Conformational diversity in purified prions produced in vitro
Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the ser...
Gespeichert in:
Veröffentlicht in: | PLoS pathogens 2023-01, Vol.19 (1), p.e1011083-e1011083 |
---|---|
Hauptverfasser: | , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
container_end_page | e1011083 |
---|---|
container_issue | 1 |
container_start_page | e1011083 |
container_title | PLoS pathogens |
container_volume | 19 |
creator | Walsh, Daniel J Schwind, Abigail M Noble, Geoffrey P Supattapone, Surachai |
description | Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the serial protein misfolding cyclic amplification (sPMCA) technique do not display this same diversity in vitro. This discrepancy has left a gap in our understanding of how conformational diversity arises at the molecular level in both types of prions. Here, we use continuous shaking instead of sPMCA to generate conformationally diverse purified prions in vitro. Using this approach, we show for the first time that wild type prions initially seeded by different native strains can propagate as metastable PrPSc conformers with distinguishable strain properties in purified reactions containing a single active cofactor. Propagation of these metastable PrPSc conformers requires appropriate shaking conditions, and changes in these conditions cause all the different PrPSc conformers to converge irreversibly into the same single conformer as that produced in sPMCA reactions. We also use continuous shaking to show that two mutant PrP molecules with different pathogenic point mutations (D177N and E199K) adopt distinguishable PrPSc conformations in reactions containing pure protein substrate without cofactors. Unlike wild-type prions, the conformations of mutant prions appear to be dictated by substrate sequence rather than seed conformation. Overall, our studies using purified substrates in shaking reactions show that wild-type and mutant prions use fundamentally different mechanisms to generate conformational diversity at the molecular level. |
doi_str_mv | 10.1371/journal.ppat.1011083 |
format | Article |
fullrecord | <record><control><sourceid>gale_plos_</sourceid><recordid>TN_cdi_plos_journals_2777449479</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A737321438</galeid><doaj_id>oai_doaj_org_article_5ea883ddd88b45e2bbf74c2e62116bae</doaj_id><sourcerecordid>A737321438</sourcerecordid><originalsourceid>FETCH-LOGICAL-c661t-e1e3ad95fc8e5d174728b39ea953e1de1a0b3bcf29f386a0884cd10ebd671f6a3</originalsourceid><addsrcrecordid>eNqVkk2P0zAQhiMEYpfCP0BQiQscWjKxYzsHkFYVH5VWIPFxthx7XFwlcdZOKvbf49LsaoP2gnywPfPM6xnPZNlzyNdAOLzd-zF0qln3vRrWkAPkgjzIzqEsyYoTTh_eOZ9lT2Lc5zkFAuxxdkYYKxip4Dx7t_Gd9aFVg_NJbWncAUN0w_XSdct-DM46NMs-JG9MmzejTvfkO7gh-KfZI6uaiM-mfZH9_Pjhx-bz6vLrp-3m4nKlGYNhhYBEmaq0WmBpgFNeiJpUqKqSIBgEldek1raoLBFM5UJQbSDH2jAOlimyyF6edPvGRzlVHmXBOae0orxKxPZEGK_2MuXbqnAtvXLyr8GHnVRhcLpBWaISghhjhKhpiUVdW051gawAYLXCpPV-em2sWzQauyGoZiY693Tul9z5g6wEz4GWSeD1JBD81YhxkK2LGptGdejHY96MEFKw1I5F9uof9P7qJmqnUgEudSy9q4-i8iL1lxRAiUjU-h4qLYOt075D65J9FvBmFpCYAX8POzXGKLffv_0H-2XO0hOrg48xoL39O8jlcXhvipTH4ZXT8KawF3f__TboZlrJH_t76xs</addsrcrecordid><sourcetype>Open Website</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2777449479</pqid></control><display><type>article</type><title>Conformational diversity in purified prions produced in vitro</title><source>Public Library of Science (PLoS) Journals Open Access</source><source>MEDLINE</source><source>DOAJ Directory of Open Access Journals</source><source>EZB-FREE-00999 freely available EZB journals</source><source>PubMed Central</source><source>PubMed Central Open Access</source><creator>Walsh, Daniel J ; Schwind, Abigail M ; Noble, Geoffrey P ; Supattapone, Surachai</creator><contributor>Woerman, Amanda L.</contributor><creatorcontrib>Walsh, Daniel J ; Schwind, Abigail M ; Noble, Geoffrey P ; Supattapone, Surachai ; Woerman, Amanda L.</creatorcontrib><description>Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the serial protein misfolding cyclic amplification (sPMCA) technique do not display this same diversity in vitro. This discrepancy has left a gap in our understanding of how conformational diversity arises at the molecular level in both types of prions. Here, we use continuous shaking instead of sPMCA to generate conformationally diverse purified prions in vitro. Using this approach, we show for the first time that wild type prions initially seeded by different native strains can propagate as metastable PrPSc conformers with distinguishable strain properties in purified reactions containing a single active cofactor. Propagation of these metastable PrPSc conformers requires appropriate shaking conditions, and changes in these conditions cause all the different PrPSc conformers to converge irreversibly into the same single conformer as that produced in sPMCA reactions. We also use continuous shaking to show that two mutant PrP molecules with different pathogenic point mutations (D177N and E199K) adopt distinguishable PrPSc conformations in reactions containing pure protein substrate without cofactors. Unlike wild-type prions, the conformations of mutant prions appear to be dictated by substrate sequence rather than seed conformation. Overall, our studies using purified substrates in shaking reactions show that wild-type and mutant prions use fundamentally different mechanisms to generate conformational diversity at the molecular level.</description><identifier>ISSN: 1553-7374</identifier><identifier>ISSN: 1553-7366</identifier><identifier>EISSN: 1553-7374</identifier><identifier>DOI: 10.1371/journal.ppat.1011083</identifier><identifier>PMID: 36626391</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Bioassays ; Biology and Life Sciences ; Brain ; Care and treatment ; Cofactors ; Conformation ; Creutzfeldt-Jakob disease ; Development and progression ; Humans ; Medicine and Health Sciences ; Molecular Conformation ; Mutants ; Mutation ; Neuropathology ; Prion diseases ; Prion Diseases - metabolism ; Prion protein ; Prion Proteins ; Prions ; Prions - metabolism ; Propagation ; Protein folding ; Protein research ; Proteins ; Research and analysis methods ; Seeds ; Self propagation ; Shaking ; Substrates</subject><ispartof>PLoS pathogens, 2023-01, Vol.19 (1), p.e1011083-e1011083</ispartof><rights>Copyright: © 2023 Walsh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</rights><rights>COPYRIGHT 2023 Public Library of Science</rights><rights>2023 Walsh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://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>2023 Walsh et al 2023 Walsh et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c661t-e1e3ad95fc8e5d174728b39ea953e1de1a0b3bcf29f386a0884cd10ebd671f6a3</citedby><cites>FETCH-LOGICAL-c661t-e1e3ad95fc8e5d174728b39ea953e1de1a0b3bcf29f386a0884cd10ebd671f6a3</cites><orcidid>0000-0001-9060-4550</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9870145/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9870145/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/36626391$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Woerman, Amanda L.</contributor><creatorcontrib>Walsh, Daniel J</creatorcontrib><creatorcontrib>Schwind, Abigail M</creatorcontrib><creatorcontrib>Noble, Geoffrey P</creatorcontrib><creatorcontrib>Supattapone, Surachai</creatorcontrib><title>Conformational diversity in purified prions produced in vitro</title><title>PLoS pathogens</title><addtitle>PLoS Pathog</addtitle><description>Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the serial protein misfolding cyclic amplification (sPMCA) technique do not display this same diversity in vitro. This discrepancy has left a gap in our understanding of how conformational diversity arises at the molecular level in both types of prions. Here, we use continuous shaking instead of sPMCA to generate conformationally diverse purified prions in vitro. Using this approach, we show for the first time that wild type prions initially seeded by different native strains can propagate as metastable PrPSc conformers with distinguishable strain properties in purified reactions containing a single active cofactor. Propagation of these metastable PrPSc conformers requires appropriate shaking conditions, and changes in these conditions cause all the different PrPSc conformers to converge irreversibly into the same single conformer as that produced in sPMCA reactions. We also use continuous shaking to show that two mutant PrP molecules with different pathogenic point mutations (D177N and E199K) adopt distinguishable PrPSc conformations in reactions containing pure protein substrate without cofactors. Unlike wild-type prions, the conformations of mutant prions appear to be dictated by substrate sequence rather than seed conformation. Overall, our studies using purified substrates in shaking reactions show that wild-type and mutant prions use fundamentally different mechanisms to generate conformational diversity at the molecular level.</description><subject>Bioassays</subject><subject>Biology and Life Sciences</subject><subject>Brain</subject><subject>Care and treatment</subject><subject>Cofactors</subject><subject>Conformation</subject><subject>Creutzfeldt-Jakob disease</subject><subject>Development and progression</subject><subject>Humans</subject><subject>Medicine and Health Sciences</subject><subject>Molecular Conformation</subject><subject>Mutants</subject><subject>Mutation</subject><subject>Neuropathology</subject><subject>Prion diseases</subject><subject>Prion Diseases - metabolism</subject><subject>Prion protein</subject><subject>Prion Proteins</subject><subject>Prions</subject><subject>Prions - metabolism</subject><subject>Propagation</subject><subject>Protein folding</subject><subject>Protein research</subject><subject>Proteins</subject><subject>Research and analysis methods</subject><subject>Seeds</subject><subject>Self propagation</subject><subject>Shaking</subject><subject>Substrates</subject><issn>1553-7374</issn><issn>1553-7366</issn><issn>1553-7374</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</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>eNqVkk2P0zAQhiMEYpfCP0BQiQscWjKxYzsHkFYVH5VWIPFxthx7XFwlcdZOKvbf49LsaoP2gnywPfPM6xnPZNlzyNdAOLzd-zF0qln3vRrWkAPkgjzIzqEsyYoTTh_eOZ9lT2Lc5zkFAuxxdkYYKxip4Dx7t_Gd9aFVg_NJbWncAUN0w_XSdct-DM46NMs-JG9MmzejTvfkO7gh-KfZI6uaiM-mfZH9_Pjhx-bz6vLrp-3m4nKlGYNhhYBEmaq0WmBpgFNeiJpUqKqSIBgEldek1raoLBFM5UJQbSDH2jAOlimyyF6edPvGRzlVHmXBOae0orxKxPZEGK_2MuXbqnAtvXLyr8GHnVRhcLpBWaISghhjhKhpiUVdW051gawAYLXCpPV-em2sWzQauyGoZiY693Tul9z5g6wEz4GWSeD1JBD81YhxkK2LGptGdejHY96MEFKw1I5F9uof9P7qJmqnUgEudSy9q4-i8iL1lxRAiUjU-h4qLYOt075D65J9FvBmFpCYAX8POzXGKLffv_0H-2XO0hOrg48xoL39O8jlcXhvipTH4ZXT8KawF3f__TboZlrJH_t76xs</recordid><startdate>20230101</startdate><enddate>20230101</enddate><creator>Walsh, Daniel J</creator><creator>Schwind, Abigail M</creator><creator>Noble, Geoffrey P</creator><creator>Supattapone, Surachai</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>ISN</scope><scope>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-9060-4550</orcidid></search><sort><creationdate>20230101</creationdate><title>Conformational diversity in purified prions produced in vitro</title><author>Walsh, Daniel J ; Schwind, Abigail M ; Noble, Geoffrey P ; Supattapone, Surachai</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c661t-e1e3ad95fc8e5d174728b39ea953e1de1a0b3bcf29f386a0884cd10ebd671f6a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Bioassays</topic><topic>Biology and Life Sciences</topic><topic>Brain</topic><topic>Care and treatment</topic><topic>Cofactors</topic><topic>Conformation</topic><topic>Creutzfeldt-Jakob disease</topic><topic>Development and progression</topic><topic>Humans</topic><topic>Medicine and Health Sciences</topic><topic>Molecular Conformation</topic><topic>Mutants</topic><topic>Mutation</topic><topic>Neuropathology</topic><topic>Prion diseases</topic><topic>Prion Diseases - metabolism</topic><topic>Prion protein</topic><topic>Prion Proteins</topic><topic>Prions</topic><topic>Prions - metabolism</topic><topic>Propagation</topic><topic>Protein folding</topic><topic>Protein research</topic><topic>Proteins</topic><topic>Research and analysis methods</topic><topic>Seeds</topic><topic>Self propagation</topic><topic>Shaking</topic><topic>Substrates</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Walsh, Daniel J</creatorcontrib><creatorcontrib>Schwind, Abigail M</creatorcontrib><creatorcontrib>Noble, Geoffrey P</creatorcontrib><creatorcontrib>Supattapone, Surachai</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: Canada</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest SciTech 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>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</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>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PLoS pathogens</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Walsh, Daniel J</au><au>Schwind, Abigail M</au><au>Noble, Geoffrey P</au><au>Supattapone, Surachai</au><au>Woerman, Amanda L.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Conformational diversity in purified prions produced in vitro</atitle><jtitle>PLoS pathogens</jtitle><addtitle>PLoS Pathog</addtitle><date>2023-01-01</date><risdate>2023</risdate><volume>19</volume><issue>1</issue><spage>e1011083</spage><epage>e1011083</epage><pages>e1011083-e1011083</pages><issn>1553-7374</issn><issn>1553-7366</issn><eissn>1553-7374</eissn><abstract>Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the serial protein misfolding cyclic amplification (sPMCA) technique do not display this same diversity in vitro. This discrepancy has left a gap in our understanding of how conformational diversity arises at the molecular level in both types of prions. Here, we use continuous shaking instead of sPMCA to generate conformationally diverse purified prions in vitro. Using this approach, we show for the first time that wild type prions initially seeded by different native strains can propagate as metastable PrPSc conformers with distinguishable strain properties in purified reactions containing a single active cofactor. Propagation of these metastable PrPSc conformers requires appropriate shaking conditions, and changes in these conditions cause all the different PrPSc conformers to converge irreversibly into the same single conformer as that produced in sPMCA reactions. We also use continuous shaking to show that two mutant PrP molecules with different pathogenic point mutations (D177N and E199K) adopt distinguishable PrPSc conformations in reactions containing pure protein substrate without cofactors. Unlike wild-type prions, the conformations of mutant prions appear to be dictated by substrate sequence rather than seed conformation. Overall, our studies using purified substrates in shaking reactions show that wild-type and mutant prions use fundamentally different mechanisms to generate conformational diversity at the molecular level.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>36626391</pmid><doi>10.1371/journal.ppat.1011083</doi><tpages>e1011083</tpages><orcidid>https://orcid.org/0000-0001-9060-4550</orcidid><oa>free_for_read</oa></addata></record> |
fulltext | fulltext |
identifier | ISSN: 1553-7374 |
ispartof | PLoS pathogens, 2023-01, Vol.19 (1), p.e1011083-e1011083 |
issn | 1553-7374 1553-7366 1553-7374 |
language | eng |
recordid | cdi_plos_journals_2777449479 |
source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; PubMed Central Open Access |
subjects | Bioassays Biology and Life Sciences Brain Care and treatment Cofactors Conformation Creutzfeldt-Jakob disease Development and progression Humans Medicine and Health Sciences Molecular Conformation Mutants Mutation Neuropathology Prion diseases Prion Diseases - metabolism Prion protein Prion Proteins Prions Prions - metabolism Propagation Protein folding Protein research Proteins Research and analysis methods Seeds Self propagation Shaking Substrates |
title | Conformational diversity in purified prions produced in vitro |
url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-25T16%3A09%3A54IST&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=Conformational%20diversity%20in%20purified%20prions%20produced%20in%20vitro&rft.jtitle=PLoS%20pathogens&rft.au=Walsh,%20Daniel%20J&rft.date=2023-01-01&rft.volume=19&rft.issue=1&rft.spage=e1011083&rft.epage=e1011083&rft.pages=e1011083-e1011083&rft.issn=1553-7374&rft.eissn=1553-7374&rft_id=info:doi/10.1371/journal.ppat.1011083&rft_dat=%3Cgale_plos_%3EA737321438%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=2777449479&rft_id=info:pmid/36626391&rft_galeid=A737321438&rft_doaj_id=oai_doaj_org_article_5ea883ddd88b45e2bbf74c2e62116bae&rfr_iscdi=true |