Exon-skipping events in candidates for clinical trials of morpholino
Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most...
Gespeichert in:
Veröffentlicht in: | Pediatrics international 2011-08, Vol.53 (4), p.524-529 |
---|---|
Hauptverfasser: | , , , , , , , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
container_end_page | 529 |
---|---|
container_issue | 4 |
container_start_page | 524 |
container_title | Pediatrics international |
container_volume | 53 |
creator | Nakano, Shiho Ozasa, Shiro Yoshioka, Kowashi Fujii, Isao Mitsui, Kouichi Nomura, Keiko Kosuge, Hirofumi Endo, Fumio Matsukura, Makoto Kimura, Shigemi |
description | Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in‐frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino‐mediated exon skipping, which changes out‐of‐frame deletions to in‐frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon‐skipping event in cells of DMD patients before the clinical trial.
Methods: Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon‐skipping event was analyzed on reverse transcription–polymerase chain reaction.
Results: Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51‐skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon‐skipping patterns, which are total or partial exon 51‐skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out‐of‐frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51‐skipping event.
Conclusion: An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino. |
doi_str_mv | 10.1111/j.1442-200X.2011.03330.x |
format | Article |
fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_884424876</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>2429473961</sourcerecordid><originalsourceid>FETCH-LOGICAL-c4900-fb08e5b5af0211a34a0a1527b842dc769d5526a1ae6b8fcd58d4e14158d37ba63</originalsourceid><addsrcrecordid>eNqNkU1v1DAQhi0Eou2Wv4AsLnBJOv6KvQcOqN1-qFWpxKJWXCwnccDbbBzsbLv77-t0yx44IHyZ0fh5X2nmRQgTyEl6R4uccE4zCnCXUyAkB8YY5OtXaH_38Tr1jKpMQSH30EGMCwBQUvG3aI8SxikTsI9OZmvfZfHe9b3rfmL7YLshYtfhynS1q81gI258wFXrOleZFg_BmTZi3-ClD_0vn-b-EL1p0tC-e6kT9P10Nj8-z66-nl0cf7nKKj4FyJoSlBWlMA1QQgzjBgwRVJaK07qSxbQWghaGGFuUqqlqoWpuCSepMlmagk3Qx61vH_zvlY2DXrpY2bY1nfWrqJVKy3MlR_LTP0kCVIFM5Ih--Atd-FXo0h6jH50qWkwTpLZQFXyMwTa6D25pwiY56TESvdDj5fV4eT1Gop8j0eskff_ivyqXtt4J_2SQgM9b4NG1dvPfxvpmdjJ2SZ9t9S4Odr3Tm3CvC8mk0LfXZ_rbDzG_vJvf6kv2BIsBqDc</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>884298269</pqid></control><display><type>article</type><title>Exon-skipping events in candidates for clinical trials of morpholino</title><source>Wiley Online Library - AutoHoldings Journals</source><source>MEDLINE</source><creator>Nakano, Shiho ; Ozasa, Shiro ; Yoshioka, Kowashi ; Fujii, Isao ; Mitsui, Kouichi ; Nomura, Keiko ; Kosuge, Hirofumi ; Endo, Fumio ; Matsukura, Makoto ; Kimura, Shigemi</creator><creatorcontrib>Nakano, Shiho ; Ozasa, Shiro ; Yoshioka, Kowashi ; Fujii, Isao ; Mitsui, Kouichi ; Nomura, Keiko ; Kosuge, Hirofumi ; Endo, Fumio ; Matsukura, Makoto ; Kimura, Shigemi</creatorcontrib><description>Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in‐frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino‐mediated exon skipping, which changes out‐of‐frame deletions to in‐frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon‐skipping event in cells of DMD patients before the clinical trial.
Methods: Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon‐skipping event was analyzed on reverse transcription–polymerase chain reaction.
Results: Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51‐skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon‐skipping patterns, which are total or partial exon 51‐skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out‐of‐frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51‐skipping event.
Conclusion: An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.</description><identifier>ISSN: 1328-8067</identifier><identifier>EISSN: 1442-200X</identifier><identifier>DOI: 10.1111/j.1442-200X.2011.03330.x</identifier><identifier>PMID: 21342350</identifier><language>eng</language><publisher>Melbourne, Australia: Blackwell Publishing Asia</publisher><subject>Antisense ; Becker's muscular dystrophy ; Bone mineral density ; Clinical trials ; Clinical Trials as Topic ; Clonal deletion ; DNA - genetics ; Duchenne muscular dystrophy ; Duchenne's muscular dystrophy ; Dystrophin ; Enhancers ; Exon skipping ; Exons ; fibroblast ; Fibroblasts ; Gene deletion ; Genetic Therapy ; Genetics ; Humans ; In Vitro Techniques ; Infection ; Language ; Molecular biology ; morpholino ; Morpholinos - therapeutic use ; Muscular dystrophy ; Muscular Dystrophy, Duchenne - genetics ; Muscular Dystrophy, Duchenne - therapy ; Oligonucleotides, Antisense - therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sequence Deletion ; Splicing ; Therapy ; Thionucleotides - therapeutic use ; Transfection</subject><ispartof>Pediatrics international, 2011-08, Vol.53 (4), p.524-529</ispartof><rights>2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society</rights><rights>2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4900-fb08e5b5af0211a34a0a1527b842dc769d5526a1ae6b8fcd58d4e14158d37ba63</citedby><cites>FETCH-LOGICAL-c4900-fb08e5b5af0211a34a0a1527b842dc769d5526a1ae6b8fcd58d4e14158d37ba63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fj.1442-200X.2011.03330.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1442-200X.2011.03330.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>315,781,785,1418,27928,27929,45578,45579</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21342350$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nakano, Shiho</creatorcontrib><creatorcontrib>Ozasa, Shiro</creatorcontrib><creatorcontrib>Yoshioka, Kowashi</creatorcontrib><creatorcontrib>Fujii, Isao</creatorcontrib><creatorcontrib>Mitsui, Kouichi</creatorcontrib><creatorcontrib>Nomura, Keiko</creatorcontrib><creatorcontrib>Kosuge, Hirofumi</creatorcontrib><creatorcontrib>Endo, Fumio</creatorcontrib><creatorcontrib>Matsukura, Makoto</creatorcontrib><creatorcontrib>Kimura, Shigemi</creatorcontrib><title>Exon-skipping events in candidates for clinical trials of morpholino</title><title>Pediatrics international</title><addtitle>Pediatr Int</addtitle><description>Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in‐frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino‐mediated exon skipping, which changes out‐of‐frame deletions to in‐frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon‐skipping event in cells of DMD patients before the clinical trial.
Methods: Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon‐skipping event was analyzed on reverse transcription–polymerase chain reaction.
Results: Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51‐skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon‐skipping patterns, which are total or partial exon 51‐skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out‐of‐frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51‐skipping event.
Conclusion: An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.</description><subject>Antisense</subject><subject>Becker's muscular dystrophy</subject><subject>Bone mineral density</subject><subject>Clinical trials</subject><subject>Clinical Trials as Topic</subject><subject>Clonal deletion</subject><subject>DNA - genetics</subject><subject>Duchenne muscular dystrophy</subject><subject>Duchenne's muscular dystrophy</subject><subject>Dystrophin</subject><subject>Enhancers</subject><subject>Exon skipping</subject><subject>Exons</subject><subject>fibroblast</subject><subject>Fibroblasts</subject><subject>Gene deletion</subject><subject>Genetic Therapy</subject><subject>Genetics</subject><subject>Humans</subject><subject>In Vitro Techniques</subject><subject>Infection</subject><subject>Language</subject><subject>Molecular biology</subject><subject>morpholino</subject><subject>Morpholinos - therapeutic use</subject><subject>Muscular dystrophy</subject><subject>Muscular Dystrophy, Duchenne - genetics</subject><subject>Muscular Dystrophy, Duchenne - therapy</subject><subject>Oligonucleotides, Antisense - therapeutic use</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Sequence Analysis, DNA</subject><subject>Sequence Deletion</subject><subject>Splicing</subject><subject>Therapy</subject><subject>Thionucleotides - therapeutic use</subject><subject>Transfection</subject><issn>1328-8067</issn><issn>1442-200X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkU1v1DAQhi0Eou2Wv4AsLnBJOv6KvQcOqN1-qFWpxKJWXCwnccDbbBzsbLv77-t0yx44IHyZ0fh5X2nmRQgTyEl6R4uccE4zCnCXUyAkB8YY5OtXaH_38Tr1jKpMQSH30EGMCwBQUvG3aI8SxikTsI9OZmvfZfHe9b3rfmL7YLshYtfhynS1q81gI258wFXrOleZFg_BmTZi3-ClD_0vn-b-EL1p0tC-e6kT9P10Nj8-z66-nl0cf7nKKj4FyJoSlBWlMA1QQgzjBgwRVJaK07qSxbQWghaGGFuUqqlqoWpuCSepMlmagk3Qx61vH_zvlY2DXrpY2bY1nfWrqJVKy3MlR_LTP0kCVIFM5Ih--Atd-FXo0h6jH50qWkwTpLZQFXyMwTa6D25pwiY56TESvdDj5fV4eT1Gop8j0eskff_ivyqXtt4J_2SQgM9b4NG1dvPfxvpmdjJ2SZ9t9S4Odr3Tm3CvC8mk0LfXZ_rbDzG_vJvf6kv2BIsBqDc</recordid><startdate>201108</startdate><enddate>201108</enddate><creator>Nakano, Shiho</creator><creator>Ozasa, Shiro</creator><creator>Yoshioka, Kowashi</creator><creator>Fujii, Isao</creator><creator>Mitsui, Kouichi</creator><creator>Nomura, Keiko</creator><creator>Kosuge, Hirofumi</creator><creator>Endo, Fumio</creator><creator>Matsukura, Makoto</creator><creator>Kimura, Shigemi</creator><general>Blackwell Publishing Asia</general><general>Blackwell Publishing Ltd</general><scope>BSCLL</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>7TK</scope><scope>7U9</scope><scope>H94</scope><scope>K9.</scope><scope>7TM</scope><scope>7X8</scope></search><sort><creationdate>201108</creationdate><title>Exon-skipping events in candidates for clinical trials of morpholino</title><author>Nakano, Shiho ; Ozasa, Shiro ; Yoshioka, Kowashi ; Fujii, Isao ; Mitsui, Kouichi ; Nomura, Keiko ; Kosuge, Hirofumi ; Endo, Fumio ; Matsukura, Makoto ; Kimura, Shigemi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4900-fb08e5b5af0211a34a0a1527b842dc769d5526a1ae6b8fcd58d4e14158d37ba63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Antisense</topic><topic>Becker's muscular dystrophy</topic><topic>Bone mineral density</topic><topic>Clinical trials</topic><topic>Clinical Trials as Topic</topic><topic>Clonal deletion</topic><topic>DNA - genetics</topic><topic>Duchenne muscular dystrophy</topic><topic>Duchenne's muscular dystrophy</topic><topic>Dystrophin</topic><topic>Enhancers</topic><topic>Exon skipping</topic><topic>Exons</topic><topic>fibroblast</topic><topic>Fibroblasts</topic><topic>Gene deletion</topic><topic>Genetic Therapy</topic><topic>Genetics</topic><topic>Humans</topic><topic>In Vitro Techniques</topic><topic>Infection</topic><topic>Language</topic><topic>Molecular biology</topic><topic>morpholino</topic><topic>Morpholinos - therapeutic use</topic><topic>Muscular dystrophy</topic><topic>Muscular Dystrophy, Duchenne - genetics</topic><topic>Muscular Dystrophy, Duchenne - therapy</topic><topic>Oligonucleotides, Antisense - therapeutic use</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Sequence Analysis, DNA</topic><topic>Sequence Deletion</topic><topic>Splicing</topic><topic>Therapy</topic><topic>Thionucleotides - therapeutic use</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nakano, Shiho</creatorcontrib><creatorcontrib>Ozasa, Shiro</creatorcontrib><creatorcontrib>Yoshioka, Kowashi</creatorcontrib><creatorcontrib>Fujii, Isao</creatorcontrib><creatorcontrib>Mitsui, Kouichi</creatorcontrib><creatorcontrib>Nomura, Keiko</creatorcontrib><creatorcontrib>Kosuge, Hirofumi</creatorcontrib><creatorcontrib>Endo, Fumio</creatorcontrib><creatorcontrib>Matsukura, Makoto</creatorcontrib><creatorcontrib>Kimura, Shigemi</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Neurosciences Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Pediatrics international</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nakano, Shiho</au><au>Ozasa, Shiro</au><au>Yoshioka, Kowashi</au><au>Fujii, Isao</au><au>Mitsui, Kouichi</au><au>Nomura, Keiko</au><au>Kosuge, Hirofumi</au><au>Endo, Fumio</au><au>Matsukura, Makoto</au><au>Kimura, Shigemi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Exon-skipping events in candidates for clinical trials of morpholino</atitle><jtitle>Pediatrics international</jtitle><addtitle>Pediatr Int</addtitle><date>2011-08</date><risdate>2011</risdate><volume>53</volume><issue>4</issue><spage>524</spage><epage>529</epage><pages>524-529</pages><issn>1328-8067</issn><eissn>1442-200X</eissn><abstract>Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in‐frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino‐mediated exon skipping, which changes out‐of‐frame deletions to in‐frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon‐skipping event in cells of DMD patients before the clinical trial.
Methods: Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon‐skipping event was analyzed on reverse transcription–polymerase chain reaction.
Results: Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51‐skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon‐skipping patterns, which are total or partial exon 51‐skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out‐of‐frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51‐skipping event.
Conclusion: An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.</abstract><cop>Melbourne, Australia</cop><pub>Blackwell Publishing Asia</pub><pmid>21342350</pmid><doi>10.1111/j.1442-200X.2011.03330.x</doi><tpages>6</tpages></addata></record> |
fulltext | fulltext |
identifier | ISSN: 1328-8067 |
ispartof | Pediatrics international, 2011-08, Vol.53 (4), p.524-529 |
issn | 1328-8067 1442-200X |
language | eng |
recordid | cdi_proquest_miscellaneous_884424876 |
source | Wiley Online Library - AutoHoldings Journals; MEDLINE |
subjects | Antisense Becker's muscular dystrophy Bone mineral density Clinical trials Clinical Trials as Topic Clonal deletion DNA - genetics Duchenne muscular dystrophy Duchenne's muscular dystrophy Dystrophin Enhancers Exon skipping Exons fibroblast Fibroblasts Gene deletion Genetic Therapy Genetics Humans In Vitro Techniques Infection Language Molecular biology morpholino Morpholinos - therapeutic use Muscular dystrophy Muscular Dystrophy, Duchenne - genetics Muscular Dystrophy, Duchenne - therapy Oligonucleotides, Antisense - therapeutic use Reverse Transcriptase Polymerase Chain Reaction Sequence Analysis, DNA Sequence Deletion Splicing Therapy Thionucleotides - therapeutic use Transfection |
title | Exon-skipping events in candidates for clinical trials of morpholino |
url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-17T07%3A15%3A22IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Exon-skipping%20events%20in%20candidates%20for%20clinical%20trials%20of%20morpholino&rft.jtitle=Pediatrics%20international&rft.au=Nakano,%20Shiho&rft.date=2011-08&rft.volume=53&rft.issue=4&rft.spage=524&rft.epage=529&rft.pages=524-529&rft.issn=1328-8067&rft.eissn=1442-200X&rft_id=info:doi/10.1111/j.1442-200X.2011.03330.x&rft_dat=%3Cproquest_cross%3E2429473961%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=884298269&rft_id=info:pmid/21342350&rfr_iscdi=true |