A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells
Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytomet...
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| Veröffentlicht in: | PloS one 2010-02, Vol.5 (2), p.e9344-e9344 |
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| creator | Banning, Carina Votteler, Jörg Hoffmann, Dirk Koppensteiner, Herwig Warmer, Martin Reimer, Rudolph Kirchhoff, Frank Schubert, Ulrich Hauber, Joachim Schindler, Michael |
| description | Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.
Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.
The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases. |
| doi_str_mv | 10.1371/journal.pone.0009344 |
| format | Article |
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Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.
The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0009344</identifier><identifier>PMID: 20179761</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animals ; Antigens, CD - genetics ; Antigens, CD - metabolism ; Assaying ; Binding Sites - genetics ; Biotechnology/Protein Chemistry and Proteomics ; Cell Biology ; Cell Line ; Cells (biology) ; Cloning ; Cytometry ; Energy transfer ; Flow cytometry ; Flow Cytometry - methods ; Fluorescence resonance energy transfer ; Fluorescence Resonance Energy Transfer - methods ; Gene Products, nef - genetics ; Gene Products, nef - metabolism ; GPI-Linked Proteins ; Health aspects ; HeLa Cells ; HIV ; HIV-1 - metabolism ; Human immunodeficiency virus ; Human Immunodeficiency Virus Proteins - genetics ; Human Immunodeficiency Virus Proteins - metabolism ; Humans ; Immunology ; Immunoprecipitation ; Infectious Diseases/HIV Infection and AIDS ; Jurkat Cells ; Luminescent Proteins - genetics ; Luminescent Proteins - metabolism ; Medical treatment ; Membrane Glycoproteins - genetics ; Membrane Glycoproteins - metabolism ; Microscopy ; Microscopy, Confocal ; Mutation ; Nef protein ; Protein Binding ; Protein interaction ; Protein Interaction Mapping - methods ; Protein-protein interactions ; Proteins ; Proteomics ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Simian Immunodeficiency Virus - metabolism ; Studies ; Target recognition ; Transfection ; Viral Proteins - genetics ; Viral Proteins - metabolism ; Viral Regulatory and Accessory Proteins - genetics ; Viral Regulatory and Accessory Proteins - metabolism ; Virology ; Virology/Virulence Factors and Mechanisms ; Viruses</subject><ispartof>PloS one, 2010-02, Vol.5 (2), p.e9344-e9344</ispartof><rights>COPYRIGHT 2010 Public Library of Science</rights><rights>2010 Banning 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>Banning et al. 2010</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c691t-3516f0bfd42127ecfc4a06854fe712e2ebe2c2a0ab1e52b6f771020743794663</citedby><cites>FETCH-LOGICAL-c691t-3516f0bfd42127ecfc4a06854fe712e2ebe2c2a0ab1e52b6f771020743794663</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/PMC2825263/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825263/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20179761$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Banning, Carina</creatorcontrib><creatorcontrib>Votteler, Jörg</creatorcontrib><creatorcontrib>Hoffmann, Dirk</creatorcontrib><creatorcontrib>Koppensteiner, Herwig</creatorcontrib><creatorcontrib>Warmer, Martin</creatorcontrib><creatorcontrib>Reimer, Rudolph</creatorcontrib><creatorcontrib>Kirchhoff, Frank</creatorcontrib><creatorcontrib>Schubert, Ulrich</creatorcontrib><creatorcontrib>Hauber, Joachim</creatorcontrib><creatorcontrib>Schindler, Michael</creatorcontrib><title>A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.
Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.
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genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Simian Immunodeficiency Virus - metabolism</subject><subject>Studies</subject><subject>Target recognition</subject><subject>Transfection</subject><subject>Viral Proteins - genetics</subject><subject>Viral Proteins - metabolism</subject><subject>Viral Regulatory and Accessory Proteins - genetics</subject><subject>Viral Regulatory and Accessory Proteins - metabolism</subject><subject>Virology</subject><subject>Virology/Virulence Factors and Mechanisms</subject><subject>Viruses</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk11r2zAUhs3YWLts_2BshsHGLpzpy5J9Mwil3QKFQhd2K2T5KFFwrMySu_nfT2ncEo9eDCP04ee8kl6dkyRvMZpjKvCXreu7VjXzvWthjhAqKWPPknNcUpJxgujzk_FZ8sr7LUI5LTh_mZwRhEUpOD5PzCI1jfud6iG4HYRuyCrloU6vbi9XqfJeDWlwqa2hDdYMqWrr2FQzeEj3nQtg22zsU9sG6JQO1rU-TtLG3tl2nWpoGv86eWFU4-HN2M-S1dXl6uJ7dn3zbXmxuM40L3HIaI65QZWpGcFEgDaaKcSLnBkQmACBCogmCqkKQ04qboTAiCDBqCgZ53SWvD_K7hvn5eiQl5gUJcnLnONILI9E7dRW7ju7U90gnbLyfsF1a6m6YHUDkpEKasM4ZYiwqmCqZrnItQCjUQVlFbW-jrv11Q5qHT3qVDMRnf5p7Uau3Z0kBckJp1Hg0yjQuV89-CB31h_8Ui243ktBKRekYEUkP_xDPn25kVqreH7bGhe31QdNuWCClpixaNUsmT9Bxa-GndUxm4yN65OAz5OAyAT4E9aq914uf9z-P3vzc8p-PGE3oJqw8a7p7zNoCrIjqDvnfQfm0WOM5KEYHtyQh2KQYzHEsHen7_MY9JD99C8EkgTJ</recordid><startdate>20100222</startdate><enddate>20100222</enddate><creator>Banning, Carina</creator><creator>Votteler, Jörg</creator><creator>Hoffmann, Dirk</creator><creator>Koppensteiner, Herwig</creator><creator>Warmer, Martin</creator><creator>Reimer, Rudolph</creator><creator>Kirchhoff, Frank</creator><creator>Schubert, Ulrich</creator><creator>Hauber, Joachim</creator><creator>Schindler, Michael</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>AEUYN</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>20100222</creationdate><title>A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells</title><author>Banning, Carina ; Votteler, Jörg ; Hoffmann, Dirk ; Koppensteiner, Herwig ; Warmer, Martin ; Reimer, Rudolph ; Kirchhoff, Frank ; Schubert, Ulrich ; Hauber, Joachim ; Schindler, Michael</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c691t-3516f0bfd42127ecfc4a06854fe712e2ebe2c2a0ab1e52b6f771020743794663</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Animals</topic><topic>Antigens, CD - genetics</topic><topic>Antigens, CD - metabolism</topic><topic>Assaying</topic><topic>Binding Sites - genetics</topic><topic>Biotechnology/Protein Chemistry and Proteomics</topic><topic>Cell Biology</topic><topic>Cell Line</topic><topic>Cells (biology)</topic><topic>Cloning</topic><topic>Cytometry</topic><topic>Energy transfer</topic><topic>Flow cytometry</topic><topic>Flow Cytometry - methods</topic><topic>Fluorescence resonance energy transfer</topic><topic>Fluorescence Resonance Energy Transfer - methods</topic><topic>Gene Products, nef - genetics</topic><topic>Gene Products, nef - metabolism</topic><topic>GPI-Linked Proteins</topic><topic>Health aspects</topic><topic>HeLa Cells</topic><topic>HIV</topic><topic>HIV-1 - metabolism</topic><topic>Human immunodeficiency virus</topic><topic>Human Immunodeficiency Virus Proteins - genetics</topic><topic>Human Immunodeficiency Virus Proteins - metabolism</topic><topic>Humans</topic><topic>Immunology</topic><topic>Immunoprecipitation</topic><topic>Infectious Diseases/HIV Infection and AIDS</topic><topic>Jurkat Cells</topic><topic>Luminescent Proteins - genetics</topic><topic>Luminescent Proteins - metabolism</topic><topic>Medical treatment</topic><topic>Membrane Glycoproteins - genetics</topic><topic>Membrane Glycoproteins - metabolism</topic><topic>Microscopy</topic><topic>Microscopy, Confocal</topic><topic>Mutation</topic><topic>Nef protein</topic><topic>Protein Binding</topic><topic>Protein interaction</topic><topic>Protein Interaction Mapping - methods</topic><topic>Protein-protein interactions</topic><topic>Proteins</topic><topic>Proteomics</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Simian Immunodeficiency Virus - metabolism</topic><topic>Studies</topic><topic>Target recognition</topic><topic>Transfection</topic><topic>Viral Proteins - genetics</topic><topic>Viral Proteins - metabolism</topic><topic>Viral Regulatory and Accessory Proteins - genetics</topic><topic>Viral Regulatory and Accessory Proteins - metabolism</topic><topic>Virology</topic><topic>Virology/Virulence Factors and Mechanisms</topic><topic>Viruses</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Banning, Carina</creatorcontrib><creatorcontrib>Votteler, Jörg</creatorcontrib><creatorcontrib>Hoffmann, Dirk</creatorcontrib><creatorcontrib>Koppensteiner, Herwig</creatorcontrib><creatorcontrib>Warmer, Martin</creatorcontrib><creatorcontrib>Reimer, Rudolph</creatorcontrib><creatorcontrib>Kirchhoff, Frank</creatorcontrib><creatorcontrib>Schubert, Ulrich</creatorcontrib><creatorcontrib>Hauber, Joachim</creatorcontrib><creatorcontrib>Schindler, Michael</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 One Sustainability</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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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Banning, Carina</au><au>Votteler, Jörg</au><au>Hoffmann, Dirk</au><au>Koppensteiner, Herwig</au><au>Warmer, Martin</au><au>Reimer, Rudolph</au><au>Kirchhoff, Frank</au><au>Schubert, Ulrich</au><au>Hauber, Joachim</au><au>Schindler, Michael</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2010-02-22</date><risdate>2010</risdate><volume>5</volume><issue>2</issue><spage>e9344</spage><epage>e9344</epage><pages>e9344-e9344</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.
Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.
The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>20179761</pmid><doi>10.1371/journal.pone.0009344</doi><tpages>e9344</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | PloS one, 2010-02, Vol.5 (2), p.e9344-e9344 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1289259561 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Animals Antigens, CD - genetics Antigens, CD - metabolism Assaying Binding Sites - genetics Biotechnology/Protein Chemistry and Proteomics Cell Biology Cell Line Cells (biology) Cloning Cytometry Energy transfer Flow cytometry Flow Cytometry - methods Fluorescence resonance energy transfer Fluorescence Resonance Energy Transfer - methods Gene Products, nef - genetics Gene Products, nef - metabolism GPI-Linked Proteins Health aspects HeLa Cells HIV HIV-1 - metabolism Human immunodeficiency virus Human Immunodeficiency Virus Proteins - genetics Human Immunodeficiency Virus Proteins - metabolism Humans Immunology Immunoprecipitation Infectious Diseases/HIV Infection and AIDS Jurkat Cells Luminescent Proteins - genetics Luminescent Proteins - metabolism Medical treatment Membrane Glycoproteins - genetics Membrane Glycoproteins - metabolism Microscopy Microscopy, Confocal Mutation Nef protein Protein Binding Protein interaction Protein Interaction Mapping - methods Protein-protein interactions Proteins Proteomics Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Simian Immunodeficiency Virus - metabolism Studies Target recognition Transfection Viral Proteins - genetics Viral Proteins - metabolism Viral Regulatory and Accessory Proteins - genetics Viral Regulatory and Accessory Proteins - metabolism Virology Virology/Virulence Factors and Mechanisms Viruses |
| title | A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-06T20%3A50%3A36IST&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=A%20flow%20cytometry-based%20FRET%20assay%20to%20identify%20and%20analyse%20protein-protein%20interactions%20in%20living%20cells&rft.jtitle=PloS%20one&rft.au=Banning,%20Carina&rft.date=2010-02-22&rft.volume=5&rft.issue=2&rft.spage=e9344&rft.epage=e9344&rft.pages=e9344-e9344&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0009344&rft_dat=%3Cgale_plos_%3EA473914474%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=1289259561&rft_id=info:pmid/20179761&rft_galeid=A473914474&rft_doaj_id=oai_doaj_org_article_42bedf4634024b84ad4575c7efc0be9b&rfr_iscdi=true |