The PS1 hairpin of Mcm3 is essential for viability and for DNA unwinding in vitro
The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative hel...
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| description | The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3(K499A)) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3(K499A) strain. The ATPase activity of Mcm2-7(3K499A) was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding. |
| doi_str_mv | 10.1371/journal.pone.0082177 |
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To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3(K499A)) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3(K499A) strain. The ATPase activity of Mcm2-7(3K499A) was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0082177</identifier><identifier>PMID: 24349215</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adenosine triphosphatase ; Adenosine Triphosphatases - metabolism ; Alanine ; Alleles ; Amino Acid Motifs ; Amino Acid Sequence ; ATPases ; Biochemistry ; Cell growth ; Chromatography, Gel ; Crosses, Genetic ; Dentistry ; Deoxyribonucleic acid ; DNA ; DNA helicase ; DNA, Fungal - metabolism ; Electrophoretic mobility ; Kinases ; Lysine ; Medicine ; Microbial Viability - drug effects ; Minichromosome Maintenance Complex Component 3 - chemistry ; Minichromosome Maintenance Complex Component 3 - metabolism ; Models, Molecular ; Molecular Sequence Data ; Mutagens - toxicity ; Mutation ; Mutation - genetics ; Nucleic Acid Conformation ; Phenotype ; Plasmids ; Presenilin 1 ; Protein Binding - drug effects ; Protein Structure, Tertiary ; Protein Subunits - chemistry ; Protein Subunits - metabolism ; Proteins ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae - cytology ; Saccharomyces cerevisiae - drug effects ; Saccharomyces cerevisiae - growth & development ; Saccharomyces cerevisiae - metabolism ; Saccharomyces cerevisiae Proteins - chemistry ; Saccharomyces cerevisiae Proteins - metabolism ; Sensors ; Single-stranded DNA ; Structure-Activity Relationship ; Temperature ; Translocation ; Unwinding ; Viability ; Yeast</subject><ispartof>PloS one, 2013-12, Vol.8 (12), p.e82177</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Lam 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>2013 Lam et al 2013 Lam et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-cbb41845a07839ebb3873d8bfa3f0226c65af2a8a3365399750514c558b1b44b3</citedby><cites>FETCH-LOGICAL-c692t-cbb41845a07839ebb3873d8bfa3f0226c65af2a8a3365399750514c558b1b44b3</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/PMC3859580/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859580/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24349215$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lam, Simon K W</creatorcontrib><creatorcontrib>Ma, Xiaoli</creatorcontrib><creatorcontrib>Sing, Tina L</creatorcontrib><creatorcontrib>Shilton, Brian H</creatorcontrib><creatorcontrib>Brandl, Christopher J</creatorcontrib><creatorcontrib>Davey, Megan J</creatorcontrib><title>The PS1 hairpin of Mcm3 is essential for viability and for DNA unwinding in vitro</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3(K499A)) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3(K499A) strain. The ATPase activity of Mcm2-7(3K499A) was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.</description><subject>Adenosine triphosphatase</subject><subject>Adenosine Triphosphatases - metabolism</subject><subject>Alanine</subject><subject>Alleles</subject><subject>Amino Acid Motifs</subject><subject>Amino Acid Sequence</subject><subject>ATPases</subject><subject>Biochemistry</subject><subject>Cell growth</subject><subject>Chromatography, Gel</subject><subject>Crosses, Genetic</subject><subject>Dentistry</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA helicase</subject><subject>DNA, Fungal - metabolism</subject><subject>Electrophoretic mobility</subject><subject>Kinases</subject><subject>Lysine</subject><subject>Medicine</subject><subject>Microbial Viability - drug effects</subject><subject>Minichromosome Maintenance Complex Component 3 - chemistry</subject><subject>Minichromosome Maintenance Complex Component 3 - metabolism</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagens - toxicity</subject><subject>Mutation</subject><subject>Mutation - genetics</subject><subject>Nucleic Acid Conformation</subject><subject>Phenotype</subject><subject>Plasmids</subject><subject>Presenilin 1</subject><subject>Protein Binding - drug effects</subject><subject>Protein Structure, Tertiary</subject><subject>Protein Subunits - chemistry</subject><subject>Protein Subunits - metabolism</subject><subject>Proteins</subject><subject>Saccharomyces cerevisiae</subject><subject>Saccharomyces cerevisiae - cytology</subject><subject>Saccharomyces cerevisiae - drug effects</subject><subject>Saccharomyces cerevisiae - growth & development</subject><subject>Saccharomyces cerevisiae - metabolism</subject><subject>Saccharomyces cerevisiae Proteins - chemistry</subject><subject>Saccharomyces cerevisiae Proteins - metabolism</subject><subject>Sensors</subject><subject>Single-stranded DNA</subject><subject>Structure-Activity Relationship</subject><subject>Temperature</subject><subject>Translocation</subject><subject>Unwinding</subject><subject>Viability</subject><subject>Yeast</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>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNkl1rFDEYhQex2Fr9B6IBQfBi13zOJDfCUr8WWqu2ehuSTGY3y2yyJjOr_fdmu9OyAwqSi4R3nnPm5XCK4hmCU0Qq9GYV-uhVO90Eb6cQcoyq6kFxggTBkxJD8vDgfVw8TmkFISO8LB8Vx5gSKjBiJ8XX66UFX64QWCoXN86D0IALsybAJWBTsr5zqgVNiGDrlHat626A8vXt5N3nGej9L-dr5xcga7eui-FJcdSoNtmnw31afP_w_vrs0-T88uP8bHY-MaXA3cRoTRGnTMGKE2G1JrwiNdeNIg3EuDQlUw1WXBFSMiJExSBD1DDGNdKUanJavNj7btqQ5JBGkoiWFa4oLXkm5nuiDmolN9GtVbyRQTl5OwhxIVXsnGmt1ILmrbS2FTGUQiFwvQsvH1EqZHZeb4e_9Xpta5ODiaodmY6_eLeUi7CVhDPBOMwGLweDGH72NnX_WHmgFipv5XwTsplZu2TkjOagCGSMZGr6Fyqf2q6dyX1oXJ6PBK9Hgsx09ne3UH1Kcn717f_Zyx9j9tUBu7Sq7ZYptH3ngk9jkO5BE0NK0Tb3ySEod3W-S0Pu6iyHOmfZ88PU70V3_SV_AOIL7Jg</recordid><startdate>20131211</startdate><enddate>20131211</enddate><creator>Lam, Simon K W</creator><creator>Ma, Xiaoli</creator><creator>Sing, Tina L</creator><creator>Shilton, Brian H</creator><creator>Brandl, Christopher J</creator><creator>Davey, Megan J</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>5PM</scope><scope>DOA</scope></search><sort><creationdate>20131211</creationdate><title>The PS1 hairpin of Mcm3 is essential for viability and for DNA unwinding in vitro</title><author>Lam, Simon K W ; Ma, Xiaoli ; Sing, Tina L ; Shilton, Brian H ; Brandl, Christopher J ; Davey, Megan J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-cbb41845a07839ebb3873d8bfa3f0226c65af2a8a3365399750514c558b1b44b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Adenosine triphosphatase</topic><topic>Adenosine Triphosphatases - metabolism</topic><topic>Alanine</topic><topic>Alleles</topic><topic>Amino Acid Motifs</topic><topic>Amino Acid Sequence</topic><topic>ATPases</topic><topic>Biochemistry</topic><topic>Cell growth</topic><topic>Chromatography, Gel</topic><topic>Crosses, Genetic</topic><topic>Dentistry</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA helicase</topic><topic>DNA, Fungal - metabolism</topic><topic>Electrophoretic mobility</topic><topic>Kinases</topic><topic>Lysine</topic><topic>Medicine</topic><topic>Microbial Viability - drug effects</topic><topic>Minichromosome Maintenance Complex Component 3 - chemistry</topic><topic>Minichromosome Maintenance Complex Component 3 - metabolism</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutagens - toxicity</topic><topic>Mutation</topic><topic>Mutation - genetics</topic><topic>Nucleic Acid Conformation</topic><topic>Phenotype</topic><topic>Plasmids</topic><topic>Presenilin 1</topic><topic>Protein Binding - drug effects</topic><topic>Protein Structure, Tertiary</topic><topic>Protein Subunits - chemistry</topic><topic>Protein Subunits - metabolism</topic><topic>Proteins</topic><topic>Saccharomyces cerevisiae</topic><topic>Saccharomyces cerevisiae - cytology</topic><topic>Saccharomyces cerevisiae - drug effects</topic><topic>Saccharomyces cerevisiae - growth & development</topic><topic>Saccharomyces cerevisiae - metabolism</topic><topic>Saccharomyces cerevisiae Proteins - chemistry</topic><topic>Saccharomyces cerevisiae Proteins - metabolism</topic><topic>Sensors</topic><topic>Single-stranded DNA</topic><topic>Structure-Activity Relationship</topic><topic>Temperature</topic><topic>Translocation</topic><topic>Unwinding</topic><topic>Viability</topic><topic>Yeast</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lam, Simon K W</creatorcontrib><creatorcontrib>Ma, Xiaoli</creatorcontrib><creatorcontrib>Sing, Tina L</creatorcontrib><creatorcontrib>Shilton, Brian H</creatorcontrib><creatorcontrib>Brandl, Christopher J</creatorcontrib><creatorcontrib>Davey, Megan J</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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To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3(K499A)) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3(K499A) strain. The ATPase activity of Mcm2-7(3K499A) was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24349215</pmid><doi>10.1371/journal.pone.0082177</doi><tpages>e82177</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Adenosine triphosphatase Adenosine Triphosphatases - metabolism Alanine Alleles Amino Acid Motifs Amino Acid Sequence ATPases Biochemistry Cell growth Chromatography, Gel Crosses, Genetic Dentistry Deoxyribonucleic acid DNA DNA helicase DNA, Fungal - metabolism Electrophoretic mobility Kinases Lysine Medicine Microbial Viability - drug effects Minichromosome Maintenance Complex Component 3 - chemistry Minichromosome Maintenance Complex Component 3 - metabolism Models, Molecular Molecular Sequence Data Mutagens - toxicity Mutation Mutation - genetics Nucleic Acid Conformation Phenotype Plasmids Presenilin 1 Protein Binding - drug effects Protein Structure, Tertiary Protein Subunits - chemistry Protein Subunits - metabolism Proteins Saccharomyces cerevisiae Saccharomyces cerevisiae - cytology Saccharomyces cerevisiae - drug effects Saccharomyces cerevisiae - growth & development Saccharomyces cerevisiae - metabolism Saccharomyces cerevisiae Proteins - chemistry Saccharomyces cerevisiae Proteins - metabolism Sensors Single-stranded DNA Structure-Activity Relationship Temperature Translocation Unwinding Viability Yeast |
| title | The PS1 hairpin of Mcm3 is essential for viability and for DNA unwinding in vitro |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-02T12%3A21%3A13IST&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=The%20PS1%20hairpin%20of%20Mcm3%20is%20essential%20for%20viability%20and%20for%20DNA%20unwinding%20in%20vitro&rft.jtitle=PloS%20one&rft.au=Lam,%20Simon%20K%20W&rft.date=2013-12-11&rft.volume=8&rft.issue=12&rft.spage=e82177&rft.pages=e82177-&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0082177&rft_dat=%3Cgale_plos_%3EA478330553%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=1467274468&rft_id=info:pmid/24349215&rft_galeid=A478330553&rft_doaj_id=oai_doaj_org_article_b94692bbe73c440992d193232396a1c8&rfr_iscdi=true |