Zoological Society of JapanActa Medica Okayama0289-00032292005Organ-Specific and Age-Dependent Expression of Insulin-like Growth Factor-I (IGF-I) mRNA Variants: IGF-IA and IB mRNAs in the Mouse10111021ENTakashiOhtsukiMarikoOtsukiYousukeMurakamiTetsuyaMaekawaTakashiYamamotoKojiAkasakaSakaeTakeuchiSumioTakahashiInsulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse IGF-IA and IGF-IB mRNAs using SYBR Green real-time RT-PCR. In the liver, IGF-I mRNA expression increased from 10 days of age to 45 days. In the uterus and ovary, IGF-I mRNA expression increased from 21 days of age, and then decreased at 45 days. In the kidney, IGF-I mRNA expression decreased from 10 days of age. IGF-IA mRNA levels were higher than IGF-IB mRNA levels in all organs examined. Estradiol-17 beta (E2) treatment in ovariectomized mice increased uterine IGF-IA and IGF-IB mRNA levels from 3 hr after injection, and highest levels for both mRNAs were detected at 6 hr, and relative increase was greater for IGF-IB mRNA than for IGF-IA mRNA. These results suggest that expression of IGF-I mRNA variants is regulated in organ-specific and age-dependent manners, and estrogen is involved in the change of IGF-I mRNA variant expression.No potential conflict of interest relevant to this article was reported.Zoological Society of JapanActa Medica Okayama0289-00032432007Alternative Leader-Exon Usage in Mouse IGF-I mRNA Variants: Class 1 and Class 2 IGF-I mRNAs241247ENTakashiOhtsukiMarikoOtsukiYousukeMurakamiKensakuHirataSakaeTakeuchiSumioTakahashiThe mouse IGF-I gene contains six exons, and exon 1 and exon 2 gene are considered to be leader exons. The regulatory mechanism of alternative usage of the leader exons is unclear in mice. The present study, was aimed at clarifying changes in class 1 (derived from exon 1) and class 2 (derived from exon 2) IGF-I mRNA expression in mice under various conditions. Both class 1 and class 2 IGF-I mRNAs were expressed in the mouse uterus, liver and kidney, and class 1 IGF-I mRNA was the major transcript in all organs studied. In the uterus, both class 1 and class 2 IGF-I mRNA expression changed markedly during the estrous cycle, with the highest level at proestrus, but in the liver and kidney there were no significant changes in IGF-I mRNA expression during the estrous cycle. Estrogen treatment increased both class 1 and class 2 IGF-I mRNA levels in the uterus of ovariectomized mice, but class 1 mRNA expression increased more in response to estrogen treatment than class 2 mRNA expression. These findings suggest that estrogen stimulates IGF-I gene expression in, uterine cells, and that a promoter involved in transcription of class 1 IGF-I mRNA is more responsive to estrogen. In conclusion, the present study revealed that two leader exons of mouse IGF-I gene are used in the uterus, liver and kidney. IGF-I mRNA levels of both classes changed during the estrous cycle in the uterus, but not in the liver or kidney. Estrogen increased IGF-I mRNA levels of both classes in the uterus.No potential conflict of interest relevant to this article was reported.Elsevier Ltd.Acta Medica Okayama0960-894X19132009Carrier PNA for shRNA delivery into cells34103413ENMizukiKitamatsuTakanoriKuboRinoMatsuzakiTamakiEndohTakashiOhtsukiMasahikoSisidoA peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as 'bridgebuilder' to connect a CPP with an shRNA. The carrier PNA successfully formed a hybrid with an shRNA bearing complementary dangling bases and the shRNA was introduced into cells by the carrier PNA, and RNAi was induced by the shRNA.No potential conflict of interest relevant to this article was reported.Pergamon-Elsevier Science Ltd.Acta Medica Okayama0040-402066512010Synthesis of pyrrolidine-based oxy-peptide nucleic acids carrying four types of nucleobases and their transport into cytoplasm96599666ENMizukiKitamatsuAkikoTakahashiTakashiOhtsukiMasahikoSisidoWe synthesized 16 pyrrolidine-based oxy-peptide nucleic acid (POPNA) monomers carrying four different nucleobases onto four different stereoisomers of pyrrolidine rings. Using these monomers, we prepared POPNA oligomers, which formed sequence-specific hybrids with DNAs. The oligomer configurations influenced the hybrid stability. The oligomers were not taken into CHO cells. However, they could enter the cell cytoplasm when mixed with the influenza virus hemagglutinin peptide-arginine heptamer conjugate.No potential conflict of interest relevant to this article was reported.Pergamon-Elsevier Science Ltd,Acta Medica Okayama0960-894X2112011Antisense effect of pyrrolidine-based oxy-peptide nucleic acids in Escherichia coli225227ENMizukiKitamatsuShunsukeKuramiTakashiOhtsukiMasahikoSisidoTo investigate the antisense effect of a pyrrolidine-based oxy-peptide nucleic acid (POPNA), we carried out the LacZ reporter assay using a 12-mer trans-l-POPNA conjugated with a cell-penetrating peptide (antisense reagent). The antisense effect of the conjugated POPNA (inhibition of LacZ activity) was comparable to that shown by a Nielsen-type peptide nucleic acid. Furthermore, the conjugated POPNA could switch the LacZ activity over a wide range of ambient temperatures.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama2045-232252015The molecular mechanism of photochemical internalization of cell penetrating peptide-cargo-photosensitizer conjugates.1857718577ENTakashiOhtsukiDepartment of Medical Bioengineering, Okayama UniversityShunyaMikiDepartment of Medical Bioengineering, Okayama UniversityShouheiKobayashiAdvanced ICT Research Institute Kobe, NICTTokukoHaraguchiAdvanced ICT Research Institute Kobe, NICTEijiNakataInstitute of Advanced Energy, Kyoto UniversityKazutakaHirakawaDepartment of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, Shizuoka UniversityKensukeSumitaDepartment of Medical Bioengineering, Okayama UniversityKazunoriWatanabeDepartment of Medical Bioengineering, Okayama UniversityShigetoshiOkazakiDepartment of Medical Spectroscopy, Hamamatsu University School of MedicineIn 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.Acta Medica Okayama2041-172372016Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs1250112501ENTakashiOhtsukiDepartment of Biomedical Engineering, Okayama UniversityShigetoKanzakiDepartment of Biomedical Engineering, Okayama UniversitySaeNishimuraDepartment of Biomedical Engineering, Okayama UniversityYoshioKunihiroDepartment of Biomedical Engineering, Okayama UniversityMasahikoSisidoDepartment of Biomedical Engineering, Okayama UniversityKazunoriWatanabeDepartment of Biomedical Engineering, Okayama UniversityThe possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl)methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at ∼405 nm at 125 mW cm(-2), DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells. No potential conflict of interest relevant to this article was reported.Acta Medica Okayama2041-172372016Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs12501ENTakashiOhtsukiDepartment of Biomedical Engineering, Okayama UniversityShigetoKanzakiDepartment of Biomedical Engineering, Okayama UniversitySaeNishimuraDepartment of Biomedical Engineering, Okayama UniversityYoshioKunihiroDepartment of Biomedical Engineering, Okayama UniversityMasahikoSisidoDepartment of Biomedical Engineering, Okayama UniversityKazunoriWatanabeDepartment of Biomedical Engineering, Okayama University The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl)methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at ∼405 nm at 125 mW cm(-2), DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0014482738322019Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1111556ENTakashiOhtsukiDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityAkiraShinaokaDepartment of Human Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKanaeKumagishi-ShinaokaDepartment of Human Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeiichiAsanoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOmer FarukHatipogluDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityJunkoInagakiDepartment of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKenTakahashiDepartment of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeiichiroNishidaDepartment of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeijiNaruseDepartment of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSatoshiHirohataDepartment of Medical Technology, Graduate School of Health Sciences, Okayama University The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1β and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama1422-00672192020Induction of CEMIP in Chondrocytes by Inflammatory Cytokines: Underlying Mechanisms and Potential Involvement in Osteoarthritis3140ENTakashiOhtsukiDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityOmer F.HatipogluDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityKeiichiAsanoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesJunkoInagakiDepartment of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeiichiroNishidaDepartment of Orthopaediac Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSatoshiHirohataDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityIn patients with osteoarthritis (OA), there is a decrease in both the concentration and molecular size of hyaluronan (HA) in the synovial fluid and cartilage. Cell migration-inducing hyaluronidase 1 (CEMIP), also known as hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), was recently reported as an HA depolymerization-related molecule expressed in the cartilage of patients with OA. However, the underlying mechanism of CEMIP regulation is not well understood. We found that CEMIP expression was transiently increased by interleukine-1 beta (IL-1 beta) stimulation in chondrocytic cells. We also observed that ERK activation and NF-kappa B nuclear translocation were involved in the induction of CEMIP by IL-1 beta. In addition, both administration of HA and mechanical strain attenuated the CEMIP induction in IL-1 beta-stimulated chondrocytes. In conclusion, we clarified the regulatory mechanism of CEMIP in chondrocytes by inflammatory cytokines and suggested the potential involvement in osteoarthritis development.No potential conflict of interest relevant to this article was reported.Nature ResearchActa Medica Okayama2045-23221012020Cell cycle dependence of apoptosis photo-triggered using peptide-photosensitizer conjugate19087ENHyungjinKimDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityShoWatanabeDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityMizukiKitamatsuDepartment of Applied Chemistry, Kindai UniversityKazunoriWatanabeDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTakashiOhtsukiDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityInvestigation 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.MDPIActa Medica Okayama1420-30492612020Photoinduced Endosomal Escape Mechanism: A View from Photochemical Internalization Mediated by CPP-Photosensitizer Conjugates36ENTet HtutSoeDepartment of Biotechnology, Mandalay Technological UniversityKazunoriWatanabeDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTakashiOhtsukiDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityEndosomal 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.MDPIActa Medica Okayama2075-17291122021A Novel 89Zr-labeled DDS Device Utilizing Human IgG Variant (scFv): “Lactosome” Nanoparticle-Based Theranostics for PET Imaging and Targeted Therapy158ENMelissa Siaw HanLimDepartment of Cell Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityTakashiOhtsukiDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityFumiakiTakenakaCollaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKazukoKobayashiCollaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityMasaruAkehiCollaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityHirotakaUjiDepartment of Material Chemistry, Graduate School of Engineering, Kyoto UniversityHirotsuguKobuchiDepartment of Cell Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityTakanoriSasakiCollaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityEiichiOzekiTechnology Research Laboratory, Shimadzu CorporationEijiMatsuuraDepartment 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.ElsevierActa Medica Okayama002235491102021Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells17881798ENMelissa Siaw HanLimYukiNishiyamaTakashiOhtsukiGraduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityKazunoriWatanabeGraduate School of Natural Science and Technology, Okayama UniversityHirotsuguKobuchiKazukoKobayashiCollaborative Research Center (OMIC), Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesEijiMatsuuraThe 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.MDPIActa Medica Okayama1422-00672292021Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress4982ENKazunoriWatanabeGraduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTakashiOhtsukiGraduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityStress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.No potential conflict of interest relevant to this article was reported.Elsevier France-Editions Scientifiques Medicales eActa Medica Okayama0753-33221392021Osteopontin silencing attenuates bleomycin-induced murine pulmonary fibrosis by regulating epithelial-mesenchymal transition111633ENOmer FarukHatipogluDepartment of Pharmacology, Faculty of Medicine, Kindai UniversityEyyupUctepeAcıbadem Labmed Ankara Tissue Typing LaboratoryGabrielOpokuDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityHidenoriWakeDepartment of Pharmacology, Faculty of Medicine, Kindai UniversityKentaroIkemuraDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityTakashiOhtsukiDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityJunkoInagakiDepartment of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMehmetGunduzDepartment of Otolaryngology, Moriya Keiyu HospitalEsraGunduzDepartment of Otolaryngology, Moriya Keiyu HospitalShogoWatanabeDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityTakashiNishinakaDepartment of Pharmacology, Faculty of Medicine, Kindai UniversityHideoTakahashiDepartment of Pharmacology, Faculty of Medicine, Kindai UniversitySatoshiHirohataDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityIdiopathic pulmonary fibrosis (IPF) is the most common and most deadly form of interstitial lung disease. Osteopontin (OPN), a matricellular protein with proinflammatory and profibrotic properties, plays a major role in several fibrotic diseases, including IPF; OPN is highly upregulated in patients' lung samples. In this study, we knocked down OPN in a bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model using small interfering RNA (siRNA) to determine whether the use of OPN siRNA is an effective therapeutic strategy for IPF. We found that fibrosing areas were significantly smaller in specimens from OPN siRNA-treated mice. The number of alveolar macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid was also reduced in OPN siRNA-treated mice. Regarding the expression of epithelial-mesenchymal transition (EMT)-related proteins, the administration of OPN-siRNA to BLM-treated mice upregulated E-cadherin expression and downregulated vimentin expression. Moreover, in vitro, we incubated the human alveolar adenocarcinoma cell line A549 with transforming growth factor (TGF)-beta 1 and subsequently transfected the cells with OPN siRNA. We found a significant upregulation of Col1A1, fibronectin, and vimentin after TGF-beta 1 stimulation in A549 cells. In contrast, a downregulation of Col1A1, fibronectin, and vimentin mRNA levels was observed in TGF-beta 1-stimulated OPN knockdown A549 cells. Therefore, the downregulation of OPN effectively reduced pulmonary fibrotic and EMT changes both in vitro and in vivo. Altogether, our results indicate that OPN siRNA exerts a protective effect on BLM-induced PF in mice. Our results provide a basis for the development of novel targeted therapeutic strategies for IPF.No potential conflict of interest relevant to this article was reported.Nature PortfolioActa Medica Okayama2045-23221112021Photocontrolled apoptosis induction using precursor miR-664a and an RNA carrier-conjugated with photosensitizer14936ENKazunoriWatanabeGraduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTomokoNawachiGraduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityRurikoOkutaniDepartment of Biomedical Engineering, Faculty of Engineering, Okayama UniversityTakashiOhtsukiGraduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityMethods to spatially induce apoptosis are useful for cancer therapy. To control the induction of apoptosis, methods using light, such as photochemical internalization (PCI), have been developed. We hypothesized that photoinduced delivery of microRNAs (miRNAs) that regulate apoptosis could spatially induce apoptosis. In this study, we identified pre-miR-664a as a novel apoptosis-inducing miRNA via mitochondrial apoptotic pathway. Further, we demonstrated the utility of photoinduced cytosolic dispersion of RNA (PCDR), which is an intracellular RNA delivery method based on PCI. Indeed, apoptosis is spatially regulated by pre-miR-664a and PCDR. In addition, we found that apoptosis induced by pre-miR-664a delivered by PCDR was more rapid than that by lipofection. These results suggest that pre-miR-664a is a nucleic acid drug candidate for cancer therapy and PCDR and pre-miR-664a-based strategies have potential therapeutic uses for diseases affecting various cell types.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama1422-00672352022Potential of a Novel Chemical Compound Targeting Matrix Metalloprotease-13 for Early Osteoarthritis: An In Vitro Study2681ENJunkoInagakiDepartment of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesAiriNakanoDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityOmer FarukHatipogluDepartment of Pharmacology, Faculty of Medicine, Kindai UniversityYukaOokaDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityYurinaTaniDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityAkaneMikiDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityKentaroIkemuraDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityGabrielOpokuDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityRyosukeAndoDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityShintaroKodamaDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityTakashiOhtsukiDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityHirosukeYamajiHeart Rhythm Center, Okayama Heart ClinicShuseiYamamotoDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityEriKatsuyamaDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityShogoWatanabeDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversitySatoshiHirohataDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityOsteoarthritis is a progressive disease characterized by cartilage destruction in the joints. Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) play key roles in osteoarthritis progression. In this study, we screened a chemical compound library to identify new drug candidates that target MMP and ADAMTS using a cytokine-stimulated OUMS-27 chondrosarcoma cells. By screening PCR-based mRNA expression, we selected 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide as a potential candidate. We found that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated IL-1 beta-induced MMP13 mRNA expression in a dose-dependent manner, without causing serious cytotoxicity. Signaling pathway analysis revealed that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated ERK- and p-38-phosphorylation as well as JNK phosphorylation. We then examined the additive effect of 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide in combination with low-dose betamethasone on IL-1 beta-stimulated cells. Combined treatment with 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide and betamethasone significantly attenuated MMP13 and ADAMTS9 mRNA expression. In conclusion, we identified a potential compound of interest that may help attenuate matrix-degrading enzymes in the early osteoarthritis-affected joints.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama2073-440911202022Novel Self-Forming Nanosized DDS Particles for BNCT: Utilizing A Hydrophobic Boron Cluster and Its Molecular Glue Effect3307ENAbdul BasithFithroniDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityKazukoKobayashiCollaborative Research Center for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityHirotakaUjiDepartment of Material Chemistry, Graduate School of Engineering, Kyoto UniversityManabuIshimotoFukushima SiC Applied Engineering Inc.MasaruAkehiCollaborative Research Center for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityTakashiOhtsukiDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityEijiMatsuuraDepartment of Cell Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityBNCT is a non-invasive cancer therapy that allows for cancer cell death without harming adjacent cells. However, the application is limited, owing to the challenges of working with clinically approved boron (B) compounds and drug delivery systems (DDS). To address the issues, we developed self-forming nanoparticles consisting of a biodegradable polymer, namely, "AB-type Lactosome (AB-Lac)" loaded with B compounds. Three carborane isomers (o-, m-, and p-carborane) and three related alkylated derivatives, i.e., 1,2-dimethy-o-carborane (diC1-Carb), 1,2-dihexyl-o-carborane (diC6-Carb), and 1,2-didodecyl-o-carborane (diC12-Carb), were separately loaded. diC6-Carb was highly loaded with AB-Lac particles, and their stability indicated the "molecular glue" effect. The efficiency of in vitro B uptake of diC6-Carb for BNCT was confirmed at non-cytotoxic concentration in several cancer cell lines. In vivo/ex vivo biodistribution studies indicated that the AB-Lac particles were remarkably accumulated within 72 h post-injection in the tumor lesions of mice bearing syngeneic breast cancer (4T1) cells, but the maximum accumulation was reached at 12 h. In ex vivo B biodistribution, the ratios of tumor/normal tissue (T/N) and tumor/blood (T/Bl) of the diC6-Carb-loaded particles remained stably high up to 72 h. Therefore, we propose the diC6-Carb-loaded AB-Lac particles as a promising candidate medicine for BNCT.No potential conflict of interest relevant to this article was reported.Elsevier BVActa Medica Okayama0960-894X682022Ultrasound-dependent RNAi using TatU1A-rose bengal conjugate128767ENNanakoSumiDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityShotaNagahiroDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityEijiNakataInstitute of Advanced Energy, Kyoto UniversityKazunoriWatanabeDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTakashiOhtsukiDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTat-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.Royal Society of Chemistry (RSC)Acta Medica Okayama0003-2654148112023FRET probe for detecting two mutations in one EGFR mRNA26262632ENMyatThuDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityKoutaYanaiDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityHajimeShigetoHealth and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)ShoheiYamamuraHealth and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)KazunoriWatanabeDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTakashiOhtsukiDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTechnologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.No potential conflict of interest relevant to this article was reported.nature portfolioActa Medica Okayama2045-23221312023Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo13050ENYukaIkawaDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTakuyaWakaiDepartment of Animal Science, Graduate of Environmental and Life Science, Okayama UniversityHiroakiFunahashiDepartment of Animal Science, Graduate of Environmental and Life Science, Okayama UniversityTet HtutSoeDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityKazunoriWatanabeDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversityTakashiOhtsukiDepartment of Interdisciplinary Science and Engineering in Health Systems, Okayama UniversitySingle-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.No potential conflict of interest relevant to this article was reported.