Real-time imaging of NADPH oxidase activity in living cells using a novel fluorescent protein reporter
Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS invo...
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| description | Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47(phox), we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47(phox) deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors. |
| doi_str_mv | 10.1371/journal.pone.0063989 |
| format | Article |
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NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47(phox), we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47(phox) deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0063989</identifier><identifier>PMID: 23704967</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Aging ; Animals ; Biology ; Biophysics ; Biosensing Techniques ; Biosensors ; Cell Survival - drug effects ; Cloning ; Computer Systems ; Degenerative diseases ; Deoxyribonucleic acid ; DNA ; Dystrophy ; Electric Stimulation ; Electrical stimuli ; Extracellular Space - drug effects ; Extracellular Space - metabolism ; Fatigue ; Fibroblasts ; Fluorescence ; Free radicals ; Genes, Reporter ; Green Fluorescent Proteins - metabolism ; Humans ; In Vitro Techniques ; Journalists ; Kinases ; Laboratory animals ; Lipopolysaccharides - pharmacology ; Macrophage Activation - drug effects ; Macrophages ; Medicine ; Membrane Glycoproteins - deficiency ; Membrane Glycoproteins - metabolism ; Mice ; Mitochondria ; Molecular Imaging - methods ; Muscle Fibers, Skeletal - drug effects ; Muscle Fibers, Skeletal - enzymology ; Muscles ; Muscular dystrophy ; Muscular fatigue ; Musculoskeletal system ; NAD(P)H oxidase ; NADPH Oxidase 2 ; NADPH Oxidases - deficiency ; NADPH Oxidases - metabolism ; Oxidases ; Oxidation ; Oxidation-Reduction - drug effects ; Oxygen ; Penicillin ; Physiology ; Proteins ; Reactive oxygen species ; Reactive Oxygen Species - metabolism ; Signaling ; Skeletal muscle ; Spatial discrimination ; Spatial resolution ; Stimulation ; Time Factors</subject><ispartof>PloS one, 2013-05, Vol.8 (5), p.e63989-e63989</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Pal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2013 Pal et al 2013 Pal et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c758t-aa29558c61cb0eee3427345303351a68d14289a7a1a3b5fb86c06cc933f74f473</citedby><cites>FETCH-LOGICAL-c758t-aa29558c61cb0eee3427345303351a68d14289a7a1a3b5fb86c06cc933f74f473</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660327/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660327/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,315,729,782,786,866,887,2104,2930,23873,27931,27932,53798,53800</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23704967$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Ushio-Fukai, Masuko</contributor><creatorcontrib>Pal, Rituraj</creatorcontrib><creatorcontrib>Basu Thakur, Poulami</creatorcontrib><creatorcontrib>Li, Shumin</creatorcontrib><creatorcontrib>Minard, Charles</creatorcontrib><creatorcontrib>Rodney, George G</creatorcontrib><title>Real-time imaging of NADPH oxidase activity in living cells using a novel fluorescent protein reporter</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47(phox), we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47(phox) deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors.</description><subject>Aging</subject><subject>Animals</subject><subject>Biology</subject><subject>Biophysics</subject><subject>Biosensing Techniques</subject><subject>Biosensors</subject><subject>Cell Survival - drug effects</subject><subject>Cloning</subject><subject>Computer Systems</subject><subject>Degenerative diseases</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Dystrophy</subject><subject>Electric Stimulation</subject><subject>Electrical stimuli</subject><subject>Extracellular Space - drug effects</subject><subject>Extracellular Space - metabolism</subject><subject>Fatigue</subject><subject>Fibroblasts</subject><subject>Fluorescence</subject><subject>Free radicals</subject><subject>Genes, Reporter</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>Humans</subject><subject>In Vitro Techniques</subject><subject>Journalists</subject><subject>Kinases</subject><subject>Laboratory animals</subject><subject>Lipopolysaccharides - pharmacology</subject><subject>Macrophage Activation - drug effects</subject><subject>Macrophages</subject><subject>Medicine</subject><subject>Membrane Glycoproteins - deficiency</subject><subject>Membrane Glycoproteins - metabolism</subject><subject>Mice</subject><subject>Mitochondria</subject><subject>Molecular Imaging - methods</subject><subject>Muscle Fibers, Skeletal - drug effects</subject><subject>Muscle Fibers, Skeletal - enzymology</subject><subject>Muscles</subject><subject>Muscular dystrophy</subject><subject>Muscular fatigue</subject><subject>Musculoskeletal system</subject><subject>NAD(P)H oxidase</subject><subject>NADPH Oxidase 2</subject><subject>NADPH Oxidases - deficiency</subject><subject>NADPH Oxidases - metabolism</subject><subject>Oxidases</subject><subject>Oxidation</subject><subject>Oxidation-Reduction - drug effects</subject><subject>Oxygen</subject><subject>Penicillin</subject><subject>Physiology</subject><subject>Proteins</subject><subject>Reactive oxygen species</subject><subject>Reactive Oxygen Species - metabolism</subject><subject>Signaling</subject><subject>Skeletal muscle</subject><subject>Spatial discrimination</subject><subject>Spatial resolution</subject><subject>Stimulation</subject><subject>Time Factors</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk0tv1DAQxyMEoqXwDRBEQkJw2MWOHTu5IK3KoytVFJXH1fI646wrb7zYzqr99jjdtNqgHpAPGTm_-c_DM1n2EqM5Jhx_uHK976Sdb10Hc4QYqav6UXaMa1LMWIHI4wP7KHsWwhVCJakYe5odFYQjWjN-nOlLkHYWzQZys5Gt6drc6fzb4tP3s9xdm0YGyKWKZmfiTW663CYrMQqsDXkfBlvmnduBzbXtnYegoIv51rsICfewdT6Cf5490dIGeDF-T7JfXz7_PD2bnV98XZ4uzmeKl1WcSVnUZVkphtUKAQChBSe0JIiQEktWNZgWVS25xJKsSr2qmEJMqZoQzammnJxkr_e6W-uCGFsUBCYlYQwhjhKx3BONk1di61PV_kY4acTthfOtkD4aZUHUTBOllCSYaSobtUoZ8RSecVrVrCyS1scxWr_aQDNU7qWdiE7_dGYtWrcTQzKkGNJ9Nwp496eHEMXGhKG3sgPX3-Zd0grjAif0zT_ow9WNVCtTAabTLsVVg6hYUF5hXtR4oOYPUOk0sDEqzZM26X7i8H7ikJgI17GVfQhi-ePy_9mL31P27QG7TqMY18HZPhrXhSlI96DyLgQP-r7JGIlhHe66IYZ1EOM6JLdXhw9073Q3_-Qvh0kEwA</recordid><startdate>20130521</startdate><enddate>20130521</enddate><creator>Pal, Rituraj</creator><creator>Basu Thakur, Poulami</creator><creator>Li, Shumin</creator><creator>Minard, Charles</creator><creator>Rodney, George G</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20130521</creationdate><title>Real-time imaging of NADPH oxidase activity in living cells using a novel fluorescent protein reporter</title><author>Pal, Rituraj ; Basu Thakur, Poulami ; Li, Shumin ; Minard, Charles ; Rodney, George G</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c758t-aa29558c61cb0eee3427345303351a68d14289a7a1a3b5fb86c06cc933f74f473</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Aging</topic><topic>Animals</topic><topic>Biology</topic><topic>Biophysics</topic><topic>Biosensing Techniques</topic><topic>Biosensors</topic><topic>Cell Survival - drug effects</topic><topic>Cloning</topic><topic>Computer Systems</topic><topic>Degenerative diseases</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>Dystrophy</topic><topic>Electric Stimulation</topic><topic>Electrical stimuli</topic><topic>Extracellular Space - drug effects</topic><topic>Extracellular Space - metabolism</topic><topic>Fatigue</topic><topic>Fibroblasts</topic><topic>Fluorescence</topic><topic>Free radicals</topic><topic>Genes, Reporter</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>Humans</topic><topic>In Vitro Techniques</topic><topic>Journalists</topic><topic>Kinases</topic><topic>Laboratory animals</topic><topic>Lipopolysaccharides - pharmacology</topic><topic>Macrophage Activation - drug effects</topic><topic>Macrophages</topic><topic>Medicine</topic><topic>Membrane Glycoproteins - deficiency</topic><topic>Membrane Glycoproteins - metabolism</topic><topic>Mice</topic><topic>Mitochondria</topic><topic>Molecular Imaging - methods</topic><topic>Muscle Fibers, Skeletal - drug effects</topic><topic>Muscle Fibers, Skeletal - enzymology</topic><topic>Muscles</topic><topic>Muscular dystrophy</topic><topic>Muscular fatigue</topic><topic>Musculoskeletal system</topic><topic>NAD(P)H oxidase</topic><topic>NADPH Oxidase 2</topic><topic>NADPH Oxidases - deficiency</topic><topic>NADPH Oxidases - metabolism</topic><topic>Oxidases</topic><topic>Oxidation</topic><topic>Oxidation-Reduction - drug effects</topic><topic>Oxygen</topic><topic>Penicillin</topic><topic>Physiology</topic><topic>Proteins</topic><topic>Reactive oxygen species</topic><topic>Reactive Oxygen Species - metabolism</topic><topic>Signaling</topic><topic>Skeletal muscle</topic><topic>Spatial discrimination</topic><topic>Spatial resolution</topic><topic>Stimulation</topic><topic>Time Factors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pal, Rituraj</creatorcontrib><creatorcontrib>Basu Thakur, Poulami</creatorcontrib><creatorcontrib>Li, Shumin</creatorcontrib><creatorcontrib>Minard, Charles</creatorcontrib><creatorcontrib>Rodney, George G</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - 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NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47(phox), we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47(phox) deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23704967</pmid><doi>10.1371/journal.pone.0063989</doi><tpages>e63989</tpages><oa>free_for_read</oa></addata></record> |
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| language | eng |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS) Journals Open Access; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Aging Animals Biology Biophysics Biosensing Techniques Biosensors Cell Survival - drug effects Cloning Computer Systems Degenerative diseases Deoxyribonucleic acid DNA Dystrophy Electric Stimulation Electrical stimuli Extracellular Space - drug effects Extracellular Space - metabolism Fatigue Fibroblasts Fluorescence Free radicals Genes, Reporter Green Fluorescent Proteins - metabolism Humans In Vitro Techniques Journalists Kinases Laboratory animals Lipopolysaccharides - pharmacology Macrophage Activation - drug effects Macrophages Medicine Membrane Glycoproteins - deficiency Membrane Glycoproteins - metabolism Mice Mitochondria Molecular Imaging - methods Muscle Fibers, Skeletal - drug effects Muscle Fibers, Skeletal - enzymology Muscles Muscular dystrophy Muscular fatigue Musculoskeletal system NAD(P)H oxidase NADPH Oxidase 2 NADPH Oxidases - deficiency NADPH Oxidases - metabolism Oxidases Oxidation Oxidation-Reduction - drug effects Oxygen Penicillin Physiology Proteins Reactive oxygen species Reactive Oxygen Species - metabolism Signaling Skeletal muscle Spatial discrimination Spatial resolution Stimulation Time Factors |
| title | Real-time imaging of NADPH oxidase activity in living cells using a novel fluorescent protein reporter |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-04T08%3A26%3A01IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Real-time%20imaging%20of%20NADPH%20oxidase%20activity%20in%20living%20cells%20using%20a%20novel%20fluorescent%20protein%20reporter&rft.jtitle=PloS%20one&rft.au=Pal,%20Rituraj&rft.date=2013-05-21&rft.volume=8&rft.issue=5&rft.spage=e63989&rft.epage=e63989&rft.pages=e63989-e63989&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0063989&rft_dat=%3Cgale_plos_%3EA478172910%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1353660070&rft_id=info:pmid/23704967&rft_galeid=A478172910&rft_doaj_id=oai_doaj_org_article_96f3ccca316f4adcbeee789a67489652&rfr_iscdi=true |