start-ver=1.4 cd-journal=joma no-vol=5 cd-vols= no-issue= article-no= start-page=18577 end-page=18577 dt-received= dt-revised= dt-accepted= dt-pub-year=2015 dt-pub=201512 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=The molecular mechanism of photochemical internalization of cell penetrating peptide-cargo-photosensitizer conjugates. en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=MikiShunya en-aut-sei=Miki en-aut-mei=Shunya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KobayashiShouhei en-aut-sei=Kobayashi en-aut-mei=Shouhei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HaraguchiTokuko en-aut-sei=Haraguchi en-aut-mei=Tokuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=NakataEiji en-aut-sei=Nakata en-aut-mei=Eiji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=HirakawaKazutaka en-aut-sei=Hirakawa en-aut-mei=Kazutaka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=SumitaKensuke en-aut-sei=Sumita en-aut-mei=Kensuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=OkazakiShigetoshi en-aut-sei=Okazaki en-aut-mei=Shigetoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil=Department of Medical Bioengineering, Okayama University kn-affil= affil-num=2 en-affil=Department of Medical Bioengineering, Okayama University kn-affil= affil-num=3 en-affil=Advanced ICT Research Institute Kobe, NICT kn-affil= affil-num=4 en-affil=Advanced ICT Research Institute Kobe, NICT kn-affil= affil-num=5 en-affil=Institute of Advanced Energy, Kyoto University kn-affil= affil-num=6 en-affil=Department of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, Shizuoka University kn-affil= affil-num=7 en-affil=Department of Medical Bioengineering, Okayama University kn-affil= affil-num=8 en-affil=Department of Medical Bioengineering, Okayama University kn-affil=‰ªŽR‘åŠw‘åŠw‰@Ž©‘R‰ÈŠwŒ¤‹†‰È affil-num=9 en-affil=Department of Medical Spectroscopy, Hamamatsu University School of Medicine kn-affil= END start-ver=1.4 cd-journal=joma no-vol=7 cd-vols= no-issue= article-no= start-page=12501 end-page=12501 dt-received= dt-revised= dt-accepted= dt-pub-year=2016 dt-pub=201608 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KanzakiShigeto en-aut-sei=Kanzaki en-aut-mei=Shigeto kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NishimuraSae en-aut-sei=Nishimura en-aut-mei=Sae kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KunihiroYoshio en-aut-sei=Kunihiro en-aut-mei=Yoshio kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=SisidoMasahiko en-aut-sei=Sisido en-aut-mei=Masahiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=2 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=3 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=4 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=5 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=6 en-affil=Department of Biomedical Engineering, Okayama University kn-affil=‰ªŽR‘åŠw‘åŠw‰@Ž©‘R‰ÈŠwŒ¤‹†‰È END start-ver=1.4 cd-journal=joma no-vol=7 cd-vols= no-issue= article-no= start-page=12501 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2016 dt-pub=201608 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs en-subtitle= kn-subtitle= en-abstract= kn-abstract= 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. en-copyright= kn-copyright= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KanzakiShigeto en-aut-sei=Kanzaki en-aut-mei=Shigeto kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NishimuraSae en-aut-sei=Nishimura en-aut-mei=Sae kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KunihiroYoshio en-aut-sei=Kunihiro en-aut-mei=Yoshio kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=SisidoMasahiko en-aut-sei=Sisido en-aut-mei=Masahiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=2 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=3 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=4 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=5 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= affil-num=6 en-affil=Department of Biomedical Engineering, Okayama University kn-affil= en-keyword=Molecular engineering kn-keyword=Molecular engineering en-keyword=Optogenetics kn-keyword=Optogenetics END start-ver=1.4 cd-journal=joma no-vol=14 cd-vols= no-issue=1 article-no= start-page=e0211505 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=20190129 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=In-stem molecular beacon targeted to a 5 '-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity en-subtitle= kn-subtitle= en-abstract= kn-abstract= Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNAMet (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5'- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies. en-copyright= kn-copyright= en-aut-name=MiyoshiYuichi en-aut-sei=Miyoshi en-aut-mei=Yuichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KashidaHiromu en-aut-sei=Kashida en-aut-mei=Hiromu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=AsanumaHiroyuki en-aut-sei=Asanuma en-aut-mei=Hiroyuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Engineering, Nagoya University kn-affil= affil-num=4 en-affil=Graduate School of Engineering, Nagoya University kn-affil= affil-num=5 en-affil=Graduate School of Natural Science and Technology, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=10 cd-vols= no-issue=1 article-no= start-page=19087 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20201105 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Cell cycle dependence of apoptosis photo-triggered using peptide-photosensitizer conjugate en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=KimHyungjin en-aut-sei=Kim en-aut-mei=Hyungjin kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=WatanabeSho en-aut-sei=Watanabe en-aut-mei=Sho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KitamatsuMizuki en-aut-sei=Kitamatsu en-aut-mei=Mizuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Department of Applied Chemistry, Kindai University kn-affil= affil-num=4 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=Biological techniques kn-keyword=Biological techniques en-keyword=Biotechnology kn-keyword=Biotechnology END start-ver=1.4 cd-journal=joma no-vol=26 cd-vols= no-issue=1 article-no= start-page=36 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20201223 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Photoinduced Endosomal Escape Mechanism: A View from Photochemical Internalization Mediated by CPP-Photosensitizer Conjugates en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=SoeTet Htut en-aut-sei=Soe en-aut-mei=Tet Htut kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= affil-num=1 en-affil=Department of Biotechnology, Mandalay Technological University kn-affil= affil-num=2 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=photochemical internalization kn-keyword=photochemical internalization en-keyword=photosensitizer kn-keyword=photosensitizer en-keyword=cell-penetrating peptide kn-keyword=cell-penetrating peptide en-keyword=endosome kn-keyword=endosome en-keyword=membrane kn-keyword=membrane END start-ver=1.4 cd-journal=joma no-vol=110 cd-vols= no-issue= article-no= start-page=1788 end-page=1798 dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20210430 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=LimMelissa Siaw Han en-aut-sei=Lim en-aut-mei=Melissa Siaw Han kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=NishiyamaYuki en-aut-sei=Nishiyama en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=KobuchiHirotsugu en-aut-sei=Kobuchi en-aut-mei=Hirotsugu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=KobayashiKazuko en-aut-sei=Kobayashi en-aut-mei=Kazuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=MatsuuraEiji en-aut-sei=Matsuura en-aut-mei=Eiji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil= kn-affil= affil-num=2 en-affil= kn-affil= affil-num=3 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=5 en-affil= kn-affil= affil-num=6 en-affil=Collaborative Research Center (OMIC), Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=7 en-affil= kn-affil= en-keyword=Lactosome kn-keyword=Lactosome en-keyword=ABCG2 kn-keyword=ABCG2 en-keyword=siRNA kn-keyword=siRNA en-keyword=Cancer kn-keyword=Cancer en-keyword=siRNA delivery kn-keyword=siRNA delivery en-keyword=Photodynamic therapy kn-keyword=Photodynamic therapy en-keyword=Polymeric micelle kn-keyword=Polymeric micelle en-keyword=Photosensitizer kn-keyword=Photosensitizer en-keyword=Photochemical internalization kn-keyword=Photochemical internalization END start-ver=1.4 cd-journal=joma no-vol=22 cd-vols= no-issue=9 article-no= start-page=4982 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20210507 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress en-subtitle= kn-subtitle= en-abstract= kn-abstract=Stress 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. en-copyright= kn-copyright= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= affil-num=1 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=heat shock response kn-keyword=heat shock response en-keyword=nuclear stress bodies kn-keyword=nuclear stress bodies en-keyword=HSF1 granules kn-keyword=HSF1 granules en-keyword=SAFB granules kn-keyword=SAFB granules en-keyword=liquid-liquid phase separation kn-keyword=liquid-liquid phase separation END start-ver=1.4 cd-journal=joma no-vol=11 cd-vols= no-issue=1 article-no= start-page=14936 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20210722 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Photocontrolled apoptosis induction using precursor miR-664a and an RNA carrier-conjugated with photosensitizer en-subtitle= kn-subtitle= en-abstract= kn-abstract=Methods 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. en-copyright= kn-copyright= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=NawachiTomoko en-aut-sei=Nawachi en-aut-mei=Tomoko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=OkutaniRuriko en-aut-sei=Okutani en-aut-mei=Ruriko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= affil-num=1 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Department of Biomedical Engineering, Faculty of Engineering, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=68 cd-vols= no-issue= article-no= start-page=128767 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20227 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Ultrasound-dependent RNAi using TatU1A-rose bengal conjugate en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=SumiNanako en-aut-sei=Sumi en-aut-mei=Nanako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=NagahiroShota en-aut-sei=Nagahiro en-aut-mei=Shota kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NakataEiji en-aut-sei=Nakata en-aut-mei=Eiji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Institute of Advanced Energy, Kyoto University kn-affil= affil-num=4 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=Ultrasound kn-keyword=Ultrasound en-keyword=Sonosensitizer kn-keyword=Sonosensitizer en-keyword=Rose Bengal kn-keyword=Rose Bengal en-keyword=RNAi kn-keyword=RNAi en-keyword=RNA delivery kn-keyword=RNA delivery END start-ver=1.4 cd-journal=joma no-vol=148 cd-vols= no-issue=11 article-no= start-page=2626 end-page=2632 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=2023 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=FRET probe for detecting two mutations in one EGFR mRNA en-subtitle= kn-subtitle= en-abstract= kn-abstract=Technologies 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. en-copyright= kn-copyright= en-aut-name=ThuMyat en-aut-sei=Thu en-aut-mei=Myat kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YanaiKouta en-aut-sei=Yanai en-aut-mei=Kouta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=ShigetoHajime en-aut-sei=Shigeto en-aut-mei=Hajime kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=YamamuraShohei en-aut-sei=Yamamura en-aut-mei=Shohei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) kn-affil= affil-num=4 en-affil=Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) kn-affil= affil-num=5 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=6 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=13 cd-vols= no-issue=1 article-no= start-page=13050 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230811 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo en-subtitle= kn-subtitle= en-abstract= kn-abstract=Single-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. en-copyright= kn-copyright= en-aut-name=IkawaYuka en-aut-sei=Ikawa en-aut-mei=Yuka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=WakaiTakuya en-aut-sei=Wakai en-aut-mei=Takuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=FunahashiHiroaki en-aut-sei=Funahashi en-aut-mei=Hiroaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=SoeTet Htut en-aut-sei=Soe en-aut-mei=Tet Htut kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=WatanabeKazunori en-aut-sei=Watanabe en-aut-mei=Kazunori kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=OhtsukiTakashi en-aut-sei=Ohtsuki en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Department of Animal Science, Graduate of Environmental and Life Science, Okayama University kn-affil= affil-num=3 en-affil=Department of Animal Science, Graduate of Environmental and Life Science, Okayama University kn-affil= affil-num=4 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=6 en-affil=Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= END