From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro
RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (...
Gespeichert in:
| Veröffentlicht in: | Genetics (Austin) 2008-10, Vol.180 (2), p.1039-1049 |
|---|---|
| Hauptverfasser: | , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 1049 |
|---|---|
| container_issue | 2 |
| container_start_page | 1039 |
| container_title | Genetics (Austin) |
| container_volume | 180 |
| creator | Torres-Barceló, Clara Martin, Susana Daros, Jose-Antonio Elena, Santiago F |
| description | RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid substitution mutants and quantified their capacity as suppressors of RNA silencing in a transient expression assay. Most mutations either had no quantitative effect or completely abolished silencing suppression (10 in each class), 3 caused a significant decrease in the activity, and 5 significantly increased it, revealing an unexpected high frequency of mutations conferring hypersuppressor activity. A representative set of the mutant alleles, containing both hypo- and hypersuppressors, was further analyzed for their effect on TEV accumulation and the strength of induced symptoms. Whereas TEV variants with hyposuppressor mutants were far less virulent than wild-type TEV, those with hypersuppressor alleles induced symptoms that were not more severe than those characteristic of the wild-type virus, suggesting that there is not a perfect match between suppression and virulence. |
| doi_str_mv | 10.1534/genetics.108.091363 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2567354</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>1584172351</sourcerecordid><originalsourceid>FETCH-LOGICAL-c520t-84c128930040f45ea01c2ded1ac9eda19383b04b136ac8753c1ad78c22f91f903</originalsourceid><addsrcrecordid>eNpdkd2O0zAQhSMEYrsLT4AEFjeIi5QZOz8OF0hVtUuRKkB099pyHaf1qo2zttOqb7GPjKOU3ytb4--c8cxJklcIU8xZ9mGjWx2M8lMEPoUKWcGeJBOsMpbSguHTZAKARVqUDC-SS-_vAaCocv48uUBeciizfJI83ji7J4tTZ1MS7HDRzvdd57T3xrYfyXXTaBWIbchsb1pLZsrUZNWvfTChDxHxxLYkbDX58XWWrsxOt8q0m4iMJtZFSTAHE06DyQDe2rVUyhId1JZ0NpwOxvWeLObpd2dfJM8aufP65fm8Su5urm_ni3T57fOX-WyZqpxCSHmmkPKKAWTQZLmWgIrWukapKl1LrBhna8jWcStS8TJnCmVdckVpU2FTAbtKPo2-Xb_e61rpNji5E50ze-lOwkoj_n1pzVZs7EHQPG40z6LB-9Fg-59sMVuKoQa0KMoK4YCRfXtu5uxDr30Q97Z3bZxPUMxwAIsIsRFSznrvdPPbFUEMiYtficcCF2PiUfX67zn-aM4RR-Dd-Z9msz0ap4Xfy90u4iiOxyNyEDQasiqSb0aykVbIjTNe3K0oIAPMC5aVJfsJ113Bdw</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>214126676</pqid></control><display><type>article</type><title>From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro</title><source>Oxford University Press Journals All Titles (1996-Current)</source><source>MEDLINE</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>Alma/SFX Local Collection</source><creator>Torres-Barceló, Clara ; Martin, Susana ; Daros, Jose-Antonio ; Elena, Santiago F</creator><creatorcontrib>Torres-Barceló, Clara ; Martin, Susana ; Daros, Jose-Antonio ; Elena, Santiago F</creatorcontrib><description>RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid substitution mutants and quantified their capacity as suppressors of RNA silencing in a transient expression assay. Most mutations either had no quantitative effect or completely abolished silencing suppression (10 in each class), 3 caused a significant decrease in the activity, and 5 significantly increased it, revealing an unexpected high frequency of mutations conferring hypersuppressor activity. A representative set of the mutant alleles, containing both hypo- and hypersuppressors, was further analyzed for their effect on TEV accumulation and the strength of induced symptoms. Whereas TEV variants with hyposuppressor mutants were far less virulent than wild-type TEV, those with hypersuppressor alleles induced symptoms that were not more severe than those characteristic of the wild-type virus, suggesting that there is not a perfect match between suppression and virulence.</description><identifier>ISSN: 0016-6731</identifier><identifier>ISSN: 1943-2631</identifier><identifier>EISSN: 1943-2631</identifier><identifier>DOI: 10.1534/genetics.108.091363</identifier><identifier>PMID: 18780745</identifier><identifier>CODEN: GENTAE</identifier><language>eng</language><publisher>United States: Genetics Soc America</publisher><subject>amino acid substitution ; Amino Acid Substitution - genetics ; Amino acids ; Cysteine Endopeptidases - genetics ; Defense mechanisms ; gene silencing ; Genome, Viral ; HC-Pro protein ; host-pathogen relationships ; infection ; Investigations ; Life Sciences ; Molecular biology ; mutants ; Mutation ; Nicotiana benthamiana ; Plant Viruses - genetics ; Potyvirus - genetics ; Potyvirus - pathogenicity ; Potyvirus - physiology ; Proteins ; RNA ; RNA Interference ; RNA silencing ; Tobacco etch virus ; viral proteins ; Viral Proteins - genetics ; virulence</subject><ispartof>Genetics (Austin), 2008-10, Vol.180 (2), p.1039-1049</ispartof><rights>Copyright Genetics Society of America Oct 2008</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><rights>Copyright © 2008 by the Genetics Society of America</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c520t-84c128930040f45ea01c2ded1ac9eda19383b04b136ac8753c1ad78c22f91f903</citedby><cites>FETCH-LOGICAL-c520t-84c128930040f45ea01c2ded1ac9eda19383b04b136ac8753c1ad78c22f91f903</cites><orcidid>0000-0003-2116-1471</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,776,780,881,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18780745$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.inrae.fr/hal-02667910$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Torres-Barceló, Clara</creatorcontrib><creatorcontrib>Martin, Susana</creatorcontrib><creatorcontrib>Daros, Jose-Antonio</creatorcontrib><creatorcontrib>Elena, Santiago F</creatorcontrib><title>From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro</title><title>Genetics (Austin)</title><addtitle>Genetics</addtitle><description>RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid substitution mutants and quantified their capacity as suppressors of RNA silencing in a transient expression assay. Most mutations either had no quantitative effect or completely abolished silencing suppression (10 in each class), 3 caused a significant decrease in the activity, and 5 significantly increased it, revealing an unexpected high frequency of mutations conferring hypersuppressor activity. A representative set of the mutant alleles, containing both hypo- and hypersuppressors, was further analyzed for their effect on TEV accumulation and the strength of induced symptoms. Whereas TEV variants with hyposuppressor mutants were far less virulent than wild-type TEV, those with hypersuppressor alleles induced symptoms that were not more severe than those characteristic of the wild-type virus, suggesting that there is not a perfect match between suppression and virulence.</description><subject>amino acid substitution</subject><subject>Amino Acid Substitution - genetics</subject><subject>Amino acids</subject><subject>Cysteine Endopeptidases - genetics</subject><subject>Defense mechanisms</subject><subject>gene silencing</subject><subject>Genome, Viral</subject><subject>HC-Pro protein</subject><subject>host-pathogen relationships</subject><subject>infection</subject><subject>Investigations</subject><subject>Life Sciences</subject><subject>Molecular biology</subject><subject>mutants</subject><subject>Mutation</subject><subject>Nicotiana benthamiana</subject><subject>Plant Viruses - genetics</subject><subject>Potyvirus - genetics</subject><subject>Potyvirus - pathogenicity</subject><subject>Potyvirus - physiology</subject><subject>Proteins</subject><subject>RNA</subject><subject>RNA Interference</subject><subject>RNA silencing</subject><subject>Tobacco etch virus</subject><subject>viral proteins</subject><subject>Viral Proteins - genetics</subject><subject>virulence</subject><issn>0016-6731</issn><issn>1943-2631</issn><issn>1943-2631</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>BENPR</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNpdkd2O0zAQhSMEYrsLT4AEFjeIi5QZOz8OF0hVtUuRKkB099pyHaf1qo2zttOqb7GPjKOU3ytb4--c8cxJklcIU8xZ9mGjWx2M8lMEPoUKWcGeJBOsMpbSguHTZAKARVqUDC-SS-_vAaCocv48uUBeciizfJI83ji7J4tTZ1MS7HDRzvdd57T3xrYfyXXTaBWIbchsb1pLZsrUZNWvfTChDxHxxLYkbDX58XWWrsxOt8q0m4iMJtZFSTAHE06DyQDe2rVUyhId1JZ0NpwOxvWeLObpd2dfJM8aufP65fm8Su5urm_ni3T57fOX-WyZqpxCSHmmkPKKAWTQZLmWgIrWukapKl1LrBhna8jWcStS8TJnCmVdckVpU2FTAbtKPo2-Xb_e61rpNji5E50ze-lOwkoj_n1pzVZs7EHQPG40z6LB-9Fg-59sMVuKoQa0KMoK4YCRfXtu5uxDr30Q97Z3bZxPUMxwAIsIsRFSznrvdPPbFUEMiYtficcCF2PiUfX67zn-aM4RR-Dd-Z9msz0ap4Xfy90u4iiOxyNyEDQasiqSb0aykVbIjTNe3K0oIAPMC5aVJfsJ113Bdw</recordid><startdate>20081001</startdate><enddate>20081001</enddate><creator>Torres-Barceló, Clara</creator><creator>Martin, Susana</creator><creator>Daros, Jose-Antonio</creator><creator>Elena, Santiago F</creator><general>Genetics Soc America</general><general>Genetics Society of America</general><general>Oxford University Press</general><scope>FBQ</scope><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>3V.</scope><scope>4T-</scope><scope>4U-</scope><scope>7QP</scope><scope>7SS</scope><scope>7TK</scope><scope>7TM</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>HCIFZ</scope><scope>K9-</scope><scope>K9.</scope><scope>LK8</scope><scope>M0K</scope><scope>M0R</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>MBDVC</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>RC3</scope><scope>1XC</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0003-2116-1471</orcidid></search><sort><creationdate>20081001</creationdate><title>From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro</title><author>Torres-Barceló, Clara ; Martin, Susana ; Daros, Jose-Antonio ; Elena, Santiago F</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c520t-84c128930040f45ea01c2ded1ac9eda19383b04b136ac8753c1ad78c22f91f903</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>amino acid substitution</topic><topic>Amino Acid Substitution - genetics</topic><topic>Amino acids</topic><topic>Cysteine Endopeptidases - genetics</topic><topic>Defense mechanisms</topic><topic>gene silencing</topic><topic>Genome, Viral</topic><topic>HC-Pro protein</topic><topic>host-pathogen relationships</topic><topic>infection</topic><topic>Investigations</topic><topic>Life Sciences</topic><topic>Molecular biology</topic><topic>mutants</topic><topic>Mutation</topic><topic>Nicotiana benthamiana</topic><topic>Plant Viruses - genetics</topic><topic>Potyvirus - genetics</topic><topic>Potyvirus - pathogenicity</topic><topic>Potyvirus - physiology</topic><topic>Proteins</topic><topic>RNA</topic><topic>RNA Interference</topic><topic>RNA silencing</topic><topic>Tobacco etch virus</topic><topic>viral proteins</topic><topic>Viral Proteins - genetics</topic><topic>virulence</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Torres-Barceló, Clara</creatorcontrib><creatorcontrib>Martin, Susana</creatorcontrib><creatorcontrib>Daros, Jose-Antonio</creatorcontrib><creatorcontrib>Elena, Santiago F</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Docstoc</collection><collection>University Readers</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science 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 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>Research Library (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</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>Research Library Prep</collection><collection>SciTech Premium Collection</collection><collection>Consumer Health Database (Alumni Edition)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Consumer Health Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Research Library</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Research Library (Corporate)</collection><collection>Biotechnology and BioEngineering Abstracts</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>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>Hyper Article en Ligne (HAL)</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Genetics (Austin)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Torres-Barceló, Clara</au><au>Martin, Susana</au><au>Daros, Jose-Antonio</au><au>Elena, Santiago F</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro</atitle><jtitle>Genetics (Austin)</jtitle><addtitle>Genetics</addtitle><date>2008-10-01</date><risdate>2008</risdate><volume>180</volume><issue>2</issue><spage>1039</spage><epage>1049</epage><pages>1039-1049</pages><issn>0016-6731</issn><issn>1943-2631</issn><eissn>1943-2631</eissn><coden>GENTAE</coden><abstract>RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid substitution mutants and quantified their capacity as suppressors of RNA silencing in a transient expression assay. Most mutations either had no quantitative effect or completely abolished silencing suppression (10 in each class), 3 caused a significant decrease in the activity, and 5 significantly increased it, revealing an unexpected high frequency of mutations conferring hypersuppressor activity. A representative set of the mutant alleles, containing both hypo- and hypersuppressors, was further analyzed for their effect on TEV accumulation and the strength of induced symptoms. Whereas TEV variants with hyposuppressor mutants were far less virulent than wild-type TEV, those with hypersuppressor alleles induced symptoms that were not more severe than those characteristic of the wild-type virus, suggesting that there is not a perfect match between suppression and virulence.</abstract><cop>United States</cop><pub>Genetics Soc America</pub><pmid>18780745</pmid><doi>10.1534/genetics.108.091363</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0003-2116-1471</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0016-6731 |
| ispartof | Genetics (Austin), 2008-10, Vol.180 (2), p.1039-1049 |
| issn | 0016-6731 1943-2631 1943-2631 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2567354 |
| source | Oxford University Press Journals All Titles (1996-Current); MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | amino acid substitution Amino Acid Substitution - genetics Amino acids Cysteine Endopeptidases - genetics Defense mechanisms gene silencing Genome, Viral HC-Pro protein host-pathogen relationships infection Investigations Life Sciences Molecular biology mutants Mutation Nicotiana benthamiana Plant Viruses - genetics Potyvirus - genetics Potyvirus - pathogenicity Potyvirus - physiology Proteins RNA RNA Interference RNA silencing Tobacco etch virus viral proteins Viral Proteins - genetics virulence |
| title | From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-09T15%3A28%3A08IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=From%20Hypo-%20to%20Hypersuppression:%20Effect%20of%20Amino%20Acid%20Substitutions%20on%20the%20RNA-Silencing%20Suppressor%20Activity%20of%20the%20Tobacco%20etch%20potyvirus%20HC-Pro&rft.jtitle=Genetics%20(Austin)&rft.au=Torres-Barcel%C3%B3,%20Clara&rft.date=2008-10-01&rft.volume=180&rft.issue=2&rft.spage=1039&rft.epage=1049&rft.pages=1039-1049&rft.issn=0016-6731&rft.eissn=1943-2631&rft.coden=GENTAE&rft_id=info:doi/10.1534/genetics.108.091363&rft_dat=%3Cproquest_pubme%3E1584172351%3C/proquest_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=214126676&rft_id=info:pmid/18780745&rfr_iscdi=true |