himi Municipal Hospital Takurou Mizuno Kanazawa Medical University Himi Municipal Hospital Keiichiro Ishigami Kanazawa Medical University Himi Municipal Hospital Kengo Kuroki Kanazawa Medical University Himi Municipal Hospital Naoto Maekawa Kanazawa Medical University Himi Municipal Hospital Daisuke Usuda Kanazawa Medical University Himi Municipal Hospital Toshihide Izumida Kanazawa Medical University Himi Municipal Hospital Shinya Yamada Kanazawa Medical University Himi Municipal Hospital Ryusho Sangen Kanazawa Medical University Himi Municipal Hospital Kazu Hamada Kanazawa Medical University Himi Municipal Hospital Jun Kiyosawa Kanazawa Medical University Himi Municipal Hospital Atsushi Saito Kanazawa Medical University Himi Municipal Hospital Masaharu Iguchi Kanazawa Medical University Himi Municipal Hospital Yuji Kasamaki Kanazawa Medical University Himi Municipal Hospital Takeshi Nakahashi Kanazawa Medical University Himi Municipal Hospital Akihiro Fukuda Kanazawa Medical University Himi Municipal Hospital Hitoshi Saito Kanazawa Medical University Himi Municipal Hospital Tsugiyasu Kanda Kanazawa Medical University Himi Municipal Hospital Masashi Okuro Department of Geriatric Medicine, Kanazawa Medical University Original Article /10.18926/AMO/64121 The sodium glucose transporter 2 (SGLT2) inhibitor tofogliflozin is a glucose-lowering drug that causes the excretion of surplus glucose by inhibiting SGLT2. Because of tofogliflozin’s osmotic diuresis mechanism, patients’ serum electrolytes, body fluid levels, and cardiac function must be monitored. We retrospectively analyzed the cases of 64 elderly Japanese patients with type 2 diabetes mellitus (T2DM) who received tofogliflozin for 3 months. Their HbA1c, serum electrolytes (sodium, potassium, chloride), hematocrit, brain natriuretic peptide (cardiac volume load marker) and renin and aldosterone (RAA; an index of regulatory hormones involved in body fluid retention) were continuously monitored during the investigation period. Renal function and cardiac function (by echocardiography) were assessed throughout the period. HbA1c significantly decreased (β1=−0.341, p<0.0001, linear regression analysis [LRA]). Most of the hormonal, electrolyte, and physiological parameters were maintained throughout the study period. In these circumstances, E/e’ tended to decrease (β1=−0.382, p=0.13, LRA). Compared to the baseline, E/e’ was significantly decreased at 1 and 3 months (p<0.01, p<0.05). In the higher E/e’ group (E/e’≥10, n=34), E/e’ decreased significantly (β1=−0.63, p<0.05, LRA). ΔE/e’ was correlated with body-weight change during treatment (r=0.64, p<0.01). The 3-month tofogliflozin treatment improved glycemic control and diastolic function represented by E/e’ in T2DM patients, without affecting serum electrolytes, renal function, or RAA. No negative impacts on the patients were observed. Three-month tofogliflozin treatment lowered glucose and improved cardiac diastolic function. 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.

Okayama University Medical School Acta Medica Okayama 0386-300X 76 5 2022 Viral Sequences Are Repurposed for Controlling Antiviral Responses as Non-Retroviral Endogenous Viral Elements 503 510 EN Hirohito Ogawa Department of Virology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomoyuki Honda Department of Virology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Review /10.18926/AMO/64025 Eukaryotic genomes contain numerous copies of endogenous viral elements (EVEs), most of which are considered endogenous retrovirus (ERV) sequences. Over the past decade, non-retroviral endogenous viral elements (nrEVEs) derived from ancient RNA viruses have been discovered. Several functions have been proposed for these elements, including antiviral defense. This review summarizes the current understanding of nrEVEs derived from RNA viruses, particularly endogenous bornavirus-like elements (EBLs) and endogenous filovirus-like elements (EFLs). EBLs are one of the most extensively studied nrEVEs. The EBL derived from bornavirus nucleoprotein (EBLN) is thought to function as a non-coding RNA or protein that regulates host gene expression or inhibits virus propagation. Ebolavirus and marburgvirus, which are filoviruses, induce severe hemorrhagic fever in humans and nonhuman primates. Although the ecology of filoviruses remains unclear, bats are believed to be potential reservoirs. Based on the knowledge from EBLs, it is postulated that EFLs in the bat genome help to maintain the balance between filovirus infection and the bat’s defense system, which may partially explain why bats act as potential reservoirs. Further research into the functions of nrEVEs could reveal novel antiviral systems and inspire novel antiviral approaches. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0959-3993 38 12 2022 Second extracellular protease mediating maturation of Vibrio mimicus hemolysin 241 EN Shin-ichi Miyoshi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Norie Toko Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tetsuya Dodo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ayako Nanko Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tamaki Mizuno Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Vibrio mimicus is a bacterium that causes gastroenteritis in humans. This pathogen produces an enterotoxic hemolysin called V. mimicus hemolysin (VMH), which is secreted extracellularly as an inactive 80-kDa protoxin and converted to a 66-kDa mature toxin through cleavage between Arg(151) and Ser(152). The 56-kDa serine protease termed V. mimicus trypsin-like protease (VmtA) is known to mediate this maturating process. However, some strains including strain ES-20 does not possess the vmtA gene. In the present study, the vmtA-negative strains were found to have a replaced gene that encodes a 43-kDa (403 aa) precursor of a serine protease designated by VmtX (V. mimicus trypsin-like protease X). To examine whether VmtX is also involved in the maturation of VMH, VmtX was isolated from the culture supernatant of V. mimicus strain NRE-20, a metalloprotease-negative mutant constructed from strain ES-20. Concretely, the culture supernatant was fractionated with 70% saturated ammonium sulfate and subjected to affinity column chromatography using a HiTrap Benzamidine FF column. The analysis of the N-terminal amino acid sequences of the proteins in the obtained VmtX preparation indicated that the 39-kDa protein was active VmtX consisting of 371 aa (Ile(33)-Ser(403)). The VmtX preparation was found to activate pro-VMH through generation of the 66-kDa protein. Additionally, treatment of the VmtX preparation with serine protease inhibitors, such as leupeptin and phenylmethylsulfonyl fluoride, significantly suppressed the activities to hydrolyze the specific peptide substrate and to synthesize the 66-kDa toxin. These findings indicate that VmtX is the second protease that mediats the maturation of VMH. No potential conflict of interest relevant to this article was reported.

Frontiers Media S.A. Acta Medica Okayama 1664-462X 13 2022 Time-series transcriptome of Brachypodium distachyon during bacterial flagellin-induced pattern-triggered immunity 1004184 EN Tsubasa Ogasahara Graduate School of Environmental and Life Science, Okayama University Yusuke Kouzai Graduate School of Environmental and Life Science, Okayama University Megumi Watanabe Graduate School of Environmental and Life Science, Okayama University Akihiro Takahashi Graduate School of Environmental and Life Science, Okayama University Kotaro Takahagi Kihara Institute for Biological Research, Yokohama City University June-Sik Kim Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University Keiichi Mochida Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of Brachypodium distachyon. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in B. distachyon. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction. 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.

MDPI Acta Medica Okayama 1999-4915 14 8 2022 A Transfectable Fusagravirus from a Japanese Strain of Cryphonectria carpinicola with Spherical Particles 1722 EN Subha Das Institute of Plant Science and Resources, Okayama University Sakae Hisano Institute of Plant Science and Resources, Okayama University Ana Eusebio-Cope Institute of Plant Science and Resources, Okayama University Hideki Kondo Institute of Plant Science and Resources, Okayama University Nobuhiro Suzuki Institute of Plant Science and Resources, Okayama University A novel dsRNA virus (Cryphonectria carpinicola fusagravirus 1, CcFGV1), isolated from a Japanese strain (JS13) of Cryphonectria carpinicola, was thoroughly characterized. The biological comparison of a set of isogenic CcFGV1-infected and -free (JS13VF) strains indicated asymptomatic infection by CcFGV1. The sequence analysis showed that the virus has a two open reading frame (ORF) genome of 9.6 kbp with the RNA-directed RNA polymerase domain encoded by ORF2. The N-terminal sequencing and peptide mass fingerprinting showed an N-terminally processed or degraded product (150 kDa) of the 5'-proximal ORF1-encoded protein (1462 amino acids) to make up the CcFGV1 spherical particles of similar to 40 nm in diameter. Interestingly, a portion of CcFGV1 dsRNA co-fractionated with a host protein of 70 kDa. The purified CcFGV1 particles were used to transfect protoplasts of JS13VF as well as the standard strain of an experimental model filamentous fungal host Cryphonectria parasitica. CcFGV1 was confirmed to be associated with asymptomatic infection of both fungi. RNA silencing was shown to target the virus in C. parasitica, resulting in reduced CcFGV1 accumulation by comparing the CcFGV1 content between RNA silencing-competent and -deficient strains. These results indicate the transfectability of spherical particles of a fusagravirus associated with asymptomatic infection. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 12 1 2022 Treatment resistance of rheumatoid arthritis relates to infection of periodontal pathogenic bacteria: a case-control cross-sectional study 12353 EN Kazu Takeuchi-Hatanaka Department of Periodontics and Endodontics, Okayama University Hospital Yoshinobu Koyama Center for Autoimmune Diseases, Division of Rheumatology, Japan Red Cross Okayama Hospital Kentaro Okamoto Department of Periodontics and Endodontics, Okayama University Hospital Kyosuke Sakaida Department of Periodontics and Endodontics, Okayama University Hospital Tadashi Yamamoto Department of Pathophysiology‑Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shogo Takashiba Department of Pathophysiology‑Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Recent studies have shown that periodontitis is associated with rheumatoid arthritis (RA) and periodontal bacteria, such as Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) are involved in the pathogenesis of RA via citrullinated proteins. Smoking has also been shown to be involved in the pathogenesis of RA; however, the extent of this involvement is still poorly understood. In addition, RA and polymyalgia rheumatica (PMR) are sometimes difficult to differentiate; however, the relationship between PMR and the factors from smoking and periodontal bacteria is unclear. The aim of this study was to clarify the relationship between periodontal pathogenic bacterial infections and smoking in patients with RA or PMR. This case-control study included 142 patients with untreated RA or PMR. This study evaluated the serum antibody titers against periodontal pathogenic bacterial antigens and an anti-citrullinated peptide antibody (ACPA). In patients with RA, the relationship between antibody titers and disease activity of RA and response after 3 months of treatment was also investigated. Additionally, the effects of smoking were evaluated. Although there was no significant difference in serum antibody titer against periodontal pathogenic bacteria between the ACPA-positive RA group and the ACPA-negative PMR group, we found an association between the elevated antibody titer against Pg and the degree of ACPA value, especially between negative group and high-value positive group (>= 100 U/mL). The antibody titers against Aa and Pg did not differ depending on disease activity score 28 (DAS28) at baseline; however, patients with high antibody titers had poor RA therapeutic response as judged by DAS28 after 3 months. We could not find any association between smoking and any of these parameters. Periodontal pathogenic bacteria, especially Pg, are associated with elevated ACPA levels. Our findings suggest that Pg and Aa infections interfere with the therapeutic response of RA. No potential conflict of interest relevant to this article was reported.

Wiley Periodicals, Inc Acta Medica Okayama 2055-5822 2022 Differences in extracellular fluid volume between acute heart failure patients with and without high systolic blood pressure EN Yusuke Namba Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kei Yunoki Department of Cardiovascular Medicine, Tsuyama Chuo Hospital Kazufumi Nakamura Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaro Ejiri Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takefumi Oka Department of Cardiovascular Medicine, Tsuyama Chuo Hospital Hiroshi Ito Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Aims Some reports have suggested that hypertensive acute heart failure (AHF) is caused by intravascular congestion, not interstitial congestion. We evaluated the differences in extracellular fluid volume assessed by bioelectrical impedance analysis (BIA) between AHF patients with and without high systolic blood pressure (sBP). <br> Methods This prospective single-centre study (UMIN000030266) included 178 patients hospitalized due to AHF between September 2017 and August 2018. We calculated extracellular water (ECW), intracellular water (ICW), total body water (TBW), and ECW-to-TBW ratio (oedema index: EI) by BIA and evaluated conventional parameters as follows: weight, N-terminal pro brain natriuretic peptide values, and echocardiography parameters on admission and before discharge. One-year outcomes included all-cause death and re-admission due to heart failure. We compared patients with sBP > 140 mmHg on admission [clinical scenario 1 (CS1) group] and with sBP of <= 140 mmHg on admission (non-CS1 group). <br> Results The mean age of the patients was 79.5 +/- 11.1 years, and 48.9% of the patients were female. EI on admission of 83 patients in the CS1 group was lower than that of 95 patients in the non-CS1 group. The change in EI from admission to before discharge was no significant in the CS1 group but was significant in the non-CS1 group. Comparing the changes from admission to before discharge between the CS1 and the non-CS1 group, delta ECW, delta ICW, delta TBW, and delta EI of the CS1 group were significantly smaller than those of the non-CS1 group. During the 1-year follow-up period after discharge of the 178 patients, the numbers of deaths and re-admissions due to acute HF were 26 (15%) and 49 (28%), respectively. Patients with high EI before discharge [> 0.408 (median)] had significantly more cardiac events than patients with low EI [hazard ratio (HR): 2.15, 95% confidence interval (CI): 1.30-3.55]. Cox regression analysis revealed that higher EI as a continuous variable was significantly associated with worse outcome in non-CS1 group (HR: 1.46, 95% CI: 1.13-1.87), but not significantly associated with worse outcome in CS1 group (HR: 1.29, 95% CI: 0.98-1.69). <br> Conclusions EI on admission in patients with high sBP was not elevated, and changes in ECW, ICW, TBW, and EI in patients with high sBP were smaller than those in patients without high sBP. EI measured by BIA could distinguish AHF with interstitial or intravascular congestion. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1420-3049 27 12 2022 Rational Design of Peptides Derived from Odorant-Binding Proteins for SARS-CoV-2-Related Volatile Organic Compounds Recognition 3917 EN Jin Wang Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kenji Sakai Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toshihiko Kiwa Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Peptides are promising molecular-binding elements and have attracted great interest in novel biosensor development. In this study, a series of peptides derived from odorant-binding proteins (OBPs) were rationally designed for recognition of SARS-CoV-2-related volatile organic compounds (VOCs). Ethanol, nonanal, benzaldehyde, acetic acid, and acetone were selected as representative VOCs in the exhaled breath during the COVID-19 infection. Computational docking and prediction tools were utilized for OBPs peptide characterization and analysis. Multiple parameters, including the docking model, binding affinity, sequence specification, and structural folding, were investigated. The results demonstrated a rational, rapid, and efficient approach for designing breath-borne VOC-recognition peptides, which could further improve the biosensor performance for pioneering COVID-19 screening and many other applications. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0960-894X 68 2022 Ultrasound-dependent RNAi using TatU1A-rose bengal conjugate 128767 EN Nanako Sumi Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shota Nagahiro Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Eiji Nakata Institute of Advanced Energy, Kyoto University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tat-U1A-rose bengal conjugate (TatU1A-RB) was prepared as an ultrasound-sensitive RNA carrier molecule. This molecule consists of Tat cell-penetrating peptide, U1A RNA-binding protein, and rose bengal as a sonosensitizer. We demonstrated that TatU1A-RB delivered RNA via the endocytosis pathway, which was followed by ultrasound-dependent endosomal escape and cytosolic dispersion of the RNA. A short hairpin RNA (shRNA) delivered by TatU1A-RB mediated RNA interference (RNAi) ultrasound-dependently. Even by ultrasound irradiation through blood cells, RNAi could be induced with TatU1A-RB and the shRNA. This ultrasound-dependent cytosolic RNA delivery method will serve as the basis for a new approach to nucleic acid therapeutics. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2227-9717 10 5 2022 Helical Foldamers and Stapled Peptides as New Modalities in Drug Discovery: Modulators of Protein-Protein Interactions 924 EN Keisuke Tsuchiya Division of Organic Chemistry, National Institute of Health Sciences Takashi Kurohara Division of Organic Chemistry, National Institute of Health Sciences Kiyoshi Fukuhara Graduate School of Pharmacy, Showa University Takashi Misawa Division of Organic Chemistry, National Institute of Health Sciences Yosuke Demizu Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University A "foldamer" is an artificial oligomeric molecule with a regular secondary or tertiary structure consisting of various building blocks. A "stapled peptide" is a peptide with stabilized secondary structures, in particular, helical structures by intramolecular covalent side-chain cross-linking. Helical foldamers and stapled peptides are potential drug candidates that can target protein-protein interactions because they enable multipoint molecular recognition, which is difficult to achieve with low-molecular-weight compounds. This mini-review describes a variety of peptide-based foldamers and stapled peptides with a view to their applications in drug discovery, including our recent progress. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2022 Study on the mode of action of CEP peptide as an endogenous suppressor in Arabidopsis thaliana EN APRILIA NUR FITRIANTI Graduate School of Environmental and Life Science, Okayama University No potential conflict of interest relevant to this article was reported.

American Association for the Advancement of Science (AAAS) Acta Medica Okayama 2375-2548 8 9 2022 Vasopressin-oxytocin–type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians eabk0331 EN Aoshi Kobayashi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Mayuko Hamada Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Masa-aki Yoshida Oki Marine Biological Station, Shimane University Yasuhisa Kobayashi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Naoaki Tsutsui Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Toshio Sekiguchi Noto Marine Laboratory, Institute of Nature and Environmental Technology, Division of Marine Environmental Studies, Kanazawa University Yuta Matsukawa Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Sho Maejima Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Joseph J. Gingell Vertex Pharmaceuticals (Europe) Ltd. Shoko Sekiguchi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ayumu Hamamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Debbie L. Hay School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland John F. Morris Department of Physiology, Anatomy, and Genetic, Le Gros Clark Building, University of Oxford Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado Vasopressin/oxytocin (VP/OT)–related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the “platytocin” system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an “antidiuretic hormone” and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses. No potential conflict of interest relevant to this article was reported.

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

MDPI Acta Medica Okayama 1422-0067 22 19 2021 Sexual Experience Induces the Expression of Gastrin-Releasing Peptide and Oxytocin Receptors in the Spinal Ejaculation Generator in Rats 10362 EN Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ryota Ueda Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ryoko Kumagai Department of Animal Sciences, Teikyo University of Science Junta Nagafuchi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takashi Ito Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasuhiko Kondo Department of Animal Sciences, Teikyo University of Science Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Male sexual function in mammals is controlled by the brain neural circuits and the spinal cord centers located in the lamina X of the lumbar spinal cord (L3-L4). Recently, we reported that hypothalamic oxytocin neurons project to the lumbar spinal cord to activate the neurons located in the dorsal lamina X of the lumbar spinal cord (dXL) via oxytocin receptors, thereby facilitating male sexual activity. Sexual experiences can influence male sexual activity in rats. However, how this experience affects the brain-spinal cord neural circuits underlying male sexual activity remains unknown. Focusing on dXL neurons that are innervated by hypothalamic oxytocinergic neurons controlling male sexual function, we examined whether sexual experience affects such neural circuits. We found that >50% of dXL neurons were activated in the first ejaculation group and similar to 30% in the control and intromission groups in sexually naive males. In contrast, in sexually experienced males, similar to 50% of dXL neurons were activated in both the intromission and ejaculation groups, compared to similar to 30% in the control group. Furthermore, sexual experience induced expressions of gastrin-releasing peptide and oxytocin receptors in the lumbar spinal cord. This is the first demonstration of the effects of sexual experience on molecular expressions in the neural circuits controlling male sexual activity in the spinal cord. No potential conflict of interest relevant to this article was reported.

Wiley Periodicals, Inc Acta Medica Okayama 2055-5822 2021 Effects of luseogliflozin on estimated plasma volume in patients with heart failure with preserved ejection fraction EN Mitsutaka Nakashima Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toru Miyoshi Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaro Ejiri Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hajime Kihara Department of Internal Medicine, Kihara Cardiovascular Clinic Yoshiki Hata Department of Cardiology, Minamino Cardiovascular Hospital Toshihiko Nagano Department of Internal Medicine, Iwasa Hospital, Atsushi Takaishi Department of Cardiology, Mitoyo General Hospital Hironobu Toda Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiji Nanba Department of Cardiology, Okayama Rosai Hospital Yoichi Nakamura Department of Cardiovascular Medicine, Specified Clinic of Soyokaze CardioVascular Medicine and Diabetes Care, Matsuyama Satoshi Akagi Department of Internal Medicine, Akaiwa Medical Association Hospital Satoru Sakuragi Department of Cardiovascular Medicine, Iwakuni Clinical Center Taro Minagawa Department of Internal Medicine, Minagawa Cardiovascular Clinic Yusuke Kawai Department of Cardiovascular Medicine, Okayama City Hospital Nobuhiro Nishii Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Soichiro Fuke Department of Cardiovascular Medicine, Japanese Red Cross Okayama Hospital Masaki Yoshikawa Department of Cardiology, Fukuyama City Hospital Kazufumi Nakamura Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroshi Ito Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences MUSCAT-HF Study Investigators Aims<br> Sodium glucose co-transporter 2 inhibitors have diuretic effects in both patients with glycosuria and with natriuresis. We sought to assess the effect of luseogliflozin on estimated plasma volume (ePV) in patients with type 2 diabetes and heart failure with preserved ejection fraction (HFpEF). <br> Methods and results<br> This study was a post-hoc analysis of the MUSCAT-HF trial (UMIN000018395), a multicentre, prospective, open-label, randomized controlled trial that assessed the effect of 12 weeks of luseogliflozin (2.5 mg, once daily, n = 83) as compared with voglibose (0.2 mg, three times daily, n = 82) on the reduction in brain natriuretic peptide (BNP) in patients with type 2 diabetes and HFpEF. The analysis compared the change in ePV calculated by the Straus formula from baseline to Weeks 4, 12, and 24, using a mixed-effects model for repeated measures. We also estimated the association between changes in ePV and changes in other clinical parameters, including BNP levels. Luseogliflozin significantly reduced ePV as compared to voglibose at Week 4 {adjusted mean group-difference -6.43% [95% confidence interval (CI): -9.11 to -3.74]}, at Week 12 [-8.73% (95%CI: -11.40 to -6.05)], and at Week 24 [-11.02% (95%CI: -13.71 to -8.33)]. The effect of luseogliflozin on these parameters was mostly consistent across various patient clinical characteristics. The change in ePV at Week 12 was significantly associated with log-transformed BNP (r = 0.197, P = 0.015) and left atrial volume index (r = 0.283, P = 0.019). <br> Conclusions<br> Luseogliflozin significantly reduced ePV in patients with type 2 diabetes and HFpEF, as compared with voglibose. The reduction of intravascular volume by luseogliflozin may provide clinical benefits to patients with type 2 diabetes and HFpEF. No potential conflict of interest relevant to this article was reported.

BMJ Acta Medica Okayama 1757-790X 14 10 2021 Idiopathic combined adrenocorticotropin and growth hormone deficiency mimicking chronic fatigue syndrome e244861 EN Kazuki Tokumasu Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kanako Ochi Center for Education in Medicine and Health Sciences, Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Fumio Otsuka Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences A 42-year-old man who had suffered from severe fatigue for 5 years was diagnosed as having chronic fatigue syndrome (CFS) and fibromyalgia. Endocrinological workup using combined anterior pituitary function tests showed that the patient had adrenocorticotropin hormone (ACTH) deficiency, with a normal pituitary MRI. Treatment with a physiologic dose of oral hydrocortisone replacement physically ameliorated his general fatigue. A secondary workup using a growth hormone-releasing peptide-2 test revealed that he also had growth hormone (GH) deficiency, and GH replacement therapy was started. His muscle pain and depression were improved by the therapy. Here, we present a rare case of combined deficiency of ACTH and GH in a middle-aged man with severe general fatigue. This case report aims to raise awareness of combined deficiency of ACTH and GH as a differential diagnosis of CFS and its mimics. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 75 6 2021 Multiple Roles of Histidine-Rich Glycoprotein in Vascular Homeostasis and Angiogenesis 671 675 EN Shangze Gao Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiro Nishibori Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Review /10.18926/AMO/62805 Histidine-rich glycoprotein (HRG) is a 75 kDa plasma protein that is synthesized in the liver of many verte-brates and present in their plasma at relatively high concentrations of 100-150 μg/mL. HRG is an abundant and well-characterized protein having a multidomain structure that enable it to interact with many ligands, func-tion as an adaptor molecule, and participate in numerous physiological and pathological processes. As a plasma protein, HRG has been reported to regulate vascular biology, including coagulation, fibrinolysis and angiogenesis, through its binding with several ligands (heparin, FXII, fibrinogen, thrombospondin, and plas-minogen) and interaction with many types of cells (endothelial cells, erythrocytes, neutrophils and platelets). This review aims to summarize the roles of HRG in maintaining vascular homeostasis and regulating angiogen-esis in various pathological conditions. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 2050-0904 9 9 2021 Longitudinal observation of insulin secretory ability before and after the onset of immune checkpoint inhibitor-induced diabetes mellitus: A report of two cases e04574 EN Noriko Fujiwara Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Mayu Watanabe Department of Primary Care and Medical Education, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Akihiro Katayama Diabetes Center, Okayama University Hospital Yohei Noda Department of Otolaryngology-Head and Neck Surgery, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Jun Eguchi Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hitomi Kataoka Department of Primary Care and Medical Education, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Shunsuke Kagawa Minimally Invasive Therapy Center, Okayama University Hospital Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Immune checkpoint inhibitor-induced diabetes mellitus is a rare immune-related adverse event. This report illustrates clinical data and insulin secretory ability before and after the onset of immune checkpoint inhibitor-induced diabetes. No potential conflict of interest relevant to this article was reported.

Frontiers Media SA Acta Medica Okayama 1664-462X 12 2021 Purification, Characterization, and Gene Expression of Rice Endo-beta-N-Acetylglucosaminidase, Endo-Os 647684 EN Megumi Maeda Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Naoko Okamoto Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Node Araki Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University Yoshinobu Kimura Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University In the endoplasmic reticulum-associated degradation system of plant and animal cells, high-mannose type free N-glycans (HMT-FNGs) are produced from misfolded glycoproteins prior to proteasomal degradation, and two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endo-beta-N-acetylglucosaminidase (endo-beta-GlcNAc-ase), are involved in the deglycosylation. Although the physiological functions of these FNGs in plant growth and development remain to be elucidated, detailed characterization of cPNGase and endo-beta-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-beta-GlcNAc-ases and preliminarily reported the gene information of rice endo-beta-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-beta-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-beta-GlcNAc-ase genes were knocked-out based on the Endo-Os gene. In this report, we describe the purification, characterization, amino acid sequence, and gene cloning of Endo-Os in detail. Purified Endo-Os, with an optimal pH of 6.5, showed high activity for high-mannose type N-glycans bearing the Man alpha 1-2Man alpha 1-3Man beta 1 unit; this substrate specificity was almost the same as that of other plant endo-beta-GlcNAc-ases, suggesting that Endo-Os plays a critical role in the production of HTM-FNGs in the cytosol. Electrospray ionization-mass spectrometry analysis of the tryptic peptides revealed 17 internal amino acid sequences, including the C terminus; the N-terminal sequence could not be identified due to chemical modification. These internal amino acid sequences were consistent with the amino acid sequence (UniProt ID: Q5W6R1) deduced from the Oryza sativa cDNA clone AK112067 (gene ID: Os05g0346500). Recombinant Endo-Os expressed in Escherichia coli using cDNA showed the same enzymatic properties as those of native Endo-Os. No potential conflict of interest relevant to this article was reported.

Nature Research Acta Medica Okayama 2045-2322 11 1 2021 The gastrin-releasing peptide/bombesin system revisited by a reverse-evolutionary study considering Xenopus 13315 EN Asuka Hirooka Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Mayuko Hamada Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Daiki Fujiyama Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keiko Takanami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasuhisa Kobayashi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yukitoshi Katayama Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the 'gut-brain peptide' systems. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2076-3417 11 10 2021 Fibril Growth Behavior of Amyloid beta on Polymer-Based Planar Membranes: Implications for the Entanglement and Hydration of Polymers 4408 EN Toshinori Shimanouchi Graduate School of Environment and Life Science, Okayama University Miki Iwamura Graduate School of Environment and Life Science, Okayama University Shintaro Deguchi Graduate School of Environment and Life Science, Okayama University Yukitaka Kimura Graduate School of Environment and Life Science, Okayama University The design of biosensors and artificial organs using biocompatible materials with a low affinity for amyloid beta peptide (A beta) would contribute to the inhibition of fibril growth causing Alzheimer's disease. We systematically studied the amyloidogenicity of A beta on various planar membranes. The planar membranes were prepared using biocompatible polymers, viz., poly(methyl methacrylate) (PMMA), polysulfone (PSf), poly(L-lactic acid) (PLLA), and polyvinylpyrrolidone (PVP). Phospholipids from biomembranes, viz., 1,2-dioleoyl-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-phosphatidylcholine (DPPC), and polyethylene glycol-graft-phosphatidyl ethanolamine (PEG-PE) were used as controls. Phospholipid- and polymer-based membranes were prepared to determine the kinetics of A beta fibril formation. Rates of A beta nucleation on the PSf- and DPPC-based membranes were significantly higher than those on the other membranes. A beta accumulation, calculated by the change in frequency of a quartz crystal microbalance (QCM), followed the order: PSf > PLLA > DOPC > PMMA, PVP, DPPC, and PEG-PE. Nucleation rates exhibited a positive correlation with the corresponding accumulation (except for the DPPC-based membrane) and a negative correlation with the molecular weight of the polymers. Strong hydration along the polymer backbone and polymer-A beta entanglement might contribute to the accumulation of A beta and subsequent fibrillation. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2075-1729 11 4 2021 Theranostics in Boron Neutron Capture Therapy 330 EN Wolfgang A. G. Sauerwein Neutron Therapy Research Center, Okayama University Lucie Sancey UGA/Inserm U 1209/CNRS UMR 5309 Joint Research Center, Institute for Advanced Biosciences Evamarie Hey-Hawkins Deutsche Gesellschaft für Bor-Neutroneneinfangtherapie DGBNCT e.V. Martin Kellert Institute of Inorganic Chemistry, Department of Chemistry and Mineralogy, University Leipzig Luigi Panza Deutsche Gesellschaft für Bor-Neutroneneinfangtherapie DGBNCT e.V. Daniela Imperio Deutsche Gesellschaft für Bor-Neutroneneinfangtherapie DGBNCT e.V. Marcin Balcerzyk Departamento de Fisiología Medica y Biofísica, Universidad de Sevilla Giovanna Rizzo Institute for Biomedical Technologies (ITB-CNR) Elisa Scalco Institute for Biomedical Technologies (ITB-CNR) Ken Herrmann Department for Nuclear Medicine, University Hospital Essen Pierluigi Mauri Deutsche Gesellschaft für Bor-Neutroneneinfangtherapie DGBNCT e.V. Antonella De Palma Proteomics and Metabolomics Laboratory, ELIXIR Infrastructure, National Research Council (ITB-CNR) Andrea Wittig Deutsche Gesellschaft für Bor-Neutroneneinfangtherapie DGBNCT e.V. Boron neutron capture therapy (BNCT) has the potential to specifically destroy tumor cells without damaging the tissues infiltrated by the tumor. BNCT is a binary treatment method based on the combination of two agents that have no effect when applied individually: B-10 and thermal neutrons. Exclusively, the combination of both produces an effect, whose extent depends on the amount of B-10 in the tumor but also on the organs at risk. It is not yet possible to determine the B-10 concentration in a specific tissue using non-invasive methods. At present, it is only possible to measure the B-10 concentration in blood and to estimate the boron concentration in tissues based on the assumption that there is a fixed uptake of B-10 from the blood into tissues. On this imprecise assumption, BNCT can hardly be developed further. A therapeutic approach, combining the boron carrier for therapeutic purposes with an imaging tool, might allow us to determine the B-10 concentration in a specific tissue using a non-invasive method. This review provides an overview of the current clinical protocols and preclinical experiments and results on how innovative drug development for boron delivery systems can also incorporate concurrent imaging. The last section focuses on the importance of proteomics for further optimization of BNCT, a highly precise and personalized therapeutic approach. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 22 7 2021 In Vivo Electrophysiology of Peptidergic Neurons in Deep Layers of the Lumbar Spinal Cord after Optogenetic Stimulation of Hypothalamic Paraventricular Oxytocin Neurons in Rats 3400 EN Daisuke Uta Department of Applied Pharmacology, Faculty of Pharmaceutical Sciences, University of Toyama Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University The spinal ejaculation generator (SEG) is located in the central gray (lamina X) of the rat lumbar spinal cord and plays a pivotal role in the ejaculatory reflex. We recently reported that SEG neurons express the oxytocin receptor and are activated by oxytocin projections from the paraventricular nucleus of hypothalamus (PVH). However, it is unknown whether the SEG responds to oxytocin in vivo. In this study, we analyzed the characteristics of the brain-spinal cord neural circuit that controls male sexual function using a newly developed in vivo electrophysiological technique. Optogenetic stimulation of the PVH of rats expressing channel rhodopsin under the oxytocin receptor promoter increased the spontaneous firing of most lamina X SEG neurons. This is the first demonstration of the in vivo electrical response from the deeper (lamina X) neurons in the spinal cord. Furthermore, we succeeded in the in vivo whole-cell recordings of lamina X neurons. In vivo whole-cell recordings may reveal the features of lamina X SEG neurons, including differences in neurotransmitters and response to stimulation. Taken together, these results suggest that in vivo electrophysiological stimulation can elucidate the neurophysiological response of a variety of spinal neurons during male sexual behavior. No potential conflict of interest relevant to this article was reported.

Royal Society of Chemistry (RSC) Acta Medica Okayama 1463-9076 23 10 2021 Two different regimes in alcohol-induced coil–helix transition: effects of 2,2,2-trifluoroethanol on proteins being either independent of or enhanced by solvent structural fluctuations 5760 5772 EN Hiroyo Ohgi Department of Chemistry, Graduate School of Science, Chiba University Hiroshi Imamura Department of Applied Chemistry, College of Life Sciences, Ritsumeikan University Tomonari Sumi Research Institute for Interdisciplinary Science, Okayama University Keiko Nishikawa Department of Chemistry, Graduate School of Science, Chiba University Yoshikata Koga Department of Chemistry, The University of British Columbia Peter Westh Department of Biotechnology and Biomedicine, Technical University of Denmark Takeshi Morita Department of Chemistry, Graduate School of Science, Chiba University Inhomogeneous distribution of constituent molecules in a mixed solvent has been known to give remarkable effects on the solute, e.g., conformational changes of biomolecules in an alcohol-water mixture. We investigated the general effects of 2,2,2-trifiuoroethanoE (TFE) on proteins/peptides in a mixture of water and TFE using melittin as a model protein. Fluctuations and Kirkwood-Buff integrals (KBIs) in the TFE-H2O mixture, quantitative descriptions of inhomogeneity, were determined by smallangle X-ray scattering investigation and compared with those in the aqueous solutions of other alcohols. The concentration fluctuation for the mixtures ranks as methanol < ethanol << TFE < tert-butanol < 1-propanol, indicating that the inhomogeneity of molecular distribution in the TFE-H2O mixture is unexpectedly comparable to those in the series of mono-ok. On the basis of the concentration dependence of KBIs between the TFE molecules, it was found that a strong attraction between the TFE molecules is not necessarily important to induce helix conformation, which is inconsistent with the previously proposed mechanism. To address this issue, by combining the KBIs and the helix contents reported by the experimental spectroscopic studies, we quantitatively evaluated the change in the preferential binding parameter of TFE to melittin attributed to the coil-helix transition. As a result, we found two different regimes on TFE-induced helix formation. In the dilute concentration region of TFE below similar to 2 M, where the TFE molecules are not aggregated among themselves, the excess preferential binding of TFE to the helix occurs due to the direct interaction between them, namely independent of the solvent fluctuation. In the higher concentration region above similar to 2 M, in addition to the former effect, the excess preferential binding is significantly enhanced by the solvent fluctuation. This scheme should be held as general cosoEvent effects of TFE on proteins/peptides. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 00223549 110 2021 Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells 1788 1798 EN Melissa Siaw Han Lim Yuki Nishiyama Takashi Ohtsuki Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Graduate School of Natural Science and Technology, Okayama University Hirotsugu Kobuchi Kazuko Kobayashi Collaborative Research Center (OMIC), Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eiji Matsuura The A3B-type Lactosome comprised of poly(sarcosine)3-block-poly(l-lactic acid), a biocompatible and biodegradable polymeric nanomicelle, was reported to accumulate in tumors in vivo via the enhanced permeability and retention (EPR) effect. Recently, the cellular uptake of Lactosome particles was enhanced through the incorporation of a cell-penetrating peptide (CPP), L7EB1. However, the ability of Lactosome as a drug delivery carrier has not been established. Herein, we have developed a method to conjugate the A3B-type Lactosome with ATP-binding cassette transporter G2 (ABCG2) siRNA for inducing in vitro apoptosis in the cancer cell lines PANC-1 and NCI-H226. The L7EB1 peptide facilitates the cellular uptake efficiency of Lactosome but does not deliver siRNA into cytosol. To establish the photoinduced cytosolic dispersion of siRNA, a photosensitizer loaded L7EB1-Lactosome was prepared, and the photosensitizer 5,10,15,20-tetra-kis(pentafluorophenyl)porphyrin (TPFPP) showed superiority in photoinduced cytosolic dispersion. We exploited the combined effects of enhanced cellular uptake by L7EB1 and photoinduced endosomal escape by TPFPP to efficiently deliver ABCG2 siRNA into the cytosol for gene silencing. Moreover, the silencing of ABCG2, a protoporphyrin IX (PpIX) transporter, also mediated photoinduced cell death via 5-aminolevulinic acid (ALA)-mediated PpIX accumulated photodynamic therapy (PDT). The synergistic capability of the L7EB1/TPFPP/siRNA-Lactosome complex enabled both gene silencing and PDT. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2075-1729 11 2 2021 A Novel 89Zr-labeled DDS Device Utilizing Human IgG Variant (scFv): “Lactosome” Nanoparticle-Based Theranostics for PET Imaging and Targeted Therapy 158 EN Melissa Siaw Han Lim Department of Cell Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Fumiaki Takenaka Collaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kazuko Kobayashi Collaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Masaru Akehi Collaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hirotaka Uji Department of Material Chemistry, Graduate School of Engineering, Kyoto University Hirotsugu Kobuchi Department of Cell Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Takanori Sasaki Collaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Eiichi Ozeki Technology Research Laboratory, Shimadzu Corporation Eiji Matsuura Department of Cell Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University “Theranostics,” a new concept of medical advances featuring a fusion of therapeutic and diagnostic systems, provides promising prospects in personalized medicine, especially cancer. The theranostics system comprises a novel 89Zr-labeled drug delivery system (DDS), derived from the novel biodegradable polymeric micelle, “Lactosome” nanoparticles conjugated with specific shortened IgG variant, and aims to successfully deliver therapeutically effective molecules, such as the apoptosis-inducing small interfering RNA (siRNA) intracellularly while offering simultaneous tumor visualization via PET imaging. A 27 kDa-human single chain variable fragment (scFv) of IgG to establish clinically applicable PET imaging and theranostics in cancer medicine was fabricated to target mesothelin (MSLN), a 40 kDa-differentiation-related cell surface glycoprotein antigen, which is frequently and highly expressed by malignant tumors. This system coupled with the cell penetrating peptide (CPP)-modified and photosensitizer (e.g., 5, 10, 15, 20-tetrakis (4-aminophenyl) porphyrin (TPP))-loaded Lactosome particles for photochemical internalized (PCI) driven intracellular siRNA delivery and the combination of 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) offers a promising nano-theranostic-based cancer therapy via its targeted apoptosis-inducing feature. This review focuses on the combined advances in nanotechnology and material sciences utilizing the “89Zr-labeled CPP and TPP-loaded Lactosome particles” and future directions based on important milestones and recent developments in this platform. No potential conflict of interest relevant to this article was reported.

Massachusetts Medical Society Acta Medica Okayama 0028-4793 384 2021 Pegcetacoplan versus Eculizumab in Paroxysmal Nocturnal Hemoglobinuria 1028 1037 EN Peter Hillmen the Department of Haematology, St. James’s University Hospital Jeff Szer the Department of Clinical Haematology, Peter MacCallum Cancer Center and Royal Melbourne Hospital Ilene Weitz Jane Anne Nohl Division of Hematology, Keck School of Medicine of USC Alexander Röth the Department of Hematology, West German Cancer Center University Hospital Essen, University of Duisburg-Essen Britta Höchsmann the Institute of Transfusion Medicine, University of Ulm and Institute of Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Transfusion Service and University Hospital Ulm Jens Panse the Department of Oncology, Hematology, Hemostaseology and Stem Cell Transplantation, University Hospital RWTH Aachen Kensuke Usuki the Department of Hematology, NTT Medical Center Tokyo Morag Griffin the Department of Haematology, St. James’s University Hospital Jean-Jacques Kiladjian Centre d’Investigations Cliniques, Hôpital Saint-Louis, Assistance Publique–Hôpitaux de Paris, Université de Paris Carlos de Castro the Department of Medicine, Division of Hematologic Malignancies and Cellular Therapy, Duke University Hisakazu Nishimori the Department of Hematology and Oncology, Okayama University Hospital Lisa Tan Lisa Tan Pharma Consulting Mohamed Hamdani Apellis Pharmaceuticals Pascal Deschatelets Apellis Pharmaceuticals Cedric Francois Apellis Pharmaceuticals Federico Grossi Apellis Pharmaceuticals Temitayo Ajayi Apellis Pharmaceuticals Antonio Risitano the Hematology and BMT Unit, AORN San Giuseppe Moscati Régis Peffault de la Tour the French Reference Center for Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria, Hôpital Saint-Louis, Assistance Publique–Hôpitaux de Paris, Université de Paris Background</br> Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease characterized by chronic complement-mediated hemolysis. C5 inhibition controls intravascular hemolysis in untreated PNH but cannot address extravascular hemolysis. Pegcetacoplan, a pegylated peptide targeting proximal complement protein C3, potentially inhibits both intravascular and extravascular hemolysis.</br> Methods</br> We conducted a phase 3 open-label, controlled trial to assess the efficacy and safety of pegcetacoplan as compared with eculizumab in adults with PNH and hemoglobin levels lower than 10.5 g per deciliter despite eculizumab therapy. After a 4-week run-in phase in which all patients received pegcetacoplan plus eculizumab, we randomly assigned patients to subcutaneous pegcetacoplan monotherapy (41 patients) or intravenous eculizumab (39 patients). The primary end point was the mean change in hemoglobin level from baseline to week 16. Additional clinical and hematologic markers of hemolysis and safety were assessed.</br> Results</br> Pegcetacoplan was superior to eculizumab with respect to the change in hemoglobin level from baseline to week 16, with an adjusted (least squares) mean difference of 3.84 g per deciliter (P<0.001). A total of 35 patients (85%) receiving pegcetacoplan as compared with 6 patients (15%) receiving eculizumab no longer required transfusions. Noninferiority of pegcetacoplan to eculizumab was shown for the change in absolute reticulocyte count but not for the change in lactate dehydrogenase level. Functional Assessment of Chronic Illness Therapy–Fatigue scores improved from baseline in the pegcetacoplan group. The most common adverse events that occurred during treatment in the pegcetacoplan and eculizumab groups were injection site reactions (37% vs. 3%), diarrhea (22% vs. 3%), breakthrough hemolysis (10% vs. 23%), headache (7% vs. 23%), and fatigue (5% vs. 15%). There were no cases of meningitis in either group.</br> Conclusions</br> Pegcetacoplan was superior to eculizumab in improving hemoglobin and clinical and hematologic outcomes in patients with PNH by providing broad hemolysis control, including control of intravascular and extravascular hemolysis. (Funded by Apellis Pharmaceuticals; PEGASUS ClinicalTrials.gov, NCT03500549. opens in new tab.) No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2079-7737 10 2 2021 Identification of an RNA Silencing Suppressor Encoded by a Symptomless Fungal Hypovirus, Cryphonectria Hypovirus 4 100 EN Annisa Aulia Institute of Plant Science and Resources (IPSR), Okayama University Kiwamu Hyodo Institute of Plant Science and Resources (IPSR), Okayama University Sakae Hisano Institute of Plant Science and Resources (IPSR), Okayama University Hideki Kondo Institute of Plant Science and Resources (IPSR), Okayama University Bradley I. Hillman Plant Biology and Pathology, Rutgers University Nobuhiro Suzuki Institute of Plant Science and Resources (IPSR), Okayama University Previously, we have reported the ability of a symptomless hypovirus Cryphonectria hypovirus 4 (CHV4) of the chestnut blight fungus to facilitate stable infection by a co-infecting mycoreovirus 2 (MyRV2)―likely through the inhibitory effect of CHV4 on RNA silencing (Aulia et al., Virology, 2019). In this study, the N-terminal portion of the CHV4 polyprotein, termed p24, is identified as an autocatalytic protease capable of suppressing host antiviral RNA silencing. Using a bacterial expression system, CHV4 p24 is shown to cleave autocatalytically at the di-glycine peptide (Gly214-Gly215) of the polyprotein through its protease activity. Transgenic expression of CHV4 p24 in Cryphonectria parasitica suppresses the induction of one of the key genes of the antiviral RNA silencing, dicer-like 2, and stabilizes the infection of RNA silencing-susceptible virus MyRV2. This study shows functional similarity between CHV4 p24 and its homolog p29, encoded by the symptomatic prototype hypovirus CHV1. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 1664-462X 11 2021 Cytosolic Free N-Glycans Are Retro-Transported Into the Endoplasmic Reticulum in Plant Cells 610124 EN Makoto Katsube Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Natsuki Ebara Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Megumi Maeda Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University Yoshinobu Kimura Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University During endoplasmic reticulum (ER)-associated degradation, free N-glycans (FNGs) are produced from misfolded nascent glycoproteins via the combination of the cytosolic peptide N-glycanase (cPNGase) and endo-beta-N-acetylglucosaminidase (ENGase) in the plant cytosol. The resulting high-mannose type (HMT)-FNGs, which carry one GlcNAc residue at the reducing end (GN1-FNGs), are ubiquitously found in developing plant cells. In a previous study, we found that HMT-FNGs assisted in protein folding and inhibited beta-amyloid fibril formation, suggesting a possible biofunction of FNGs involved in the protein folding system. However, whether these HMT-FNGs occur in the ER, an organelle involved in protein folding, remained unclear. On the contrary, we also reported the presence of plant complex type (PCT)-GN1-FNGs, which carry the Lewis(a) epitope at the non-reducing end, indicating that these FNGs had been fully processed in the Golgi apparatus. Since plant ENGase was active toward HMT-N-glycans but not PCT-N-glycans that carry beta 1-2xylosyl and/or alpha 1-3 fucosyl residue(s), these PCT-GN1-FNGs did not appear to be produced from fully processed glycoproteins that harbored PCT-N-glycans via ENGase activity. Interestingly, PCT-GN1-FNGs were found in the extracellular space, suggesting that HMT-GN1-FNGs formed in the cytosol might be transported back to the ER and processed in the Golgi apparatus through the protein secretion pathway. As the first step in elucidating the production mechanism of PCT-GN1-FNGs, we analyzed the structures of free oligosaccharides in plant microsomes and proved that HMT-FNGs (Man(9-7)GlcNAc(1) and Man(9-8)GlcNAc(2)) could be found in microsomes, which almost consist of the ER compartments. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 75 1 2021 Marked Hypertriglyceridemia in a Patient with type 2 Diabetes Receiving SGLT2 Inhibitors 103 107 EN Mayumi Senoo Department of Internal Medicine, Diabetes Center, Okayama Saiseikai General Hospital Atsuhito Tone Department of Internal Medicine, Diabetes Center, Okayama Saiseikai General Hospital Yusuke Imai Department of Internal Medicine, Diabetes Center, Okayama Saiseikai General Hospital Satoko Watanabe Department of Internal Medicine, Diabetes Center, Okayama Saiseikai General Hospital Mitsuhiro Kaneto Department of Internal Medicine, Diabetes Center, Okayama Saiseikai General Hospital Yasuyuki Shimomura Department of Internal Medicine, Diabetes Center, Okayama Saiseikai General Hospital Sanae Teshigawara Department of Internal Medicine, Diabetes Center, Okayama Saiseikai General Hospital Tatsuaki Nakatou Department of Internal Medicine, Diabetes Center, Okayama Saiseikai General Hospital Case Report 10.18926/AMO/61441 A 43-year-old male with type 2 diabetes, under treatment with 5 mg/day of dapagliflozin, was referred to our hospital with upper left abdominal pain and marked hypertriglyceridemia (triglycerides [TGs], 5,960 mg/dl). He was also on a low-carbohydrate diet that promoted ketosis under sodium glucose cotransporter 2 (SGLT2) inhibitor administration. Polyacrylamide gel electrophoresis revealed a remarkable increase in very-low-den-sity lipoprotein, a TG-rich lipoprotein particle synthesized in the liver using free fatty acids derived from adi-pose tissue. Although SGLT2 inhibitors generally improve the lipid profile, under certain conditions such as a low-carbohydrate diet, they may adversely exacerbate the lipid profile via ketosis. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 2186-7755 110 2021 遺伝子比較解析による珪藻類特異的なSET ドメインタンパク質ファミリーの同定 1 6 EN Michiko Nemoto Graduate School of Environmental and Life Science, Okayama University 　The silica cell walls of diatoms, which exhibit species-spe-cific micro- and nano- patterned structures are promising candidates for applications in nanotechnology. Previous studies revealed a number of silica cell wall-associated proteins involved in silica formation. However, molecular biological analyses toward understanding of diatom cell wall formation have been mostly limited to model diatom species and general silica formation process in diatoms is still incompletely understood. In this study, to gain a compre-hensive insight into diatom silica biomineralization, tran-scriptome data of three diatom species, Nitzschia palea, Achnanthes kuwaitensis and Pseudoleyanella lunata, were newly developed. The reads obtained from RNA sequencing were assembled into 31,946, 60,767 and 38,314 unique transcripts for N. palea, A. kuwaitensis and P. lunata, respectively. In order to identify the diatom-specific genes, three transcriptome data sets developed in this study and the protein-coding gene sets of five genome-sequenced diatoms were compared. The proteins shared only by eight diatom species that are predicted to possess an endoplasmic reticulum (ER)-targeting signal peptide were selected for further analyses. These include proteins showing homology to silicanin-1, a recently reported diatom-specific protein involved in silica formation, as well as a number of SET domain proteins. The SET domain proteins might be novel diatom-specific family of methyltransferases that may reg-ulate the function of silica formation related proteins or long chain polyamines. The genes encoding the diatom-specific SET domain proteins identified in this study, which were shown to respond to silicon were suggested to be implicated in silica biomineralization. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1420-3049 26 1 2020 Photoinduced Endosomal Escape Mechanism: A View from Photochemical Internalization Mediated by CPP-Photosensitizer Conjugates 36 EN Tet Htut Soe Department of Biotechnology, Mandalay Technological University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Endosomal escape in cell-penetrating peptide (CPP)-based drug/macromolecule delivery systems is frequently insufficient. The CPP-fused molecules tend to remain trapped inside endosomes and end up being degraded rather than delivered into the cytosol. One of the methods for endosomal escape of CPP-fused molecules is photochemical internalization (PCI), which is based on the use of light and a photosensitizer and relies on photoinduced endosomal membrane destabilization to release the cargo molecule. Currently, it remains unclear how this delivery strategy behaves after photostimulation. Recent findings, including our studies using CPP-cargo-photosensitizer conjugates, have shed light on the photoinduced endosomal escape mechanism. In this review, we discuss the structural design of CPP-photosensitizer and CPP-cargo-photosensitizer conjugates, and the PCI mechanism underlying their application. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0953-8194 32 8 2020 Vasopressin gene products are colocalised with corticotrophin‐releasing factor within neurosecretory vesicles in the external zone of the median eminence of the Japanese macaque monkey (Macaca fuscata) e12875 EN Akito Otubo Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Natsuko Kawakami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Sho Maejima Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasumasa Ueda Department of Physiology, Kyoto Prefectural University of Medicine John F. Morris Department of Physiology, Anatomy & Genetics, University of Oxford Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Arginine vasopressin (AVP), when released into portal capillaries with corticotrophin‐releasing factor (CRF) from terminals of parvocellular neurones of the hypothalamic paraventricular nucleus (PVH), facilitates the secretion of adrenocorticotrophic hormone (ACTH) in stressed rodents. The AVP gene encodes a propeptide precursor containing AVP, AVP‐associated neurophysin II (NPII), and a glycopeptide copeptin, although it is currently unclear whether copeptin is always cleaved from the neurophysin and whether the NPII and/or copeptin have any functional role in the pituitary. Furthermore, for primates, it is unknown whether CRF, AVP, NPII and copeptin are all colocalised in neurosecretory vesicles in the terminal region of the paraventricular CRF neurone axons. Therefore, we investigated, by fluorescence and immunogold immunocytochemistry, the cellular and subcellular relationships of these peptides in the CRF‐ and AVP‐producing cells in unstressed Japanese macaque monkeys (Macaca fuscata). Reverse transcription‐polymerase chain reaction analysis showed the expression of both CRF and AVP mRNAs in the monkey PVH. As expected, in the magnocellular neurones of the PVH and supraoptic nucleus, essentially no CRF immunoreactivity could be detected in NPII‐immunoreactive (AVP‐producing) neurones. Immunofluorescence showed that, in the parvocellular part of the PVH, NPII was detectable in a subpopulation (approximately 39%) of the numerous CRF‐immunoreactive neuronal perikarya, whereas, in the outer median eminence, NPII was more prominent (approximately 52%) in the CRF varicosities. Triple immunoelectron microscopy in the median eminence demonstrated the presence of both NPII and copeptin immunoreactivity in dense‐cored vesicles of CRF‐containing axons. The results are consistent with an idea that the AVP propeptide is processed and NPII and copeptin are colocalised in hypothalamic‐pituitary CRF axons in the median eminence of a primate. The CRF, AVP and copeptin are all co‐packaged in neurosecretory vesicles in monkeys and are thus likely to be co‐released into the portal capillary blood to amplify ACTH release from the primate anterior pituitary. No potential conflict of interest relevant to this article was reported.

IOP Press Acta Medica Okayama 1387-2877 73 1 2020 A New Serum Biomarker Set to Detect Mild Cognitive Impairment and Alzheimer’s Disease by Peptidome Technology 217 227 EN Koji Abe Department of Neurology, Okayama University Jingwei Shang Department of Neurology, Okayama University Xiaowen Shi Department of Neurology, Okayama University Toru Yamashita Department of Neurology, Okayama University Nozomi Hishikawa Department of Neurology, Okayama University Mami Takemoto Department of Neurology, Okayama University Ryuta Morihara Department of Neurology, Okayama University Yumiko Nakano Department of Neurology, Okayama University Yasuyuki Ohta Department of Neurology, Okayama University Kentaro Deguchi Department of Neurology, Okayama City Hospital, Okayama Masaki Ikeda Department of Neurology, Gunma University, Graduate School of Medicine Yoshio Ikeda Department of Neurology, Gunma University, Graduate School of Medicine Koichi Okamoto Department of Neurology, Geriatrics Research Institute and Hospital Mikio Shoji Department of Neurology, Geriatrics Research Institute and Hospital Masamitsu Takatama Department of Neurology, Geriatrics Research Institute and Hospital Motohisa Kojo Department of Neurology, Ako Chuo Hospital Takeshi Kuroda Division of Neurology, Department of Medicine, Showa University, School of Medicine Kenjiro Ono Division of Neurology, Department of Medicine, Showa University, School of Medicine Noriyuki Kimura Department of Neurology, Faculty of Medicine, Oita University Etsuro Matsubara Department of Neurology, Faculty of Medicine, Oita University Yosuke Osakada Department of Neurology, Kurashiki Heisei Hospital Yosuke Wakutani Department of Neurology, Kurashiki Heisei Hospital Yoshiki Takao Department of Neurology, Kurashiki Heisei Hospital Yasuto Higashi Department of Neurology, Himeji Central Hospital Kyoichi Asada Membrane Protein and Ligand Analysis Center, Protosera Inc., Takehito Senga Membrane Protein and Ligand Analysis Center, Protosera Inc., Lyang-Ja Lee Membrane Protein and Ligand Analysis Center, Protosera Inc., Kenji Tanaka Membrane Protein and Ligand Analysis Center, Protosera Inc., Background:</br> Because dementia is an emerging problem in the world, biochemical markers of cerebrospinal fluid (CSF) and radio-isotopic analyses are helpful for diagnosing Alzheimer’s disease (AD). Although blood sample is more feasible and plausible than CSF or radiological biomarkers for screening potential AD, measurements of serum amyloid- β (Aβ), plasma tau, and serum antibodies for Aβ1 - 42 are not yet well established.</br> Objective:</br> We aimed to identify a new serum biomarker to detect mild cognitive impairment (MCI) and AD in comparison to cognitively healthy control by a new peptidome technology.</br> Methods:</br> With only 1.5μl of serum, we examined a new target plate “BLOTCHIP®” plus a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) to discriminate control (n = 100), MCI (n = 60), and AD (n = 99). In some subjects, cognitive Mini-Mental State Examination (MMSE) were compared to positron emission tomography (PET) with Pittsburgh compound B (PiB) and the serum probability of dementia (SPD). The mother proteins of candidate serum peptides were examined in autopsied AD brains.</br> Results:</br> Apart from Aβ or tau, the present study discovered a new diagnostic 4-peptides-set biomarker for discriminating control, MCI, and AD with 87% of sensitivity and 65% of specificity between control and AD (***p < 0.001). MMSE score was well correlated to brain Aβ deposition and to SPD of AD. The mother proteins of the four peptides were upregulated for coagulation, complement, and plasticity (three proteins), and was downregulated for anti-inflammation (one protein) in AD brains.</br> Conclusion:</br> The present serum biomarker set provides a new, rapid, non-invasive, highly quantitative and low-cost clinical application for dementia screening, and also suggests an alternative pathomechanism of AD for neuroinflammation and neurovascular unit damage. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0960-9822 31 1 2020 Oxytocin Influences Male Sexual Activity via Non-synaptic Axonal Release in the Spinal Cord 103 114.e5 EN Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keita Satoh Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Daisuke Uta Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama Junta Nagafuchi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Sayaka Tateishi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ryota Ueda Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keiko Takanami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Larry J. Young Center for Translational Social Neuroscience, Silvio O. Conte Center for Oxytocin and Social Cognition, Department of Psychiatry and Behavioral Sciences, Yerkes National Primate Research Center, Emory University Antony Galione Department of Pharmacology, University of Oxford John F. Morris Department of Physiology, Anatomy & Genetics, University of Oxford Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Oxytocinergic neurons in the paraventricular nucleus of the hypothalamus that project to extrahypothalamic brain areas and the lumbar spinal cord play an important role in the control of erectile function and male sexual behavior in mammals. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the “spinal ejaculation generator (SEG).” We have examined the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system in rats. Here, we show that SEG/GRP neurons express oxytocin receptors and are activated by oxytocin during male sexual behavior. Intrathecal injection of oxytocin receptor antagonist not only attenuates ejaculation but also affects pre-ejaculatory behavior during normal sexual activity. Electron microscopy of potassium-stimulated acute slices of the lumbar cord showed that oxytocin-neurophysin-immunoreactivity was detected in large numbers of neurosecretory dense-cored vesicles, many of which are located close to the plasmalemma of axonal varicosities in which no electron-lucent microvesicles or synaptic membrane thickenings were visible. These results suggested that, in rats, release of oxytocin in the lumbar spinal cord is not limited to conventional synapses but occurs by exocytosis of the dense-cored vesicles from axonal varicosities and acts by diffusion―a localized volume transmission―to reach oxytocin receptors on GRP neurons and facilitate male sexual function. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0168-3659 330 2020 Self-assembling A6K peptide nanotubes as a mercaptoundecahydrododecaborate (BSH) delivery system for boron neutron capture t (BNCT) 788 196 EN Hiroyuki Michiue Neutron Therapy Research Center, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Kindai University Asami Fukunaga Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nobushige Tsuboi Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Atsushi Fujimura Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroaki Matsushita Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuyo Igawa Neutron Therapy Research Center, Okayama University Tomonari Kasai Neutron Therapy Research Center, Okayama University Natsuko Kondo Institute for Integrated Radiation and Nuclear Science, Kyoto University Hideki Matsui Neutron Therapy Research Center, Okayama University Shuichi Furuya Neutron Therapy Research Center, Okayama University Boron neutron capture therapy (BNCT) is a tumor selective therapy, the effectiveness of which depends on sufficient 10B delivery to and accumulation in tumors. In this study, we used self-assembling A6K peptide nanotubes as boron carriers and prepared new boron agents by simple mixing of A6K and BSH. BSH has been used to treat malignant glioma patients in clinical trials and its drug safety and availability have been confirmed; however, its contribution to BNCT efficacy is low. A6K nanotube delivery improved two major limitations of BSH, including absence of intracellular transduction and non-specific drug delivery to tumor tissue. Varying the A6K peptide and BSH mixture ratio produced materials with different morphologies―determined by electron microscopy―and intracellular transduction efficiencies. We investigated the A6K/BSH 1:10 mixture ratio and found high intracellular boron uptake with no toxicity. Microscopy observation showed intracellular localization of A6K/BSH in the perinuclear region and endosome in human glioma cells. The intracellular boron concentration using A6K/BSH was almost 10 times higher than that of BSH. The systematic administration of A6K/BSH via mouse tail vein showed tumor specific accumulation in a mouse brain tumor model with immunohistochemistry and pharmacokinetic study. Neutron irradiation of glioma cells treated with A6K/BSH showed the inhibition of cell proliferation in a colony formation assay. Boron delivery using A6K peptide provides a unique and simple strategy for next generation BNCT drugs. No potential conflict of interest relevant to this article was reported.

The Genetics Society of Japan Acta Medica Okayama 1341-7568 95 3 2020 Ribosome rescue activity of an Arabidopsis thaliana ArfB homolog 119 131 EN Michiaki Nagao Graduate School of Natural Science and Technology, Okayama University Fumina Tsuchiya Graduate School of Integrated Science and Technology, Shizuoka University Reiko Motohashi Graduate School of Integrated Science and Technology, Shizuoka University Tatsuhiko Abo Graduate School of Natural Science and Technology, Okayama University A homolog of the bacterial ribosome rescue factor ArfB was identified in Arabidopsis thaliana. The factor, named AtArfB for Arabidopsis thaliana ArfB, showed ribosome rescue activity in both in vivo and in vitro assays based on the bacterial translation system. As has been shown for ArfB, the ribosome rescue activity of AtArfB was dependent on the GGQ motif, the crucial motif for the function of class I release factors and ArfB. The C-terminal region of AtArfB was also important for its function. The N-terminal region of AtArfB, which is absent in bacterial ArfB, functioned as a transit peptide for chloroplast targeting in tobacco cells. These results strongly suggest that AtArfB is a ribosome rescue factor that functions in chloroplasts. No potential conflict of interest relevant to this article was reported.

Nature Research Acta Medica Okayama 2045-2322 10 1 2020 Cell cycle dependence of apoptosis photo-triggered using peptide-photosensitizer conjugate 19087 EN Hyungjin Kim Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Sho Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Kindai University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Investigation of the relevance between cell cycle status and the bioactivity of exogenously delivered biomacromolecules is hindered by their time-consuming cell internalization and the cytotoxicity of transfection methods. In this study, we addressed these problems by utilizing the photochemical internalization (PCI) method using a peptide/protein-photosensitizer conjugate, which enables immediate cytoplasmic internalization of the bioactive peptides/proteins in a light-dependent manner with low cytotoxicity. To identify the cell-cycle dependent apoptosis, a TatBim peptide-photosensitizer conjugate (TatBim-PS) with apoptotic activity was photo-dependently internalized into HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (Fucci2). Upon irradiation, cytoplasmic TatBim-PS internalization exceeded 95% for all cells classified in the G(1), S, and G(2)/M cell cycle phases with no significant differences between groups. TatBim-PS-mediated apoptosis was more efficiently triggered by photoirradiation in the G(1)/S transition than in the G(1) and S/G(2)/M phases, suggesting high sensitivity of the former phase to Bim-induced apoptosis. Thus, the cell cycle dependence of Bim peptide-induced apoptosis was successfully investigated using Fucci2 indicator and the PCI method. Since PCI-mediated cytoplasmic internalization of peptides is rapid and does not span multiple cell cycle phases, the Fucci-PCI method constitutes a promising tool for analyzing the cell cycle dependence of peptides/protein functions. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 2047-9980 9 16 2020 Effect of Luseogliflozin on Heart Failure With Preserved Ejection Fraction in Patients With Diabetes Mellitus e015103 EN Kentaro Ejiri Okayama University Graduate School of Medicine, Density and Pharmaceutical Sciences Toru Miyoshi Okayama University Graduate School of Medicine, Density and Pharmaceutical Sciences Hajime Kihara Department of Internal Medicine, Kihara Cardiovascular Clinic Yoshiki Hata Department of Cardiology, Minamino Cardiovascular Hospital Toshihiko Nagano Department of Internal Medicine, Iwasa Hospital Atsushi Takaishi Department of Cardiology, Mitoyo General Hospital Hironobu Toda Okayama University Graduate School of Medicine, Density and Pharmaceutical Sciences Seiji Nanba Department of Cardiology, Okayama Rosai Hospital Yoichi Nakamura Department of Cardiovascular Medicine, Specified Clinic of Soyokaze Cardiovascular Medicine and Diabetes Care Satoshi Akagi Okayama University Graduate School of Medicine, Density and Pharmaceutical Sciences Satoru Sakuragi Department of Cardiovascular Medicine, Iwakuni Clinical Center Taro Minagawa Department of Internal Medicine, Minagawa Cardiovascular Clinic Yusuke Kawai Department of Cardiovascular Medicine, Okayama City Hospital Nobuhiro Nishii Okayama University Graduate School of Medicine, Density and Pharmaceutical Sciences Soichiro Fuke Department of Cardiovascular Medicine, Japanese Red Cross Okayama Hospital Masaki Yoshikawa Department of Cardiology, Fukuyama City Hospital Kazufumi Nakamura Okayama University Graduate School of Medicine, Density and Pharmaceutical Sciences Hiroshi Ito Okayama University Graduate School of Medicine, Density and Pharmaceutical Sciences Background</br> Effects of sodium‐glucose cotransporter 2 inhibitors on reducing hospitalization for heart failure have been reported in randomized controlled trials, but their effects on patients with heart failure with preserved ejection fraction (HFpEF) are unknown. This study aimed to evaluate the drug efficacy of luseogliflozin, a sodium‐glucose cotransporter 2 inhibitor, in patients with type 2 diabetes mellitus and HFpEF.</br> Methods and Results</br> We performed a multicenter, open‐label, randomized, controlled trial for comparing luseogliflozin 2.5 mg once daily with voglibose 0.2 mg 3 times daily in patients with type 2 diabetes mellitus suffering from HFpEF (left ventricular ejection fraction >45% and BNP [B‐type natriuretic peptide] concentrations ≥35 pg/mL) in a 1:1 randomization fashion. The primary outcome was the difference from baseline in BNP levels after 12 weeks of treatment between the 2 drugs. A total of 173 patients with diabetes mellitus and HFpEF were included. Of these, 83 patients were assigned to receive luseogliflozin and 82 to receive voglibose. There was no significant difference in the reduction in BNP concentrations after 12 weeks from baseline between the 2 groups. The ratio of the mean BNP value at week 12 to the baseline value was 0.79 in the luseogliflozin group and 0.87 in the voglibose group (percent change, −9.0% versus −1.9%; ratio of change with luseogliflozin versus voglibose, 0.93; 95% CI, 0.78–1.10; P=0.26).</br> Conclusion</br> In patients with type 2 diabetes mellitus and HFpEF, there is no significant difference in the degree of reduction in BNP concentrations after 12 weeks between luseogliflozin and voglibose. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 9 10 2020 In Vitro Studies to Define the Cell-Surface and Intracellular Targets of Polyarginine-Conjugated Sodium Borocaptate as a Potential Delivery Agent for Boron Neutron Capture Therapy 2149 EN Atsushi Fujimura Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiji Yasui Neutron Therapy Research Center, Okayama University Kazuyo Igawa Neutron Therapy Research Center, Okayama University Ai Ueda Neutron Therapy Research Center, Okayama University Kaori Watanabe Neutron Therapy Research Center, Okayama University Tadashi Hanafusa Neutron Therapy Research Center, Okayama University Yasuaki Ichikawa Neutron Therapy Research Center, Okayama University Sachiko Yoshihashi Graduate School of Engineering, Nagoya University Kazuki Tsuchida Graduate School of Engineering, Nagoya University Atsunori Kamiya Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shuichi Furuya Neutron Therapy Research Center, Okayama University Boron neutron capture therapy (BNCT) requires pharmaceutical innovations and molecular-based evidence of effectiveness to become a standard cancer therapeutic in the future. Recently, in Japan, 4-borono-L-phenylalanine (BPA) was approved as a boron agent for BNCT against head and neck (H&N) cancers. H&N cancer appears to be a suitable target for BPA-BNCT, because the expression levels of L-type amino acid transporter 1 (LAT1), one of the amino acid transporters responsible for BPA uptake, are elevated in most cases of H&N cancer. However, in other types of cancer including malignant brain tumors, LAT1 is not always highly expressed. To expand the possibility of BNCT for these cases, we previously developed poly-arginine peptide (polyR)-conjugated mercaptoundecahydrododecaborate (BSH). PolyR confers the cell membrane permeability and tumor selectivity of BSH. However, the molecular determinants for the properties are not fully understood. In this present study, we have identified the cluster of differentiation 44 (CD44) protein and translational machinery proteins as a major cell surface target and intracellular targets of BSH-polyR, respectively. CD44, also known as a stem cell-associated maker in various types of cancer, is required for the cellular uptake of polyR-conjugated molecules. We showed that BSH-polyR was predominantly delivered to a CD44(High) cell population of cancer cells. Once delivered, BSH-polyR interacted with the translational machinery components, including the initiation factors, termination factors, and poly(A)-biding protein (PABP). As a proof of principle, we performed BSH-polyR-based BNCT against glioma stem-like cells and revealed that BSH-polyR successfully induced BNCT-dependent cell death specifically in CD44(High) cells. Bioinformatics analysis indicated that BSH-polyR would be suitable for certain types of malignant tumors. Our results shed light on the biochemical properties of BSH-polyR, which may further contribute to the therapeutic optimization of BSH-BNCT in the future. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 5 2020 Seronegative Oligoarthritis Preceding Psoriasis by 9.5 Years 449 453 EN Junpei Tsuchiya Division of Orthopedic Surgery, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences Naoki Kondo Division of Orthopedic Surgery, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences Atsushi Fujimoto Division of Dermatology, Niigata University Graduate School of Medical and Dental Sciences Rie Kondo Division of Respiratory Medicine, Niigata University Graduate School of Medical and Dental Sciences Masahiko Yamada Division of Orthopedic Surgery, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences Tatsuya Kuraishi Division of Orthopedic Surgery, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences Takuya Yoda Division of Orthopedic Surgery, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences Riichiro Abe Division of Dermatology, Niigata University Graduate School of Medical and Dental Sciences Naoto Endo Division of Orthopedic Surgery, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences Case Report /10.18926/AMO/60807 We report a case of psoriatic arthritis where oligoarthritis preceded the skin lesions. A 57-year-old man complained of left third-finger pain. Laboratory examinations were negative for anti-cyclic citrullinated peptide antibodies and rheumatoid factor; he was treated for suspected rheumatoid arthritis. Six years later, X-ray revealed enthesitis of his fingers and wrist joint. At 9.5 years after the initial visit, skin lesions appeared in the left auricular region and buttock and dermatopathology findings indicated psoriasis vulgaris. The final diagnosis was psoriatic arthritis. In cases of seronegative oligoarthritis, psoriatic arthritis must be considered because some patients demonstrate osteoarticular lesions preceding skin lesions. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 5 2020 Clinical Relevance of Serum Prolactin Levels to Inflammatory Reaction in Male Patients 381 389 EN Koichiro Yamamoto Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshihisa Hanayama Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kou Hasegawa Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuki Tokumasu Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomoko Miyoshi 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 Hiroko Ogawa Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mikako Obika Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koichi Itoshima Department of Laboratory Medicine, Okayama University Hospital Fumio Otsuka Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/60797 To clarify the relevance of prolactin (PRL) to clinical parameters in patients who visited our general medicine department, medical records of 353 patients in whom serum PRL levels were measured during the period from 2016 to 2018 were retrospectively reviewed. Data for 140 patients (M/F: 42/98) were analyzed after excluding patients lacking detailed records and patients taking dopaminergic agents. Median serum PRL levels were significantly lower in males than females: 6.5 ng/ml (IQR: 4.2-10.3) versus 8.1 ng/ml (5.9-12.9), respectively. Pain and general fatigue were the major symptoms at the first visit, and past histories of hypertension and dyslipidemia were frequent. Male patients with relatively high PRL levels (≥ 10 ng/ml) had significantly lower levels of serum albumin and significantly higher levels of serum LDH than those with low PRL (< 10 ng/ml). There were significant correlations of male PRL level with the erythrocyte sedimentation rate (R=0.62), serum LDH level (R=0.39) and serum albumin level (R=−0.52), while the level of serum CRP (R=0.33) showed an insignificant but weak positive correlation with PRL level. Collectively, these results show that PRL levels had gender-specific relevance to various clinical factors, with PRL levels in males being significantly related to inflammatory status. No potential conflict of interest relevant to this article was reported.

Nature Research Acta Medica Okayama 2045-2322 10 1 2020 Dysfunction of CD8+PD-1+T cells in type 2 diabetes caused by the impairment of metabolism-immune axis 14928 EN Ichiro Nojima Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shingo Eikawa Department of Hematology/Oncology, Hess Cancer Institute, Icahn School of Medicine At Mount Sinai Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshiko Hada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nobuo Kajitani Department of Internal Medicine, Diabetes Center, Okayama City Hospital Sanae Teshigawara Diabetes Center, Okayama S Satoshi Miyamoto Center for Innovative Clinical Medicine, Okayama University Hospital Atsuhito Tone Diabetes Center, Okayama Saiseikai General Hospital Haruhito A. Uchida Department of Chronic Kidney Disease and Cardiovascular Disease, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Atsuko Nakatsuka Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jun Eguchi Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kenichi Shikata Center for Innovative Clinical Medicine, Okayama University Hospital Heiichiro Udono Department of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The metabolic changes and dysfunction in CD8+T cells may be involved in tumor progression and susceptibility to virus infection in type 2 diabetes (T2D). In C57BL/6JJcl mice fed with high fat-high sucrose chow (HFS), multifunctionality of CD8+splenic and tumor-infiltrating lymphocytes (TILs) was impaired and associated with enhanced tumor growth, which were inhibited by metformin. In CD8+splenic T cells from the HFS mice, glycolysis/basal respiration ratio was significantly reduced and reversed by metformin. In the patients with T2D (DM), multifunctionality of circulating CD8+PD-1+T cells stimulated with PMA/ionomycin as well as with HLA-A*24:02 CMV peptide was dampened, while metformin recovered multifunctionality. Both glycolysis and basal respiration were reduced in DM, and glycolysis was increased by metformin. The disturbance of the link between metabolism and immune function in CD8+PD-1+T cells in T2D was proved by recovery of antigen-specific and non-specific cytokine production via metformin-mediated increase in glycolytic activity. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0021-9967 529 7 2020 Variation of pro‐vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin‐related glycopeptide copeptin 1372 1390 EN Natsuko Kawakami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Akito Otubo Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Sho Maejima Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ashraf H. Talukder Laboratory of Information Biology, Graduate School of Information Sciences, Tohoku University Keita Satoh Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keiko Takanami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasumasa Ueda Department of Physiology, Kyoto Prefectural University of Medicine Keiichi Itoi Laboratory of Information Biology, Graduate School of Information Sciences, Tohoku University John F. Morris Department of Physiology, Anatomy and Genetics, University of Oxford Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Arginine vasopressin (AVP) is synthesized in parvocellular‐ and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre‐pro‐AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N‐terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII‐expressing neurons exhibited strong N‐terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII‐immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin‐immunoreactivity co‐localized with NPII‐immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa‐d‐arginine amide resulted in a marked reduction of copeptin‐immunoreactivity in the NPII‐immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro‐AVP could explain the disproportionally low levels of N‐terminal copeptin expression in rodent AVP (NPII)‐expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 1664-2392 11 2020 Transcriptomic Analysis of the Kuruma PrawnMarsupenaeus japonicusReveals Possible Peripheral Regulation of the Ovary 541 EN Naoaki Tsutsui Faculty of Science, Ushimado Marine Institute, Okayama University Yasuhisa Kobayashi Faculty of Science, Ushimado Marine Institute, Okayama University Kouichi Izumikawa Research Institute for Fisheries Science, Okayama Prefectural Technology Center for Agriculture, Forestry, and Fisheries Tatsuya Sakamoto Faculty of Science, Ushimado Marine Institute, Okayama University Crustacean reproduction has been hypothesized to be under complex endocrinological regulation by peptide hormones. To further improve our understanding of the mechanisms underlying this complex regulation, knowledge is needed regarding the hormones not only of the central nervous system (CNS) such as the X-organ/sinus gland (XOSG), brain, and thoracic ganglia, but also the peripheral gonadal tissues. For example, in vertebrates, some gonadal peptide hormones including activin, inhibin, follistatin, and relaxin are known to be involved in the reproductive physiology. Therefore, it is highly likely that some peptide factors from the ovary are serving as the signals among peripheral tissues and central nervous tissues in crustaceans. In this work, we sought to find gonadal peptide hormones and peptide hormone receptors by analyzing the transcriptome of the ovary of the kuruma prawnMarsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-alpha and -beta, the crustacean hyperglycemic hormone (CHH)-like peptide, insulin-like peptide (ILP), and neuroparsin-like peptide (NPLP). Dominant gene expressions for the bursicons were observed in the thoracic ganglia and the ovary, in the CNS for the CHH-like peptide, in the heart for NPLP, and in the ovary for ILP. Since the gene expressions of CHH-like peptide and NPLP were affected by a CHH (Penaeus japonicussinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP usingEscherichia coliexpression system to examine their possible peripheral regulation. As a result, we found that the recombinant NPLP increased vitellogenin gene expression in incubated ovarian tissue fragments. Moreover, contigs encoding putative receptors for insulin-like androgenic gland factor, insulin, neuroparsin, and neuropeptide Y/F, as well as several contigs encoding orphan G-protein coupled receptors and receptor-type guanylyl cyclases were also identified in the ovarian transcriptome. These results suggest that reproductive physiology in crustaceans is regulated by various gonadal peptide hormones, akin to vertebrates. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1661-6596 21 12 2020 The Effects of Mutual Interaction of Orexin-A and Glucagon-Like Peptide-1 on Reflex Swallowing Induced by SLN Afferents in Rats 4422 EN Motoi Kobashi Department of Oral Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuichi Shimatani Department of Medical Engineering, Faculty of Science and Engineering, Tokyo City University Masako Fujita Department of Oral Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshihiro Mitoh Department of Oral Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryusuke Yoshida Department of Oral Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryuji Matsuo Department of Oral Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (1) Background: Our previous studies revealed that orexin-A, an appetite-increasing peptide, suppressed reflex swallowing via the commissural part of the nucleus tractus solitarius (cNTS), and that glucagon-like peptide-1 (GLP-1), an appetite-reducing peptide, also suppressed reflex swallowing via the medial nucleus of the NTS (mNTS). In this study, we examined the mutual interaction between orexin-A and GLP-1 in reflex swallowing. (2) Methods: Sprague-Dawley rats under urethane-chloralose anesthesia were used. Swallowing was induced by electrical stimulation of the superior laryngeal nerve (SLN) and was identified by the electromyographic (EMG) signals obtained from the mylohyoid muscle. (3) Results: The injection of GLP-1 (20 pmol) into the mNTS reduced the swallowing frequency and extended the latency of the first swallow. These suppressive effects of GLP-1 were not observed after the fourth ventricular administration of orexin-A. After the injection of an orexin-1 receptor antagonist (SB334867) into the cNTS, an ineffective dose of GLP-1 (6 pmol) into the mNTS suppressed reflex swallowing. Similarly, the suppressive effects of orexin-A (1 nmol) were not observed after the injection of GLP-1 (6 pmol) into the mNTS. After the administration of a GLP-1 receptor antagonist (exendin-4(5-39)), an ineffective dose of orexin-A (0.3 nmol) suppressed reflex swallowing. (4) Conclusions: The presence of reciprocal inhibitory connections between GLP-1 receptive neurons and orexin-A receptive neurons in the NTS was strongly suggested. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 0914-7187 34 2020 Improvement of biodistribution profile of a radiogallium-labeled, αvβ6 integrin-targeting peptide probe by incorporation of negatively charged amino acids 575 582 EN Shunsuke Nakamura Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Aya Matsuno Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Masashi Ueda Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Objective</br> Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Since αvβ6 integrin has been reported as a promising target for PDAC diagnosis, we previously developed H-Cys(mal-NOTA-67Ga)-(Gly)6-A20FMDV2-NH2 ([67Ga]CG6) as an αvβ6 integrin-targeting probe. Although [67Ga]CG6 specifically binds to αvβ6 integrin-positive xenografts, the uptake of [67Ga]CG6 in the organs surrounding the pancreas, such as the liver and spleen, was comparable to that in the αvβ6 integrin-positive xenografts. We hypothesized that the undesirable accumulation of [67Ga]CG6 in those organs was caused by the positive charges of [67Ga]CG6 (+ 3). In this study, we aimed to decrease [67Ga]CG6 uptake in the liver and spleen by reducing the electric charges of the probe.</br> Methods</br> We synthesized H-Cys(mal-NOTA-67Ga)-(Asp)6-A20FMDV2-NH2 ([67Ga]CD6) and evaluated its affinity to αvβ6 integrin via in vitro competitive binding assay. Isoelectric points of the probes were determined by electrophoresis. Biodistribution study, autoradiography, and immunostaining for β6 integrin were conducted using αvβ6 integrin-positive and negative tumor-bearing mice.</br> Results</br> In vitro competitive binding assay showed that the alteration of the linker had a negligible impact on the affinity of [67Ga]CG6 to αvβ6 integrin. The results of electrophoresis revealed that [67Ga]CG6 was positively charged whereas [67Ga]CD6 was negatively charged. In the biodistribution study, the uptake of [67Ga]CD6 in the αvβ6 integrin-positive xenografts was significantly higher than that in the αvβ6 integrin-negative ones at 60 and 120 min. The uptake of [67Ga]CD6 in the liver and spleen was more than two-fold lower than that of [67Ga]CG6 at both time points. In the immunohistochemistry study, the radioactivity accumulated areas in the autoradiogram of the αvβ6 integrin-positive xenograft roughly coincided with β6 integrin-expressing areas.</br> Conclusion</br> We have successfully reduced the nonspecific uptake in the liver and spleen by altering the linker amino acid from G6 to D6. [67Ga]CD6 overcame the drawbacks of [67Ga]CG6 in its biodistribution. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1553-7366 16 4 2020 Non-pathogenic Escherichia coli acquires virulence by mutating a growth-essential LPS transporter e1008469 EN Chikara Kaito Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hirono Yoshikai Graduate School of Pharmaceutical Sciences, The University of Tokyo Ai Wakamatsu Japan Biological Informatics Consortium (JBIC) Atsushi Miyashita Graduate School of Pharmaceutical Sciences, The University of Tokyo Yasuhiko Matsumoto Department of Microbiology, Meiji Pharmaceutical University Tomoko Fujiyuki The Institute of Medical Science, The University of Tokyo Masaru Kato Devision of Bioanalytical Chemistry, School of Pharmacy,Showa University Yoshitoshi Ogura Department of Bacteriology, Faculty of Medical Sciences,Kyushu University Tetsuya Hayashi Department of Bacteriology, Faculty of Medical Sciences,Kyushu University Takao Isogai Translational Research Center, Fukushima Medical University Kazuhisa Sekimizu Institute of Medical Mycology, Teikyo University The molecular mechanisms that allow pathogenic bacteria to infect animals have been intensively studied. On the other hand, the molecular mechanisms by which bacteria acquire virulence functions are not fully understood. In the present study, we experimentally evaluated the evolution of a non-pathogenic strain of Escherichia coli in a silkworm infection model and obtained pathogenic mutant strains. As one cause of the high virulence properties of E. coli mutants, we identified amino acid substitutions in LptD (G580S) and LptE (T95I) constituting the lipopolysaccharide (LPS) transporter, which translocates LPS from the inner to the outer membrane and is essential for E. coli growth. The growth of the LptD and LptE mutants obtained in this study was indistinguishable from that of the parent strain. The LptD and LptE mutants exhibited increased secretion of outer membrane vesicles containing LPS and resistance against various antibiotics, antimicrobial peptides, and host complement. In vivo cross-linking studies revealed that the conformation of the LptD-LptE complex was altered in the LptD and LptE mutants. Furthermore, several clinical isolates of E. coli carried amino acid substitutions of LptD and LptE that conferred resistance against antimicrobial substances. This study demonstrated an experimental evolution of bacterial virulence properties in an animal infection model and identified functional alterations of the growth-essential LPS transporter that led to high bacterial virulence by conferring resistance against antimicrobial substances. These findings suggest that non-pathogenic bacteria can gain virulence traits by changing the functions of essential genes, and provide new insight to bacterial evolution in a host environment. Author summary Pathogenic bacteria developed their virulence properties by changing the functions of various genes after the emergence of the host animals on earth. The types of gene function alterations that confer bacterial virulence properties, however, have remained unclear. We utilized a silkworm infection model to perform an experimental evolution of bacterial virulence activity. From a non-pathogenic strain of Escherichia coli, we obtained a mutant strain that exhibited 500-fold higher virulence than the original strain and identified mutations of the lipopolysaccharide (LPS) transporter, which translocates LPS onto the bacterial surface, as one cause of the high virulence. The mutations changed the structure of the LPS transporter, increased the secretion of outer membrane vesicles, and enabled bacterial survival in the presence of host antimicrobial substances. This mechanism to gain high virulence occurs naturally, as several E. coli clinical isolates carried mutations of the LPS transporter that confer resistance against antimicrobial substances. Our study unveiled a novel mechanism by which bacteria increase their virulence through modifying their gene function. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0968-0896 28 1 2020 Development and characterization of a 68Ga-labeled A20FMDV2 peptide probe for the PET imaging of αvβ6 integrin-positive pancreatic ductal adenocarcinoma 115189 EN Takashi Ui Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Masashi Ueda Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yusuke Higaki Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama Universit Shinichiro Kamino Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kohei Sano Graduate School of Pharmaceutical Sciences, Kyoto University Hiroyuki Kimura Graduate School of Pharmaceutical Sciences, Kyoto University Hideo Saji Graduate School of Pharmaceutical Sciences, Kyoto University Shuichi Enomoto RIKEN Center for Life Science Technologies Pancreatic ductal adenocarcinoma (PDAC) is known to be one of the most lethal cancers. Since the majority of patients are diagnosed at an advanced stage, development of a detection method for PDAC at an earlier stage of disease progression is strongly desirable. Integrin αVβ6 is a promising target for early PDAC detection because its expression increases during precancerous changes. The present study aimed to develop an imaging probe for positron emission tomography (PET) which targets αVβ6 integrin-positive PDAC. We selected A20FMDV2 peptide, which binds specifically to αvβ6 integrin, as a probe scaffold, and 68Ga as a radioisotope. A20FMDV2 peptide has not been previously labeled with 68Ga. A cysteine residue was introduced to the N-terminus of the probe at a site-specific conjugation of maleimide-NOTA (mal-NOTA) chelate. Different numbers of glycine residues were also introduced between cysteine and the A20FMDV2 sequence as a spacer in order to reduce the steric hindrance of the mal-NOTA on the binding probe to αVβ6 integrin. In vitro, the competitive binding assay revealed that probes containing a 6-glycine linker ([natGa]CG6 and [natGa]Ac-CG6) showed high affinity to αVβ6 integrin. Both probes could be labeled by 67/68Ga with high radiochemical yield (>50%) and purity (>98%). On biodistribution analysis, [67Ga]Ac-CG6 showed higher tumor accumulation, faster blood clearance, and lower accumulation in the surrounding organs of pancreas than did [67Ga]CG6. The αVβ6 integrin-positive xenografts were clearly visualized by PET imaging with [68Ga]Ac-CG6. The intratumoral distribution of [68Ga]Ac-CG6 coincided with the αVβ6 integrin-positive regions detected by immunohistochemistry. Thus, [68Ga]Ac-CG6 is a useful peptide probe for the imaging of αVβ6 integrin in PDAC. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 2 2020 Increased Plasma Levels of Platelet Factor 4 and β-thromboglobulin in Women with Recurrent Pregnancy Loss 115 122 EN Sayoko Kotani Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasuhiko Kamada Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keiko Shimizu Okayama Rosai Hospital Ai Sakamoto Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mikiya Nakatsuka Graduate School of Health Sciences, Okayama University Yuji Hiramatsu Okayama City Hospital Hisashi Masuyama Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/58269 Thrombosis in decidual vessels is one of the mechanisms of pregnancy loss. However, few studies have assessed the relation between platelet activation, which is known to cause of thrombosis, and recurrent pregnancy loss (RPL). We investigated platelet activation in women with RPL compared to controls by measuring plasma levels of platelet factor 4 (PF4) and β-thromboglobulin (βTG), and assessed correlations between PF4/βTG and coagulative risk factors associated with RPL. The study group included 135 women who had experienced two or more consecutive pregnancy losses. The control group included 28 age-matched healthy women who had never experienced pregnancy loss. PF4 and βTG plasma levels were significantly higher in the women with RPL than controls (PF4: 14.0 [8.0-20.0] vs. 9.0 [6.0-12.0] ng/ml, p=0.043; βTG: 42.0 [24.3-59.8] vs. 31.5 [26.6-36.4] ng/ml, p=0.002). There was a significant association between βTG and anti-phosphatidylethanolamine antibody immunoglobulin M (aPE IgM) (p=0.048). Among the women with RPL, 18 of those who were positive for PF4 (45%) and 18 of those who were positive for βTG (37%) were negative for all known coagulative risk factors associated with RPL. Measurements of PF4 and βTG may be important because they help identify women who are at risk of RPL. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1467-7644 18 2 2019 Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening 415 428 EN Pingyu Wang Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Shaolun Yao Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Ken-ichi Kosami Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Ting Guo Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Jing Li Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Yuanyuan Zhang Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Yoichiro Fukao Department of Bioinformatics, Ritsumeikan University Takako Kaneko-Kawano College of Pharmaceutical Sciences, Ritsumeikan University Heng Zhang Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Yi-Min She Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Pengcheng Wang Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences Weiman Xing Biomolecular Structure and Design, Shanghai Center for Plant Stress Biology Kousuke Hanada Department of Bioscience and Bioinformatics, Kyushu Institute of Technology Renyi Liu Center for Agroforestry Mega Data Science and FAFU-UCR Joint Center for Horticultural Biology and Metabolomics, Haixia Institute of Science and Technology,Fujian Agriculture and Forestry University Yoji Kawano Institute of Plant Science and Resources, Okayama University Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1420-3049 24 8 2019 Melittin Exerts Beneficial Effects on Paraquat-Induced Lung Injuries in Mice by Modifying Oxidative Stress and Apoptosis EN Bishoy El-Aarag Division of Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University Mohamed Magdy Department of Chemistry, Faculty of Science, Menoufia University Mohamed F. AlAjmi Department of Pharmacognosy, College of Pharmacy, King Saud University Shaden A. M. Khalifa Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University Hesham R. El-Seedi Department of Chemistry, Faculty of Science, Menoufia University Melittin (MEL) is a 26-amino acid peptide with numerous biological activities. Paraquat (PQ) is one of the most widely used herbicides, although it is extremely toxic to humans. To date, PQ poisoning has no effective treatment, and therefore the current study aimed to assess for the first time the possible effects of MEL on PQ-induced lung injuries in mice. Mice received a single intraperitoneal (IP) injection of PQ (30 mg/kg), followed by IP treatment with MEL (0.1 and 0.5 mg/kg) twice per week for four consecutive weeks. Histological alterations, oxidative stress, and apoptosis in the lungs were studied. Hematoxylin and eosin (H&E) staining indicated that MEL markedly reduced lung injuries induced by PQ. Furthermore, treatment with MEL increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity, and decreased malonaldehyde (MDA) and nitric oxide (NO) levels in lung tissue homogenates. Moreover, immunohistochemical staining showed that B-cell lymphoma-2 (Bcl-2) and survivin expressions were upregulated after MEL treatment, while Ki-67 expression was downregulated. The high dose of MEL was more effective than the low dose in all experiments. In summary, MEL efficiently reduced PQ-induced lung injuries in mice. Specific pharmacological examinations are required to determine the effectiveness of MEL in cases of human PQ poisoning. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2045-2322 9 2019 Imaging Amyloplasts in the Developing Endosperm of Barley and Rice 3745 EN Ryo Matsushima Institute of Plant Science and Resources, Okayama University Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Amyloplasts are plant-specific organelles responsible for starch biosynthesis and storage. Inside amyloplasts, starch forms insoluble particles, referred to as starch grains (SGs). SG morphology differs between species and SG morphology is particularly diverse in the endosperm of Poaceae plants, such as rice (Oryza sativa) and barley (Hordeum vulgare), which form compound SGs and simple SGs, respectively. SG morphology has been extensively imaged, but the comparative imaging of amyloplast morphology has been limited. In this study, SG-containing amyloplasts in the developing endosperm were visualized using stable transgenic barley and rice lines expressing amyloplast stroma-targeted green fluorescent protein fused to the transit peptide (TP) of granule-bound starch synthase I (TP-GFP). The TP-GFP barley and rice plants had elongated amyloplasts containing multiple SGs, with constrictions between the SGs. In barley, some amyloplasts were connected by narrow protrusions extending from their surfaces. Transgenic rice lines producing amyloplast membrane-localized SUBSTANDARD STARCH GRAIN6 (SSG6)-GFP were used to demonstrate that the developing amyloplasts contained multiple compound SGs. TP-GFP barley can be used to visualize the chloroplasts in leaves and other plastids in pollen and root in addition to the endosperm, therefore it provides as a useful tool to observe diverse plastids. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 73 5 2019 Relationship between Intracellular Signaling of the (Pro)renin Receptor and the Pathogenesis of Preeclampsia 433 440 EN Shoko Tamada Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takashi Mitsui Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akiko Ohira Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazumasa Tani Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jota Maki Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takeshi Eguchi Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eriko Eto Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kei Hayata Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hisashi Masuyama Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article /10.18926/AMO/57374 An association between preeclampsia and (pro)renin was recently reported. Intracellular signaling of the (pro) renin receptor [(P)RR] increases the expressions of TGF-β and PAI-1. In this study we sought to clarify the involvement of (pro)renin in the pathogenesis of preeclampsia via the intracellular signaling of (P)RR on preeclampsia placentas. Activated (pro)renin plasma concentrations were compared between pregnant women with (n=15) and without (n=28) preeclampsia. The placentas were immunohistochemically evaluated with anti-HIF-1α and anti-(P)RR antibodies. HTR-8/SVneo cells were cultured under hypoxic conditions and treated with human recombinant (pro)renin. The mRNA expressions of HIF-1α, (P)RR, PAI-1, TGF-β, and ET-1 were also examined by real-time RCR. The activated (pro)renin plasma concentration was significantly higher in the third vs. the second trimester in the preeclampsia patients. HIF-1α and (P)RR expressions were significantly increased in the preeclampsia placentas. The mRNA expressions of PAI-1, TGF-β, and ET-1 were significantly increased in the experiments using recombinant (pro)renin vs. hypoxic conditions. (P)RR expression in preeclampsia placentas is increased by persistent hypoxia through the second and third trimesters, and PAI-1, TGF-β, and ET-1 production is increased via (P)RR. Our results suggest that ET-1 production via the intracellular signaling of (P)RR is important in the pathogenesis of preeclampsia. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 73 5 2019 Histidine-rich Glycoprotein Modulates the Blood-vascular System in Septic Condition 379 382 EN Hidenori Wake Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Review /10.18926/AMO/57366 Histidine-rich glycoprotein (HRG) is a 75 kDa glycoprotein synthesized in the liver whose plasma concentration is 100-150 μg/ml. HRG has been shown to modulate sepsis-related biological reactions by binding to several substances and cells, including heparin, factor XII, fibrinogen, thrombospondin, plasminogen, C1q, IgG, heme, LPS, dead cells, bacteria, and fungi. Therefore, reduction of plasma HRG levels in sepsis leads to dysregulation of coagulation, fibrinolysis, and immune response, resulting in disseminated intravascular coagulation and multiple organ failure. This review summarizes the binding and functional properties of HRG in sepsis. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2019 Orexin A modulates prolactin production by regulating BMP-4 activity in rat pituitary lactotorope cells EN Satoshi Fujisawa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 7 11 2012 Mitochondrial localization of ABC transporter ABCG2 and its function in 5-aminolevulinic acid-mediated protoporphyrin IX accumulation e50082 EN Hirotsugu Kobuchi 1 Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koko Moriya Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University Tetsuya Ogino Department of Nursing Science, Faculty of Health and Welfare Science, Okayama Prefectural University Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Keiji Inoue Department of Urology, Kochi Medical School Taro Shuin Department of Urology, Kochi Medical School Tatsuji Yasuda 1 Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kozo Utsumi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Toshihiko Utsumi Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 73 4 2019 Dynamic Reorganization of Microtubule and Glioma Invasion 285 297 EN Yoshihiro Otani Department of Neurosurgery, The University of Texas Health Science Center at Houston Tomotsugu Ichikawa Department of Neurosurgery, Kagawa Prefectural Central Hospital Kazuhiko Kurozumi Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Isao Date Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Review /10.18926/AMO/56930 Gliomas are characterized as highly diffuse infiltrating tumors, and currently available treatments such as surgery, radiation and chemotherapy are unfeasible or show limited efficacy against these tumors. Recent genetic and epigenetic analyses of glioma have revealed increasing evidence of the role of driver genetic alterations in glioma development and led to the identification of prognostic factors. Despite these findings, the survival rates of glioma patients remain low, and alternative treatments and novel targets are needed. Recent studies identified neural stem cells as the possible origin of gliomas, and some evidence has revealed shared functions and mechanisms between glioma cells and neurons, also supporting their similarity. The cytoskeleton plays important roles in the migration of normal cells as well as cancer cells. Recent reports have described a role for microtubules, a component of the cytoskeleton, in glioma invasion. Notably, several factors that regulate microtubule functions, such as microtubule-associated proteins, plus-end tracking proteins, or motor proteins, are upregulated in glioma tissues compared with normal tissue, and upregulation of these factors is associated with high invasiveness of glioma cells. In this review, we describe the mechanism of microtubules in glioma invasion and discuss the possibility of microtubule-targeted therapy to inhibit glioma invasion. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 73 2 2019 Collagen XVIII Deposition in the Basement Membrane Zone beneath the Newly Forming Epidermis during Wound Healing in Mice 135 146 EN Takahiro Maeba Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Tomoko Yonezawa Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Mitsuaki Ono Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Yasuko Tomono Shigei Medical Research Institute Ritva Heljasvaara Center for Cancer Biomarkers CCBIO, Department of Biomedicine, University of Bergen Taina Pihlajaniemi Oulu Center for Cell-Matrix Research, Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, University of Oulu Kiichi Inagawa Department of Plastic and Reconstructive Surgery, Kawasaki Medical School, Kurashiki Toshitaka Oohashi Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Original Article 10.18926/AMO/56649 The basement membrane (BM) is composed of various extracellular molecules and regulates tissue regeneration and maintenance. Here, we demonstrate that collagen XVIII was spatiotemporally expressed in the BM during skin wound healing in a mouse excisional wound-splinting model. Re-epithelialization was detected at days 3 and 6 post-wounding. The ultrastructure of epidermal BM was discontinuous at day 3, whereas on day 6 a continuous BM was observed in the region proximal to the wound edge. Immunohistochemistry demonstrated that collagen XVIII was deposited in the BM zone beneath newly forming epidermis in day 3 and 6 wounds. Laminin-332, known to be the earliest BM component appearing in wounds, was colocalized with collagen XVIII in the epidermal BM zone at days 3 and 6. The deposition of α1(IV) collagen and nidogen-1 in the epidermal BM zone occurred later than that of collagen XVIII. We also observed the short isoform of collagen XVIII in the epidermal BM zone at day 3 post-wounding. Collectively, our results suggested that collagen XVIII plays a role in the formation of the dermal-epidermal junction during re-epithelialization, and that it is the short isoform that is involved in the early phase of re-epithelialization. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 73 1 2019 Histidine-rich Glycoprotein Could Be an Early Predictor of Vasospasm after Aneurysmal Subarachnoid Hemorrhage 29 39 EN Atsushi Matsumoto Department of Neurological Surgery, Kagawa University Faculty of Medicine Takehiro Nakamura Department of Medical Technology, Kagawa Prefectural University of Health Sciences Aya Shinomiya Department of Neurological Surgery, Kagawa University Faculty of Medicine Kenya Kawakita Emergency Medical Center, Kagawa University Hospital Masahiko Kawanishi Department of Neurological Surgery, Kagawa University Faculty of Medicine Keisuke Miyake Department of Neurological Surgery, Kagawa University Faculty of Medicine Yasuhiro Kuroda Emergency Medical Center, Kagawa University Hospital Richard F. Keep Department of Neurosurgery, University of Michigan Takashi Tamiya Department of Neurological Surgery, Kagawa University Faculty of Medicine Original Article /10.18926/AMO/56456 Cerebral vasospasm (CVS) is a major contributor to the high morbidity and mortality of aneurysmal subarachnoid hemorrhage (aSAH) patients. We measured histidine-rich glycoprotein (HRG), a new biomarker of aSAH, in cerebrospinal fluid (CSF) to investigate whether HRG might be an early predictor of CVS. A total of seven controls and 14 aSAH patients (8 males, 6 females aged 53.4±15.4 years) were enrolled, and serial CSF and serum samples were taken. We allocated these samples to three phases (T1-T3) and measured HRG, interleukin (IL)-6, fibrinopeptide A (FpA), and 8-hydroxy-2’-deoxyguanosine (8OHdG) in the CSF, and the HRG in serum. We also examined the release of HRG in rat blood incubated in artificial CSF. In contrast to the other biomarkers examined, the change in the CSF HRG concentration was significantly different between the nonspasm and spasm groups (p<0.01). The rat blood/CSF model revealed a time course similar to that of the human CSF samples in the non-spasm group. HRG thus appears to have the potential to become an early predictor of CVS. In addition, the interaction of HRG with IL-6, FpA, and 8OHdG may form the pathology of CVS. 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.

Acta Medica Okayama 2018 Functional peptide KP24 enhances submandibular gland tissue growth in vitro EN Atsushi Ikeda Okayama University No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2017 Regulatory role of melatonin and BMP-4 in prolactin production by rat pituitary lactotrope GH3 cells EN Kanako Ochi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 71 6 2017 Signal Diversity of Receptor for Advanced Glycation End Products 459 465 EN Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Endy Widya Putranto Faculty of Medicine, Universitas Gadjah Mada I Made Winarsa Ruma Faculty of Medicine, Udayana University I Wayan Sumardika Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Chen Youyi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Naoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Review /10.18926/AMO/55582 The receptor for advanced glycation end products (RAGE) is involved in inflammatory pathogenesis. It functions as a receptor to multiple ligands such as AGEs, HMGB1 and S100 proteins, activating multiple intracellular signaling pathways with each ligand binding. The molecular events by which ligand-activated RAGE controls diverse signaling are not well understood, but some progress was made recently. Accumulating evidence revealed that RAGE has multiple binding partners within the cytoplasm and on the plasma membrane. It was first pointed out in 2008 that RAGE’s cytoplasmic tail is able to recruit Diaphanous-1 (Dia-1), resulting in the acquisition of increased cellular motility through Rac1/Cdc42 activation. We also observed that within the cytosol, RAGE’s cytoplasmic tail behaves similarly to a Toll-like receptor (TLR4)-TIR domain, interacting with TIRAP and MyD88 adaptor molecules that in turn activate multiple downstream signals. Subsequent studies demonstrated the presence of an alternative adaptor molecule, DAP10, on the plasma membrane. The coupling of RAGE with DAP10 is critical for enhancing the RAGE-mediated survival signal. Interestingly, RAGE interaction on the membrane was not restricted to DAP10 alone. The chemotactic G-protein-coupled receptors (GPCRs) formyl peptide receptors1 and 2 (FPR1 and FPR2) also interacted with RAGE on the plasma membrane. Binding interaction between leukotriene B4 receptor 1 (BLT1) and RAGE was also demonstrated. All of the interactions affected the RAGE signal polarity. These findings indicate that functional interactions between RAGE and various molecules within the cytoplasmic area or on the membrane area coordinately regulate multiple ligand-mediated RAGE responses, leading to typical cellular phenotypes in several pathological settings. Here we review RAGE’s signaling diversity, to contribute to the understanding of the elaborate functions of RAGE in physiological and pathological contexts. No potential conflict of interest relevant to this article was reported.

CSIRO Publishing Acta Medica Okayama 1031-3613 28 10 2015 Multiple roles of hypoxia in ovarian function: roles of hypoxia-inducible factor-related and -unrelated signals during the luteal phase 1479 1486 EN Ryo Nishimura Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University Kiyoshi Okuda Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University There is increasing interest in the role of oxygen conditions in the microenvironment of organs because of the discovery of a hypoxia-specific transcription factor, namely hypoxia-inducible factor (HIF) 1. Ovarian function has several phases that change day by day, including ovulation, follicular growth and corpus luteum formation and regression. These phases are regulated by many factors, including pituitary hormones and local hormones, such as steroids, peptides and cytokines, as well as oxygen conditions. Hypoxia strongly induces angiogenesis because transcription of the potent angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF1. Follicular development and luteal formation are accompanied by a marked increase in angiogenesis assisted by HIF1-VEGF signalling. Hypoxia is also one of the factors that induces luteolysis by suppressing progesterone synthesis and by promoting apoptosis of luteal cells. The present review focuses on recent studies of hypoxic conditions, as well as HIF1-regulated genes and proteins, in the regulation of ovarian function. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 7 11 2012 Mitochondrial localization of ABC transporter ABCG2 and its function in 5-aminolevulinic acid-mediated protoporphyrin IX accumulation e50082 EN Hirotsugu Kobuchi Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koko Moriya Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tetsuya Ogino Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hirofumi Fujita Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keiji Inoue Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Taro Shuin Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tatsuji Yasuda Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kozo Utsumi Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshihiko Utsumi Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol. No potential conflict of interest relevant to this article was reported.

Wistar Institute of Anatomy and Biology Acta Medica Okayama 0021-9967 525 7 2017 Identification of the sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that controls male reproductive function in the mouse and Asian house musk shrew (Suncus murinus) 1586 1598 EN Kei Tamura Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasuhisa Kobayashi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Asuka Hirooka Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keiko Takanami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takamichi Jogahara Laboratory of Animal Management and Resources, Department of Zoology, Okayama University of Science Sen-ichi Oda Laboratory of Animal Management and Resources, Department of Zoology, Okayama University of Science Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 129 1 2017 薬物相互作用 （ 38 - 新規睡眠薬 : ラメルテオン, スボレキサントの相互作用） 53 57 EN Kazuko Kubo Department of Pharmacy, Okayama University Hospital Satoru Esumi Department of Pharmacy, Okayama University Hospital Yoshihisa Kitamura Department of Pharmacy, Okayama University Hospital Toshiaki Sendo Department of Pharmacy, Okayama University Hospital No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2016 BMP-6 modulates somatostatin effects on luteinizing hormone production by gonadrotrope cells EN Kishio Toma No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2045-2322 5 2015 The molecular mechanism of photochemical internalization of cell penetrating peptide-cargo-photosensitizer conjugates. 18577 18577 EN Takashi Ohtsuki Department of Medical Bioengineering, Okayama University Shunya Miki Department of Medical Bioengineering, Okayama University Shouhei Kobayashi Advanced ICT Research Institute Kobe, NICT Tokuko Haraguchi Advanced ICT Research Institute Kobe, NICT Eiji Nakata Institute of Advanced Energy, Kyoto University Kazutaka Hirakawa Department of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, Shizuoka University Kensuke Sumita Department of Medical Bioengineering, Okayama University Kazunori Watanabe Department of Medical Bioengineering, Okayama University Shigetoshi Okazaki Department of Medical Spectroscopy, Hamamatsu University School of Medicine In many drug delivery strategies, an inefficient transfer of macromolecules such as proteins and nucleic acids to the cytosol often occurs because of their endosomal entrapment. One of the methods to overcome this problem is photochemical internalization, which is achieved using a photosensitizer and light to facilitate the endosomal escape of the macromolecule. In this study, we examined the molecular mechanism of photochemical internalization of cell penetrating peptide-cargo (macromolecule)-photosensitizer conjugates. We measured the photophysical properties of eight dyes (photosensitizer candidates) and determined the respective endosomal escape efficiencies using these dyes. Correlation plots between these factors indicated that the photogenerated (1)O2 molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of (1)O2 was confirmed using (1)O2 quenchers. In addition, time-lapse fluorescence imaging showed that the photoinduced endosomal escape occurred at a few seconds to a few minutes after irradiation (much longer than (1)O2 lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 128 2 2016 平成27年度岡山医学会賞 総合研究奨励賞（結城賞） 91 94 EN Ai Kajita Department of Dermatology, Okayama University Hospital No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2016 Studies on the sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that controls male reproductive function EN Takumi Ochi No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2016 Regulatory effects of fibroblast growth factor-8 and tumor necrosis factor-α on osteoblast marker expression induced by bone morphogenetic protein-2 EN Takayuki Katsuyama No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2015 Postprandial serum C-peptide value is the optimal index to identify patients with non-obese type 2 diabetes who require multiple daily insulin injection: Analysis of C-peptide values before and after short-term intensive insulin therapy EN Daisuke Fujiwara No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2045-2322 5 2015 Peptide-modified Substrate for Modulating Gland Tissue Growth and Morphology In Vitro 11468 EN Hiroaki Taketa Ara Sathi Gulsan Farahat Mahmoud Anisur Rahman Kazi Takayoshi Sakai Yoshiaki Hirano Takuo Kuboki Yasuhiro Torii Takuya Matsumoto In vitro fabricated biological tissue would be a valuable tool to screen newly synthesized drugs or understand the tissue development process. Several studies have attempted to fabricate biological tissue in vitro. However, controlling the growth and morphology of the fabricated tissue remains a challenge. Therefore, new techniques are required to modulate tissue growth. RGD (arginine-glycine-aspartic acid), which is an integrin-binding domain of fibronectin, has been found to enhance cell adhesion and survival; it has been used to modify substrates for in vitro cell culture studies or used as tissue engineering scaffolds. In addition, this study shows novel functions of the RGD peptide, which enhances tissue growth and modulates tissue morphology in vitro. When an isolated submandibular gland (SMG) was cultured on an RGD-modified alginate hydrogel sheet, SMG growth including bud expansion and cleft formation was dramatically enhanced. Furthermore, we prepared small RGD-modified alginate beads and placed them on the growing SMG tissue. These RGD-modified beads successfully induced cleft formation at the bead position, guiding the desired SMG morphology. Thus, this RGD-modified material might be a promising tool to modulate tissue growth and morphology in vitro for biological tissue fabrication. No potential conflict of interest relevant to this article was reported.

Okayama University Acta Medica Okayama 13 2015 Peptide directs artificial tissue growth EN Takuya Matsumoto No potential conflict of interest relevant to this article was reported.

the Allergy and Immunology Society of Thailand Acta Medica Okayama 0125-877X 31 4 2013 Cytokine expression in human dermal fibroblasts stimulated with eosinophil cationic protein measured by protein array 271 276 EN Takamaro Sato Yoshihiko Soga Tomoko Yamaguchi Michio Meguro Hiroshi Maeda Joji Tada Takayuki Otani Masaharu Seno Shogo Takashiba [Background] : Eosinophil cationic protein (ECP) was reported previously to be involved in allergic inflammation with cytotoxic activity. On the other hand, recent studies showed that ECP did not induce cell death but inhibited the growth of cancer-derived cells. Our previous study indicated that human ECP enhanced differentiation of rat neonatal cardiomyocytes and stress fiber formation in Balb/c 3T3 mouse fibroblasts, while the effects of human ECP on human fibroblasts are unknown. [Objective] : The present study was performed to determine the effects of human ECP on cytokine expression in human fibroblasts by protein array. [Methods] : The effects of recombinant human ECP (rhECP) on normal human dermal fibroblasts (NHDF) were examined by assaying cell growth. Furthermore, cytokine expression of NHDF stimulated by ECP, which could influence cell growth, was evaluated by protein array. [Results] : ECP was not cytotoxic but enhanced the growth of NHDF. The peak rhECP concentration that enhanced the cell counts by 1.56-fold was 100 ng/mL, which was significantly different from cultures without ECP stimulation (ANOVA/Scheffe’s test, P < 0.05). Array analyses indicated that ciliary neurotrophic factor (CNTF), neutrophilactivating peptide (NAP)-2, and neurotrophin (NT)-3 were significantly upregulated in NHDF stimulated with 100 ng/mL ECP compared to those without stimulation. [Conclusion] : ECP is not cytotoxic but enhances the growth of NHDF. CNTF, NAP-2, and NT-3 were suggested to be involved in enhancing the growth of NHDF. These findings will contribute to determination of the role of ECP in allergic inflammation. (Asian Pac J Allergy Immunol 2013;31:271-6) No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 127 3 2015 透析シャント心不全―非過大シャント心不全 “Non-High-Output Cardiac Failure”の病態― 203 207 EN Toyomu Ugawa Background: Hemodialysis-related heart failure has been considered to be associated with excessive blood flow through the arteriovenous (AV) shunt used for vascular access. However, some patients undergoing dialysis have heart failure in the absence of an increase in cardiac output (CO) related to shunt blood-flow loading because the loading cannot be compensated for by increasing CO. This condition may be challenging to manage ; thus, early diagnosis is important. Methods and Results: Twelve patients (mean age, 71 years ; 9 men) with end-stage renal disease, dialysis-related heart failure, a high brain natriuretic peptide (BNP) level, and a mean New York Heart Association (NYHA) class of II underwent AV shunt closure. Their cardiac index (CI), pre- and post-dialysis BNP levels, and several cardiac variables were assessed pre- and postoperatively. All patients achieved relief of heart failure symptoms and a reduction in NYHA class after AV closure, but six patients had a postoperative increase in CI (the "non-high-output" cardiac failure group), whereas the other six had a decrease in CI (the "high-output" cardiac failure group). The high-output patients had greater improvements in BNP levels and most cardiac variables compared to the non-high-output group ; therefore, the heart failure in the non-high-output patients was considered more serious than that in the high-output group. Conclusions: The selection of effective strategies for treating dialysis-related heart failure may depend partly on identifying which patients have non-high-output failure. Such identification requires serial measurements of BNP levels and evaluations of cardiac variables other than the ejection fraction. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 127 2 2015 腹膜透析Update 111 115 EN Hitoshi Sugiyama No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 127 2 2015 平成26年度岡山医学会賞 総合研究奨励賞（結城賞） 87 90 EN Nanako Ookubo No potential conflict of interest relevant to this article was reported.

日本薬学会 Acta Medica Okayama 09186158 31 11 2008 Royal jelly ameliorates insulin resistance in fructose-drinking rats 2103 2107 EN Yoshito Zamami Shingo Takatori Mitsuhiro Goda Toshihiro Koyama Yukiko Iwatani Xin Jin Shima Takada-Doi Hiromu Kawasaki Royal jelly (RJ) is known to contain excellent nutrition and a variety of biological activities. The present study was designed to investigate the effects of RJ on insulin resistance (hyperinsulinemia) in fructose-drinking rats (FDR; insulin resistance animal model). Male Wistar rats (6 weeks old) received 15% fructose solution in drinking water for 8 weeks. FDR showed significant increases in plasma levels of insulin and triglyceride, Homeostasis Model Assessment ratio (HOMA-R, an index of insulin resistance), and systolic blood pressure, but not blood glucose levels, when compared with control rats. RJ (100, 300mg/kg, p.o.) treatment for 8 weeks significantly decreased the plasma levels of insulin and triglyceride, HOMA-R, without affecting blood glucose or total cholesterol levels and tended to lower systolic blood pressure. In isolated and perfused mesenteric vascular beds of FDR, RJ treatment resulted in a significant reduction in sympathetic nerve-mediated vasoconstrictor response to periarterial nerve stimulation (PNS) and tended to increase the calcitonin gene-related peptide (CGRP) nervemediated vasodilator response to PNS, compared with those in untreated FDR. However, RJ treatment did not significantly affect norepinephrine-induced vasoconstriction or CGRP-induced vasodilation. These results suggest that RJ could be an effective functional food to prevent insulin resistance associated with the development of hypertension. No potential conflict of interest relevant to this article was reported.

日本薬学会 Acta Medica Okayama 00316903 127 12 2007 フルクトース負荷インスリン抵抗性モデル(ラット)におけるPropolisによるインスリン抵抗性改善作用 2065 2073 EN Yoshito Zamami Shingo Takatori Toshihiro Koayama Mitsuhiro Goda Yukiko Iwatani Shima Doi Hiromu Kawasaki Propolis, a honeybee product, contains a variety of biologically active substances. The present study was designed to investigate the effects of propolis on insulin resistance induced by fructose-drinking rats (FDR; type 2 diabetic animal model). Male Wistar rats (6 weeks old) received 15% fructose solution in drinking water for 8 weeks. FDR showed significant increases in plasma levels of insulin, Homeostasis Model Assessment ratio (HOMA-R, an index of insulin resistance), body weight, and systolic blood pressure but not blood glucose levels, when compared with control rats. Brazilian propolis extract (100 and 300mg/kg, p. o.) treatment for 8 weeks significantly decreased the plasma level of insulin, HOMA-R, and body weight, increased plasma triglyceride levels without affecting blood glucose and total cholesterol levels, and tended to decrease systolic blood pressure. In isolated and perfused mesenteric vascular beds of FDR, propolis treatment resulted in a significant reduction of sympathetic nerve-mediated vasoconstrictor response to periarterial nerve stimulation (PNS; 8Hz) and tended to increase the calcitonin gene-related peptide (CGRP) nerve-mediated vasodilator response to PNS, compared with those in untreated FDR. However, propolis treatment did not significantly affect norepinephrine-induced vasoconstriction and CGRP-induced vasodilation. These results suggest that propolis could be an effective functional food to prevent the development of insulin resistance. No potential conflict of interest relevant to this article was reported.

日本薬学会 Acta Medica Okayama 00316903 128 3 2008 食後高血糖が血管反応性に及ぼす影響 419 424 EN Yoshito Zamami Shingo Takatori Yukiko Iwatani Kosuke Yamawaki Satoko Miyashita Nana Yabumae Fusako Takayama Mitsunobu Mio Hiromu Kawasaki Recent clinical studies demonstrated that transient postprandial hyperglycemia and hyperinsulinemia may contribute to the development of hypertension. Therefore, we investigated influence of acute hyperglycemia and/or hyperinsulinemia induced by glucose or insulin infusion on neuronal and humoral control of vascular tone in rats. Euglycemic male Wistar rats were pithed under anesthesia and arterial blood pressure was measured. Changes in vascular responses to spinal cord stimulation (SCS) and intravenous bolus injections of noradrenaline, angiotensin II, calcitonin generelated peptide (CGRP), acetylcholine and sodium nitroprusside (SNP) were studied by infusing various concentration of glucose or insulin. Continuous glucose infusion, which increased both blood glucose and serum insulin levels, significantly augmented adrenergic nerve-mediated pressor responses to SCS without affecting injection of pressor responses to noradrenaline or angiotensin II. In pithed rats with artificially increased blood pressure and blockade of autonomic outflow, glucose infusion attenuated CGRPergic nerve-depressor responses to SCS without affecting depressor responses to injection of CGRP, acetylcholine or SNP. In pithed rats treated with octreotide, which increased blood glucose without increasing serum insulin levels, glucose infusion caused only significant augmentation of adrenergic nervemediated pressor responses. Combined infusion of insulin and glucose, which resulted in increased serum insulin levels with euglycemic, significantly augmented adrenergic nerve-mediated pressor responses and attenuated CGRPergic nerve-mediated depressor responses. The present results suggest that acute hyperglycemia and hyperinsulinemia increases adrenergic nerve-mediated vasoconstriction, which is partly associated with the blunted CGRPergic nerve function, and that plasma insulin concentration associated with hyperglycemia may be responsible for alteration of neuronal vascular regulation. No potential conflict of interest relevant to this article was reported.

日本薬学会 Acta Medica Okayama 0031-6903 130 6 2010 Propolis 長期投与による Otsuka Long-Evans Tokushima Fatty (OLETF) ラットの インスリン抵抗性改善作用 833 840 EN Yoshito Zamami Hiroki Fujiwara Miho Hosoda Hayato Hino Kazuhiro Hirai Kazuaki Okamoto Hiromu Kawasaki Propolis is known to have abundant bioactive constituents and a variety of biological activities. To investigate the effect of Brazilian propolis on insulin resistance, 10-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a non-insulin-dependent type 2 diabetic model, were treated for 4 weeks with propolis (100 and 300 mg/kg, p.o.) or vehicle (control). Propolis treatment significantly decreased the plasma levels of insulin and insulin resistance index (Homeostasis Model Assessment-Insulin Resistance; HOM-IR), without affecting blood glucose levels and tended to lower systolic blood pressure compared with the control. In isolated and perfused mesenteric vascular beds of OLETF rats, propolis treatment resulted in significant reduction of sympathetic nerve-mediated vasoconstrictor response to periarterial nerve stimulation (PNS) and tended to increase calcitonin gene-related peptide (CGRP) nerve-mediated vasodilator response to PNS compared with in vehicle-treated OLETF rats. However, propolis treatment did not significantly affect the vasoconstrictor and vasodilator response to noradrenaline, CGRP, acetylcholine, and sodium nitroprusside. These results suggest that propolis could be an effective and functional food to prevent development of insulin resistance. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0172-6390 61 130 2014 Management Of Peritoneal Effusion by Sealing with a Self-Assembling Nanofiber Polypeptide Following Pelvic Surgery 349 353 EN Yoshitaka Kondo Takeshi Nagasaka Satoru Kobayashi Naoya Kobayashi Toshiyoshi Fujiwara Background/Aims: PuraMatrix is a synthetic material consisting of 16-amino acid peptides that self-assemble into nanofibers, previously used as a scaffold for functional cell cultures. We conducted a clinical study to determine the safety and sealing properties of PuraMatrix in post-operative lymphorrhea following pelvic surgery in humans. Methodology: A total of 20 patients who underwent rectal cancer resection were analyzed. The study group (n = 10) consisted of patients who received PuraMatrix, matched with a control group (n = 10) of patients operated on conventionally. Results: During the 2 to 3 month follow-up period, there were no abnormal findings or adverse events in any the patients who received PuraMatrix. We found that the patients who received PuraMatrix had significantly reduced post-operative drainage volumes compared with the patients in the control group. Conclusions: PuraMatrix is a safe and effective bio-compatible sealing material for the management of post-operative peritoneal effusion following pelvic surgery. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0007-0963 171 3 2014 Cathelicidin antimicrobial peptide LL-37 augments interferon-beta expression and antiviral activity induced by double-stranded RNA in keratinocytes 492 498 EN T Takiguchi S Morizane T Yamamoto A Kajita K Ikeda K Iwatsuki Background Cathelicidin antimicrobial peptide LL-37 has the capacity to kill a wide range of microbes and to modify host immunity. Recently, our group observed that the activation of keratinocytes by LL-37 and DNA greatly increases interferon (IFN)-beta through Toll-like receptor (TLR) 9. However, the effect of LL-37 on the induction of IFN-beta through TLR3, a sensor of double-stranded (ds) RNA, in keratinocytes is not well known. Objectives To investigate whether LL-37 could affect TLR3 signalling and antiviral activity in normal human epidermal keratinocytes (NHEKs). Methods We investigated the production of IFN-beta in NHEKs stimulated with a TLR3 ligand, poly (I:C), in the presence of LL-37. To examine the effect of LL-37 and poly (I:C) on antiviral activity, a virus plaque assay using herpes simplex (HS) virus type-1 was carried out. The uptake of poly (I:C) conjugated with fluorescein isothiocyanate (FITC) into the keratinocytes was observed in the presence of LL-37. Immunostaining for TLR3 and LL-37 was performed using skin samples from HS. Results LL-37 and poly (I:C) synergistically induced the expression of IFN-beta in NHEKs. Furthermore, co-stimulation with LL-37 and poly (I:C) significantly decreased the viral plaque numbers compared with poly (I:C) or LL-37 alone. LL-37 enhanced the uptake of FITC-conjugated poly (I:C) into cells. Immunohistochemical analysis demonstrated that the expression of TLR3 and LL-37 is up-regulated in HS lesions. Conclusions Our findings suggest that LL-37 augments the antiviral activity induced by dsRNA in keratinocytes, which may contribute to the innate immune response to cutaneous viral infections such as HS. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2014 Cathelicidin antimicrobial peptide LL-37 augments IFN-β expression and anti-viral activity induced by double-stranded RNA in keratinocytes EN Tetsuya Takiguchi No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2014 A New Classification System for Evaluating Patients with Severe Trauma Using B-type Natriuretic Peptide Levels and Estimated Glomerular Filtration Rate EN Sunao Morisada No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 68 5 2014 Prompt Resolution of Hypoglycemia by Hepatic Transarterial Embolization for Malignant Insulinoma with Multiple Liver Metastases 307 311 EN Shinichiro Muro Junichiro Nasu Ryo Harada Minoru Matsubara Asuka Nakarai Hiromitsu Kanzaki Kouichiro Tsutsumi Hironari Kato Takehiro Tanaka Hiroyasu Fujiwara Masatoshi Uno Hiroyuki Okada Kazuhide Yamamoto Case Report 10.18926/AMO/52900 A 45-year-old female who presented with loss of consciousness and a cold sweat was found to have a pancreatic tumor and multiple liver metastases. Laboratory studies showed marked hypoglycemia and inappropriately elevated serum insulin, C-peptide, and serum tumor markers. Fine needle aspiration revealed Grade 3 small-cell type primary pancreatic neuroendocrine carcinoma. Consequently, the diagnosis of malignant insulinoma was made. Transarterial embolization (TAE) for hepatic metastases resulted in the reduction of tumor volume and prompt resolution of hypoglycemic attacks, whereas diazoxide and systemic chemotherapy had been ineffective for controlling blood glucose levels, and octreotide was unavailable due to the allergic effect. This case report highlights the potential usefulness of TAE for malignant insulinomas in the management of hypoglycemia. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 68 5 2014 A New Classification System for Evaluating Patients with Severe Trauma Using B-type Natriuretic Peptide Levels and Estimated Glomerular Filtration Rate 285 290 EN Sunao Morisada Toyomu Ugawa Nobuyuki Nosaka Yoshihito Ujike Original Article 10.18926/AMO/52897 Current systems for the evaluation of trauma severity are tedious and difficult to apply in an actual emergency setting. We aimed to develop and assess the accuracy of a more efficient severity evaluation system, termed the Ugawa classification, using brain-type natriuretic peptide (BNP) measurement and the estimated glomerular filtration rate (eGFR). Two-hundred trauma patients were divided into 2 groups using an eGFR cut-off value of 90ml/min/1.73m2 as an indicator of normal renal function and 2 additional groups according to whether the BNP values were greater or less than the age in years. This resulted in 4 subject groups with different combinations of eGFR and BNP. The mean SOFA score, injury severity scores (ISS), trauma and injury severity scores (TRISS), and Acute Physiology and Chronic Health Evaluation II (APACHE II) scores of the groups were compared by Kruskal-Wallis test, and the mortality rate after 90 days was calculated. Significant intergroup differences were found in SOFA scores, ISS scores, and APACHE II-predicted mortality rates. Although no significant differences were found in the mortality rate after 90 days or TRISS-predicted mortality rate among the 4 groups, there was a trend toward increasing trauma severity from group 1 to 4. Thus, the Ugawa classification is as accurate as existing systems, has greater efficiency, and is user-friendly. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0303-7207 381 1-2 2013 Mutual interaction of kisspeptin, estrogen and bone morphogenetic protein-4 activity in GnRH regulation by GT1-7 cells 8 15 EN Tomohiro Terasaka Fumio Otsuka Naoko Tsukamoto Eri Nakamura Kenichi Inagaki Kishio Toma Kanako Ogura-Ochi Christine Glidewell-Kenney Mark A. Lawson Hirofumi Makino Reproduction is integrated by interaction of neural and hormonal signals converging on hypothalamic neurons for controlling gonadotropin-releasing hormone (GnRH). Kisspeptin, the peptide product of the kiss1 gene and the endogenous agonist for the GRP54 receptor, plays a key role in the regulation of GnRH secretion. In the present study, we investigated the interaction between kisspeptin, estrogen and BMPs in the regulation of GnRH production by using mouse hypothalamic GT1-7 cells. Treatment with kisspeptin increased GnRH mRNA expression and GnRH protein production in a concentration-dependent manner. The expression levels of kiss1 and GPR54 were not changed by kisspeptin stimulation. Kisspeptin induction of GnRH was suppressed by co-treatment with BMPs, with BMP-4 action being the most potent for suppressing the kisspeptin effect. The expression of kisspeptin receptor, GPR54, was suppressed by BMPs, and this effect was reversed in the presence of kisspeptin. It was also revealed that BMP-induced Smad1/5/8 phosphorylation and Id-1 expression were suppressed and inhibitory Smad6/7 was induced by kisspeptin. In addition, estrogen induced GPR54 expression, while kisspeptin increased the expression levels of ER alpha. and ER beta, suggesting that the actions of estrogen and kisspeptin are mutually enhanced in GT1-7 cells. Moreover, kisspeptin stimulated MAPKs and AKT signaling, and ERK signaling was functionally involved in the kisspeptin-induced GnRH expression. BMP-4 was found to suppress kisspeptin-induced GnRH expression by reducing ERK signaling activity. Collectively, the results indicate that the axis of kisspeptin-induced GnRH production is bi-directionally controlled, being augmented by an interaction between ER alpha/beta and GPR54 signaling and suppressed by BMP-4 action in GT1-7 neuron cells. No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 9 7 2014 The Neutral Self-Assembling Peptide Hydrogel SPG-178 as a Topical Hemostatic Agent EN Seiji Komatsu Yusuke Nagai Keiji Naruse Yoshihiro Kimata Conventional self-assembling peptide hydrogels are effective as topical hemostatic agents. However, there is a possibility to harm living tissues due to their low pH. The aim of the present study was to demonstrate the efficacy of SPG-178, a neutral self-assembling peptide hydrogel, as a topical hemostatic agent. First, we measured the bleeding duration of incisions made on rat livers after application of SPG-178 (1.0% w/v), SPG-178 (1.5% w/v), RADA16 (1.0% w/v), and saline (n = 12/group). Second, we observed the bleeding surfaces by transmission electron microscopy immediately after hemostasis. Third, we measured the elastic and viscous responses (G′ and G″, respectively) of the hydrogels using a rheometer. Our results showed that bleeding duration was significantly shorter in the SPG-178 group than in the RADA16 group and that there were no significant differences in transmission electron microscopy findings between the groups. The greater the G′ value of a hydrogel, the shorter was the bleeding duration. We concluded that SPG-178 is more effective and has several advantages: it is non-biological, transparent, nonadherent, and neutral and can be sterilized by autoclaving. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 126 2 2014 平成25年度岡山医学会賞 胸部・循環研究奨励賞（砂田賞） 95 98 EN Atsuko Nakatsuka No potential conflict of interest relevant to this article was reported.

Spandidos Publications Ltd. Acta Medica Okayama 1107-3756 32 4 2013 Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization 938 944 EN Endy Widya Putranto Hitoshi Murata Ken-Ichi Yamamoto Ken Kataoka Hidenori Yamada Jun-Ichiro Futami Masakiyo Sakaguchi Nam-Ho Huh The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid beta. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 126 1 2014 自己集合性ペプチドハイドロゲル内で三次元培養された細胞への機械刺激 7 10 EN Yusuke Nagai Hidenori Yokoi Keiko Kaihara Keiji Naruse No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 126 1 2014 糖尿病の腎臓においてcholecystokininはマクロファージに対する抗炎症作用を介した新規の保護効果を発揮する 1 6 EN Satoshi Miyamoto Kenichi Shikata Kyoko Miyasaka Shinichi Okada Motofumi Sasaki Ryo Kodera Daisho Hirota Nobuo Kajitani Tetsuharu Takatsuka Hitomi Kataoka Usui Shingo Nishishita Chikage Horiguchi Sato Akihiro Funakoshi Hisakazu Nishimori Haruhito Adam Uchida Daisuke Ogawa Hirofumi Makino No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2013 Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization EN Endy Widya Putranto No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 2186-7755 103 2014 Changes in Glycinin‒Digesting Protease Activity During Soybean Germination. 1 4 EN Md. Akhtaruzzaman Megumi Maeda Keiko Kitagawa Shigeaki Takagi Yoshinobu Kimura 　Changes in glycinin-digesting protease activity during soybean germination have been investigated. The glycinin-digesting protease activities of imbibed or germinated soybean seed were assayed by RP‒HPLC using a tryptic peptide from CM‒glycinin or by SDS‒PAGE using CM‒glycinin as the endogenous substrate. Proteolytic activities of the germinated soybean seeds were found through the whole period of germination, the activities were maintained significantly unchanged during germination for 4 days, and then those specific activities declined slowly. AE‒HPLC analysis of the glycinin-digesting protease in the imbibed or germinated soybean seeds showed unchanged peaks corresponding to glycinin- digesting activity, suggesting that the glycinin-digesting protease was not induced during germination but had already been synthesized during seed maturation. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 37 11 2013 Effects of atrial natriuretic Peptide after prolonged hypothermic storage of the isolated rat heart 1003 1008 EN Hitoshi Kanamitsu Yasuhiro Fujii Hideya Mitsui Shunji Sano Primary graft failure (PGF) caused by ischemia-reperfusion injury (IRI) is the strongest determinant of perioperative mortality after heart transplantation. Atrial natriuretic peptide (ANP) has been found to reduce the IRI of cardiomyocytes and may be beneficial in alleviating PGF after heart transplantation, although there is a lack of evidence to support this issue. The purpose of this study was to investigate the cardioprotective effects of ANP after prolonged hypothermic storage. For this purpose, an isolated working-heart rat model was used. After the preparation, the hearts were arrested with and stored in an extracellular-based cardioplegic solution at 3-4°C for 6 h and followed by 25 min of reperfusion. The hearts were divided into four groups (n = 7 in each group) according to the timing of ANP administration: Group 1 (in perfusate before storage), Group 2 (in cardioplegia), Group 3 (in reperfusate), and control (no administration of ANP). Left ventricular functional recovery and the incidence of ventricular fibrillation (VF) were compared. ANP administration at the time of reperfusion improved the percent recovery of left ventricular developed pressure (control, 45.5 ± 10.2; Group 1, 47.4 ± 8.8; Group 2, 45.3 ± 12 vs. Group 3, 76.3 ± 7; P < 0.05) and maximum first derivative of the left ventricular pressure (control, 47.9 ± 8.7; Group 1, 46.7 ± 8.8; Group 2, 49.6 ± 10.8 vs. Group 3, 76.6 ± 7.5; P < 0.05). The incidence of VF after reperfusion did not differ significantly among these four groups (71.4, 85.7, 57.1, and 85.7% in Groups 1, 2, 3, and control, respectively). This result suggests that the administration of ANP at the time of reperfusion may have the potential to decrease the incidence of PGF after heart transplantation. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2013 Effects of Atrial Natriuretic Peptide After Prolonged Hypothermic Storage of the Isolated Rat Heart EN Hitoshi Kanemitsu No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 7 11 2012 Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation EN Hirotsugu Kobuchi Koko Moriya Tetsuya Ogino Hirofumi Fujita Keiji Inoue Taro Shuin Tatsuji Yasuda Kozo Utsumi Toshihiko Utsumi Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0340-7004 62 4 2013 Intratumoral peptide injection enhances tumor cell antigenicity recognized by cytotoxic T lymphocytes: a potential option for improvement in antigen-specific cancer immunotherapy 639 652 EN Daisuke Nobuoka Toshiaki Yoshikawa Mari Takahashi Tatsuaki Iwama Kazutaka Horie Manami Shimomura Shiro Suzuki Noriko Sakemura Munehide Nakatsugawa Hiroshi Sadamori Takahito Yagi Toshiyoshi Fujiwara Tetsuya Nakatsura Antigen-specific cancer immunotherapy is a promising strategy for improving cancer treatment. Recently, many tumor-associated antigens and their epitopes recognized by cytotoxic T lymphocytes (CTLs) have been identified. However, the density of endogenously presented antigen-derived peptides on tumor cells is generally sparse, resulting in the inability of antigen-specific CTLs to work effectively. We hypothesize that increasing the density of an antigen-derived peptide would enhance antigen-specific cancer immunotherapy. Here, we demonstrated that intratumoral peptide injection leads to additional peptide loading onto major histocompatibility complex class I molecules of tumor cells, enhancing tumor cell recognition by antigen-specific CTLs. In in vitro studies, human leukocyte antigen (HLA)-A*02:01-restricted glypican-3(144-152) (FVGEFFTDV) and cytomegalovirus(495-503) (NLVPMVATV) peptide-specific CTLs showed strong activity against all peptide-pulsed cell lines, regardless of whether the tumor cells expressed the antigen. In in vivo studies using immunodeficient mice, glypican-3(144-152) and cytomegalovirus(495-503) peptides injected into a solid mass were loaded onto HLA class I molecules of tumor cells. In a peptide vaccine model and an adoptive cell transfer model using C57BL/6 mice, intratumoral injection of ovalbumin(257-264) peptide (SIINFEKL) was effective for tumor growth inhibition and survival against ovalbumin-negative tumors without adverse reactions. Moreover, we demonstrated an antigen-spreading effect that occurred after intratumoral peptide injection. Intratumoral peptide injection enhances tumor cell antigenicity and may be a useful option for improvement in antigen-specific cancer immunotherapy against solid tumors. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2013 Intratumoral peptide injection enhances tumor cell antigenicity recognized by cytotoxic T lymphocytes: a potential option for improvement in antigen-specific cancer immunotherapy EN Daisuke Nobuoka No potential conflict of interest relevant to this article was reported.

Elsevier Sci Ltd Acta Medica Okayama 0264-410X 28 32 2010 Three novel NY-ESO-1 epitopes bound to DRB1*0803, DQB1*0401 and DRB1*0901 recognized by CD4 T cells from CHP-NY-ESO-1-vaccinated patients 5338 5346 EN Yu Mizote Taku Taniguchi Kei Tanaka Midori Isobe Hisashi Wada Takashi Saika Shoichi Kita Yukari Koide Akiko Uenaka Eiichi Nakayama Three novel NY-ESO-1 CD4 T cell epitopes were identified using PBMC obtained from patients who were vaccinated with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1). The restriction molecules were determined by antibody blocking and using various EBV-B cells with different HLA alleles as APC to present peptides to CD4 T cells. The minimal epitope peptides were determined using various N- and C-termini truncated peptides deduced from 18-mer overlapping peptides originally identified for recognition. Those epitopes were DRB1*0901-restricted NY-ESO-1 87-100. DQB1*0401-restricted NY-ESO-1 95-107 and DRB1*0803-restricted NY-ESO-1 124-134. CD4 T cells used to determine those epitope peptides recognized EBV-B cells or DC that were treated with recombinant NY-ESO-1 protein or NY-ESO-1-expressing tumor cell lysate, suggesting that the epitope peptides are naturally processed. These CD4 T cells showed a cytokine profile with Th1 characteristics. Furthermore, NY-ESO-1 87-100 peptide/HLA-DRB1*0901 tetramer staining was observed. Multiple Th1-type CD4 T cell responses are beneficial for inducing effective anti-tumor responses after NY-ESO-1 protein vaccination. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0020-7136 131 11 2012 Runt-related transcription factor 3 reverses epithelial-mesenchymal transition in hepatocellular carcinoma 2537 2546 EN Shigetomi Tanaka Hidenori Shiraha Yutaka Nakanishi Shin-Ichi Nishina Minoru Matsubara Shigeru Horiguchi Nobuyuki Takaoka Masaya Iwamuro Junro Kataoka Kenji Kuwaki Hiroaki Hagihara Junichi Toshimori Hideki Ohnishi Akinobu Takaki Shinichiro Nakamura Kazuhiro Nouso Takahito Yagi Kazuhide Yamamoto Loss or decreased expression of runt-related transcription factor 3 (RUNX3), a tumor suppressor gene involved in gastric and other cancers, has been frequently observed in hepatocellular carcinoma (HCC). The objective of this study was to identify the regulatory mechanism of the epithelialmesenchymal transition (EMT) by RUNX3 in HCC. Human HCC cell lines, Hep3B, Huh7, HLF and SK-Hep1, were divided into low- and high-EMT lines, based on their expression of TWIST1 and SNAI2, and were used in this in vitro study. Ectopic RUNX3 expression had an anti-EMT effect in low-EMT HCC cell lines characterized by increased E-cadherin expression and decreased N-cadherin and vimentin expression. RUNX3 expression has previously been reported to reduce jagged-1 (JAG1) expression; therefore, JAG1 ligand peptide was used to reinduce EMT in RUNX3-expressing low-EMT HCC cells. Immunohistochemical analyses were performed for RUNX3, E-cadherin, N-cadherin and TWIST1 in 33 human HCC tissues, also divided into low- and high-EMT HCC, based on TWIST1 expression. E-cadherin expression was correlated positively and N-cadherin expression was correlated negatively with RUNX3 expression in low-EMT HCC tissues. Correlations between EMT markers and RUNX3 mRNA expression were analyzed using Oncomine datasets. Similarly, mRNA expression of E-cadherin was also significantly correlated with that of RUNX3 in low-EMT HCC, while mRNA expression of JAG1 was negatively correlated with that of RUNX3. These results suggest a novel mechanism by which loss or decreased expression of RUNX3 induces EMT via induction of JAG1 expression in low-EMT HCC. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 67 1 2013 Phospholipase C Produced by Clostridium botulinum Types C and D:Comparison of Gene, Enzymatic, and Biological Activities with Those of Clostridium perfringens Alpha-toxin 9 18 EN Ni Nengah Dwi Fatmawati Yoshihiko Sakaguchi Tomonori Suzuki Masataka Oda Kenta Shimizu Yumiko Yamamoto Jun Sakurai Osamu Matsushita Keiji Oguma Original Article 10.18926/AMO/49252 Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 124 2 2012 熱ショックタンパク質と抗腫瘍免疫 175 177 EN Shusaku Mizukami No potential conflict of interest relevant to this article was reported.

Elsevier Ltd. Acta Medica Okayama 0142-9612 33 4 2012 The mechanical stimulation of cells in 3D culture within a self-assembling peptide hydrogel 1044 1051 EN Yusuke Nagai The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 +/- 1.5 times during 8 days of incubation; mean +/- SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 +/- 0.2 times; mean +/- SEM). These results demonstrated that the developed self-assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2012 The mechanical stimulation of cells in 3D culture within a self-assembling peptide hydrogel EN Yusuke Nagai No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 124 1 2012 ニガウリ抽出物の血糖降下作用に関する文献的考察 15 26 EN Mitsumasa Mankura Yasuko Noda Akitane Mori Diabetes mellitus (DM) represents a global health and economical problem. Many patients with DM in Asia, South America, India and East Africa have traditionally used the water extract of unripe fruits of Momordica charantia (bitter melon) as some form of complementary and alternative medicine. Studies of laboratory animals have shown the beneficial blood-glucose lowering and anti-diabetic effects of this remedy. Some oral components that bring lower blood glucose level have been isolated : charantin (sterol glycosides), charantin (polypeptide) and cucurbine-type triterpenes. Part of their actions are related to AMP-activated kinase and repression of the oxidative stress that is increased in DM. Most clinical reports are not fully convincing due to the lack of randomized control studies. The present article reviews the pharmacological and clinical effects of bitter melon with special emphasis on the anti-diabetic effects, and some effects that would require caution in the context of human trials. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 66 1 2012 Heat Shock Protein Magic in Antigen Trafficking within Dendritic Cells:Implications in Antigen Cross-presentation in Immunity 1 6 EN Heiichiro Udono Review /10.18926/AMO/48075 Dendritic cells (DCs) take up soluble- or cell-associated antigens and digest them, delivering fragments to the MHC class I pathway to display antigenic peptides to CD8＋ T cells, a process known as cross-presentation. The pathway requires that, in order to be degraded by proteosomes, the extracellular antigens must have access to the cytosol across the endosomal membranes. Although the cross-presentation phenomena was first identified in the 1970s, the molecular mechanism responsible for the translocation is still not fully understood. In this context, we have recently found that cytosolic heat shock protein (HSP)90 translocates internalized antigen to the cytosol in DCs. Our results revealed the important role that cytosolic HSP90 plays in cross-presentation by pulling out endosomal antigen to the cytosol. 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 Lactobacillus gasseri の生産するバクテリオシンの食品利用へ向けた検討 65 70 EN Kensuke Arakawa Bacteriocins are natural antibacterial peptides ribosomally biosynthesized by bacteria. Gassericin T which is a bacteriocin produced by many strains of Lactobacillus gasseri has a broad antibacterial spectrum against food spoilage and pathogenic Gram-positive bacteria; therefore, the bacteriocin and its producers are predicted for use as safe food preservatives. However, probiotic Lb. gasseri strains show poor growth and minimally produce gassericin T in food-grade natural media such as reconstituted cheese whey, unlike in MRS broth. The growth of Lb. gasseri in reconstituted cheese whey was improved by adding proteose peptone as a nutrient factor. The production of gassericin T in MRS broth was specifically inhibited by adding divalent metal cations, and then the inhibition was removed using a chelator of divalent cations, trisodium citrate dihydrate. The production of gassericin T in reconstituted cheese whey was also restored by adding trisodium citrate dihydrate, and the activity was enhanced by a surfactant, Tween 80. In addition, trisodium citrate dihydrate led to over-production of and synergistic antibacterial effect with gassericin T. In this study, we developed a cheese whey-based medium containing proteose peptone, trisodium citrate dihydrate and Tween 80 for gassericin T production. The developed food-grade medium may contribute to the effective use of some bacteriocins from probiotic lactic acid bacteria for biopreservation of foods. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 20 11 2003 Phase Shifts of the Circadian Locomotor Rhythm Induced by Pigment-Dispersing Factor in the Cricket Gryllus bimaculatus 1347 1354 EN Muniyandi Singaravel Yuko Fujisawa Miki Hisada ASM Saifullah Kenji Tomioka Pigment-dispersing factors (PDFs) are octadeca-peptides widely distributed in insect optic lobes and brain. In this study, we have purified PDF and determined its amino acid sequence in the cricket Gryllus bimaculatus. Its primary structure was NSEIINSLLGLPKVLNDA-NH2, homologous to other PDH family members so far reported. When injected into the optic lobe of experimentally blinded adult male crickets, Gryllus-PDF induced phase shifts in their activity rhythms in a phase dependent and dose dependent manner. The resulted phase response curve (PRC) showed delays during the late subjective night to early subjective day and advances during the mid subjective day to mid subjective night. The PRC was different in shape from those for light, serotonin and temperature. These results suggest that PDF plays a role in phase regulation of the circadian clock through a separate pathway from those of other known phase regulating agents. No potential conflict of interest relevant to this article was reported.