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Enrichment of in vivo transcription data from dietary intervention studies with in vitro data provides improved insight into gene regulation mechanisms in the intestinal mucosa
Gene expression profiles of intestinal mucosa of chickens and pigs fed over long-term periods (days/weeks) with a diet rich in rye and a diet supplemented with zinc, respectively, or of chickens after a one-day amoxicillin treatment of chickens, were recorded recently. Such dietary interventions are...
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Published in: | Genes & nutrition 2017-04, Vol.12 (1), p.11-11, Article 11 |
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description | Gene expression profiles of intestinal mucosa of chickens and pigs fed over long-term periods (days/weeks) with a diet rich in rye and a diet supplemented with zinc, respectively, or of chickens after a one-day amoxicillin treatment of chickens, were recorded recently. Such dietary interventions are frequently used to modulate animal performance or therapeutically for monogastric livestock. In this study, changes in gene expression induced by these three interventions in cultured "Intestinal Porcine Epithelial Cells" (IPEC-J2) recorded after a short-term period of 2 and 6 hours, were compared to the in vivo gene expression profiles in order to evaluate the capability of this in vitro bioassay in predicting in vivo responses.
Lists of response genes were analysed with bioinformatics programs to identify common biological pathways induced in vivo as well as in vitro. Furthermore, overlapping genes and pathways were evaluated for possible involvement in the biological processes induced in vivo by datamining and consulting literature.
For all three interventions, only a limited number of identical genes and a few common biological processes/pathways were found to be affected by the respective interventions. However, several enterocyte-specific regulatory and secreted effector proteins that responded in vitro could be related to processes regulated in vivo, i.e. processes related to mineral absorption, (epithelial) cell adherence and tight junction formation for zinc, microtubule and cytoskeleton integrity for amoxicillin, and cell-cycle progression and mucus production for rye.
Short-term gene expression responses to dietary interventions as measured in the in vitro bioassay have a low predictability for long-term responses as measured in the intestinal mucosa in vivo. The short-term responses of a set regulatory and effector genes, as measured in this bioassay, however, provided additional insight into how specific processes in piglets and broilers may be modulated by "early" signalling molecules produced by enterocytes. The relevance of this set of regulatory/effector genes and cognate biological processes for zinc deficiency and supplementation, gluten allergy (rye), and amoxicillin administration in humans is discussed. |
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Lists of response genes were analysed with bioinformatics programs to identify common biological pathways induced in vivo as well as in vitro. Furthermore, overlapping genes and pathways were evaluated for possible involvement in the biological processes induced in vivo by datamining and consulting literature.
For all three interventions, only a limited number of identical genes and a few common biological processes/pathways were found to be affected by the respective interventions. However, several enterocyte-specific regulatory and secreted effector proteins that responded in vitro could be related to processes regulated in vivo, i.e. processes related to mineral absorption, (epithelial) cell adherence and tight junction formation for zinc, microtubule and cytoskeleton integrity for amoxicillin, and cell-cycle progression and mucus production for rye.
Short-term gene expression responses to dietary interventions as measured in the in vitro bioassay have a low predictability for long-term responses as measured in the intestinal mucosa in vivo. The short-term responses of a set regulatory and effector genes, as measured in this bioassay, however, provided additional insight into how specific processes in piglets and broilers may be modulated by "early" signalling molecules produced by enterocytes. The relevance of this set of regulatory/effector genes and cognate biological processes for zinc deficiency and supplementation, gluten allergy (rye), and amoxicillin administration in humans is discussed.</description><identifier>ISSN: 1555-8932</identifier><identifier>EISSN: 1865-3499</identifier><identifier>DOI: 10.1186/s12263-017-0559-1</identifier><identifier>PMID: 28413565</identifier><language>eng</language><publisher>Germany: BioMed Central</publisher><subject>Additives ; Allergies ; Amoxicillin ; Animal Breeding & Genomics ; Animal Breeding and Genetics ; Animal Nutrition ; Animal welfare ; Antibiotics ; Bioassays ; Bioinformatics ; Broilers ; CVI Infectiebiologie ; CVI Infection Biology ; Cytokines ; Cytoskeleton ; Datasets ; Diarrhea ; Diervoeding ; Diet ; Enterocytes ; Epithelial cells ; Fokkerij & Genomica ; Fokkerij en Genetica ; Gene expression ; Gene regulation ; Gluten ; Immune system ; Infectiebiologie ; Infection Biology ; Intervention ; Intestinal mucosa ; Intestine ; Laboratory animals ; Livestock ; LR - Animal Breeding & Genomics ; LR - Animal Nutrition ; LR - Diervoeding ; LR - Genomica ; Microbiota ; Mucosa ; Mucus ; Nutrient deficiency ; Pathogens ; Penicillin ; Pigs ; Rye ; Signal transduction ; Small intestine ; Transcription ; WIAS ; Zinc ; Zinc oxides</subject><ispartof>Genes & nutrition, 2017-04, Vol.12 (1), p.11-11, Article 11</ispartof><rights>2017. This work is licensed under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.</rights><rights>The Author(s) 2017</rights><rights>Wageningen University & Research</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c544t-b614a92c46ab89b8560deb82e219e6cf3f116283e996b6eec8ed9425860d839f3</citedby><cites>FETCH-LOGICAL-c544t-b614a92c46ab89b8560deb82e219e6cf3f116283e996b6eec8ed9425860d839f3</cites><orcidid>0000-0001-8020-8853</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/2575275373/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/2575275373?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25753,27924,27925,37012,37013,44590,53791,53793,75126</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28413565$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hulst, Marcel</creatorcontrib><creatorcontrib>Jansman, Alfons</creatorcontrib><creatorcontrib>Wijers, Ilonka</creatorcontrib><creatorcontrib>Hoekman, Arjan</creatorcontrib><creatorcontrib>Vastenhouw, Stéphanie</creatorcontrib><creatorcontrib>van Krimpen, Marinus</creatorcontrib><creatorcontrib>Smits, Mari</creatorcontrib><creatorcontrib>Schokker, Dirkjan</creatorcontrib><title>Enrichment of in vivo transcription data from dietary intervention studies with in vitro data provides improved insight into gene regulation mechanisms in the intestinal mucosa</title><title>Genes & nutrition</title><addtitle>Genes Nutr</addtitle><description>Gene expression profiles of intestinal mucosa of chickens and pigs fed over long-term periods (days/weeks) with a diet rich in rye and a diet supplemented with zinc, respectively, or of chickens after a one-day amoxicillin treatment of chickens, were recorded recently. Such dietary interventions are frequently used to modulate animal performance or therapeutically for monogastric livestock. In this study, changes in gene expression induced by these three interventions in cultured "Intestinal Porcine Epithelial Cells" (IPEC-J2) recorded after a short-term period of 2 and 6 hours, were compared to the in vivo gene expression profiles in order to evaluate the capability of this in vitro bioassay in predicting in vivo responses.
Lists of response genes were analysed with bioinformatics programs to identify common biological pathways induced in vivo as well as in vitro. Furthermore, overlapping genes and pathways were evaluated for possible involvement in the biological processes induced in vivo by datamining and consulting literature.
For all three interventions, only a limited number of identical genes and a few common biological processes/pathways were found to be affected by the respective interventions. However, several enterocyte-specific regulatory and secreted effector proteins that responded in vitro could be related to processes regulated in vivo, i.e. processes related to mineral absorption, (epithelial) cell adherence and tight junction formation for zinc, microtubule and cytoskeleton integrity for amoxicillin, and cell-cycle progression and mucus production for rye.
Short-term gene expression responses to dietary interventions as measured in the in vitro bioassay have a low predictability for long-term responses as measured in the intestinal mucosa in vivo. The short-term responses of a set regulatory and effector genes, as measured in this bioassay, however, provided additional insight into how specific processes in piglets and broilers may be modulated by "early" signalling molecules produced by enterocytes. The relevance of this set of regulatory/effector genes and cognate biological processes for zinc deficiency and supplementation, gluten allergy (rye), and amoxicillin administration in humans is discussed.</description><subject>Additives</subject><subject>Allergies</subject><subject>Amoxicillin</subject><subject>Animal Breeding & Genomics</subject><subject>Animal Breeding and Genetics</subject><subject>Animal Nutrition</subject><subject>Animal welfare</subject><subject>Antibiotics</subject><subject>Bioassays</subject><subject>Bioinformatics</subject><subject>Broilers</subject><subject>CVI Infectiebiologie</subject><subject>CVI Infection Biology</subject><subject>Cytokines</subject><subject>Cytoskeleton</subject><subject>Datasets</subject><subject>Diarrhea</subject><subject>Diervoeding</subject><subject>Diet</subject><subject>Enterocytes</subject><subject>Epithelial cells</subject><subject>Fokkerij & Genomica</subject><subject>Fokkerij en Genetica</subject><subject>Gene expression</subject><subject>Gene regulation</subject><subject>Gluten</subject><subject>Immune system</subject><subject>Infectiebiologie</subject><subject>Infection Biology</subject><subject>Intervention</subject><subject>Intestinal mucosa</subject><subject>Intestine</subject><subject>Laboratory animals</subject><subject>Livestock</subject><subject>LR - Animal Breeding & Genomics</subject><subject>LR - Animal Nutrition</subject><subject>LR - Diervoeding</subject><subject>LR - Genomica</subject><subject>Microbiota</subject><subject>Mucosa</subject><subject>Mucus</subject><subject>Nutrient deficiency</subject><subject>Pathogens</subject><subject>Penicillin</subject><subject>Pigs</subject><subject>Rye</subject><subject>Signal transduction</subject><subject>Small intestine</subject><subject>Transcription</subject><subject>WIAS</subject><subject>Zinc</subject><subject>Zinc oxides</subject><issn>1555-8932</issn><issn>1865-3499</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>PIMPY</sourceid><sourceid>DOA</sourceid><recordid>eNpdksFu1DAQhiMEoqXwAFxQJC5cArEdOzYHpKoqUKkSFzhbjjPZuErsxXZ2xVvxiEw2paJcYmvm_794Rn9RvCb1e0Kk-JAIpYJVNWmrmnNVkSfFOdZ5xRqlnuKdc15JxehZ8SKlu7rmirH6eXFGZUMYF_y8-H3to7PjDD6XYSidLw_uEMocjU82un12wZe9yaYcYpjL3kE28RfqMsQDmtZ2ygvWU3l0edwIOYbNtI_h4HrsuXm9Qo_95HZjXgmh3IGHMsJumcyJNIMdjXdpTisnj3D6UcrOm6mcFxuSeVk8G8yU4NX9eVH8-Hz9_eprdfvty83V5W1ledPkqhOkMYraRphOqk5yUffQSQqUKBB2YAMhgkoGSolOAFgJvWool6iTTA3sorjZuH0wd3of3Yxz62CcPhVC3GkTs7MTaGjbFvrOctb1DQjkSQNU2nqQIEVfI-vjxjoanNh5_GhvonXpBJxcF1f4cYnaT-uxX7qkOaUtbdD8aTNjcYbe4tKjmR696HHHu1HvwkFzpupGSAS8uwfE8HPBberZJQvTZDyEJWkipVSCt6JF6dv_pHdhibj8pClvOW05axmqyKayMaQUYXh4DKn1Gky9BVNjMPUaTE3Q8-bfKR4cf5PI_gDkUOZg</recordid><startdate>20170413</startdate><enddate>20170413</enddate><creator>Hulst, Marcel</creator><creator>Jansman, Alfons</creator><creator>Wijers, Ilonka</creator><creator>Hoekman, Arjan</creator><creator>Vastenhouw, Stéphanie</creator><creator>van Krimpen, Marinus</creator><creator>Smits, Mari</creator><creator>Schokker, Dirkjan</creator><general>BioMed Central</general><general>BMC</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7RV</scope><scope>7X7</scope><scope>7XB</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>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB0</scope><scope>LK8</scope><scope>M0S</scope><scope>M7P</scope><scope>NAPCQ</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>5PM</scope><scope>QVL</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-8020-8853</orcidid></search><sort><creationdate>20170413</creationdate><title>Enrichment of in vivo transcription data from dietary intervention studies with in vitro data provides improved insight into gene regulation mechanisms in the intestinal mucosa</title><author>Hulst, Marcel ; Jansman, Alfons ; Wijers, Ilonka ; Hoekman, Arjan ; Vastenhouw, Stéphanie ; van Krimpen, Marinus ; Smits, Mari ; Schokker, Dirkjan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c544t-b614a92c46ab89b8560deb82e219e6cf3f116283e996b6eec8ed9425860d839f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Additives</topic><topic>Allergies</topic><topic>Amoxicillin</topic><topic>Animal Breeding & Genomics</topic><topic>Animal Breeding and Genetics</topic><topic>Animal Nutrition</topic><topic>Animal welfare</topic><topic>Antibiotics</topic><topic>Bioassays</topic><topic>Bioinformatics</topic><topic>Broilers</topic><topic>CVI Infectiebiologie</topic><topic>CVI Infection Biology</topic><topic>Cytokines</topic><topic>Cytoskeleton</topic><topic>Datasets</topic><topic>Diarrhea</topic><topic>Diervoeding</topic><topic>Diet</topic><topic>Enterocytes</topic><topic>Epithelial cells</topic><topic>Fokkerij & Genomica</topic><topic>Fokkerij en Genetica</topic><topic>Gene expression</topic><topic>Gene regulation</topic><topic>Gluten</topic><topic>Immune system</topic><topic>Infectiebiologie</topic><topic>Infection Biology</topic><topic>Intervention</topic><topic>Intestinal mucosa</topic><topic>Intestine</topic><topic>Laboratory animals</topic><topic>Livestock</topic><topic>LR - Animal Breeding & Genomics</topic><topic>LR - Animal Nutrition</topic><topic>LR - Diervoeding</topic><topic>LR - Genomica</topic><topic>Microbiota</topic><topic>Mucosa</topic><topic>Mucus</topic><topic>Nutrient deficiency</topic><topic>Pathogens</topic><topic>Penicillin</topic><topic>Pigs</topic><topic>Rye</topic><topic>Signal transduction</topic><topic>Small intestine</topic><topic>Transcription</topic><topic>WIAS</topic><topic>Zinc</topic><topic>Zinc oxides</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hulst, Marcel</creatorcontrib><creatorcontrib>Jansman, Alfons</creatorcontrib><creatorcontrib>Wijers, Ilonka</creatorcontrib><creatorcontrib>Hoekman, Arjan</creatorcontrib><creatorcontrib>Vastenhouw, Stéphanie</creatorcontrib><creatorcontrib>van Krimpen, Marinus</creatorcontrib><creatorcontrib>Smits, Mari</creatorcontrib><creatorcontrib>Schokker, Dirkjan</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>ProQuest Nursing and Allied Health Journals</collection><collection>ProQuest Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</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)</collection><collection>ProQuest Central</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>AUTh Library subscriptions: ProQuest Central</collection><collection>ProQuest Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection (Proquest) (PQ_SDU_P3)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Biological Sciences</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>ProQuest Biological Science Journals</collection><collection>Nursing & Allied Health Premium</collection><collection>Publicly Available Content Database (Proquest) (PQ_SDU_P3)</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>NARCIS:Publications</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>Genes & nutrition</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hulst, Marcel</au><au>Jansman, Alfons</au><au>Wijers, Ilonka</au><au>Hoekman, Arjan</au><au>Vastenhouw, Stéphanie</au><au>van Krimpen, Marinus</au><au>Smits, Mari</au><au>Schokker, Dirkjan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Enrichment of in vivo transcription data from dietary intervention studies with in vitro data provides improved insight into gene regulation mechanisms in the intestinal mucosa</atitle><jtitle>Genes & nutrition</jtitle><addtitle>Genes Nutr</addtitle><date>2017-04-13</date><risdate>2017</risdate><volume>12</volume><issue>1</issue><spage>11</spage><epage>11</epage><pages>11-11</pages><artnum>11</artnum><issn>1555-8932</issn><eissn>1865-3499</eissn><abstract>Gene expression profiles of intestinal mucosa of chickens and pigs fed over long-term periods (days/weeks) with a diet rich in rye and a diet supplemented with zinc, respectively, or of chickens after a one-day amoxicillin treatment of chickens, were recorded recently. Such dietary interventions are frequently used to modulate animal performance or therapeutically for monogastric livestock. In this study, changes in gene expression induced by these three interventions in cultured "Intestinal Porcine Epithelial Cells" (IPEC-J2) recorded after a short-term period of 2 and 6 hours, were compared to the in vivo gene expression profiles in order to evaluate the capability of this in vitro bioassay in predicting in vivo responses.
Lists of response genes were analysed with bioinformatics programs to identify common biological pathways induced in vivo as well as in vitro. Furthermore, overlapping genes and pathways were evaluated for possible involvement in the biological processes induced in vivo by datamining and consulting literature.
For all three interventions, only a limited number of identical genes and a few common biological processes/pathways were found to be affected by the respective interventions. However, several enterocyte-specific regulatory and secreted effector proteins that responded in vitro could be related to processes regulated in vivo, i.e. processes related to mineral absorption, (epithelial) cell adherence and tight junction formation for zinc, microtubule and cytoskeleton integrity for amoxicillin, and cell-cycle progression and mucus production for rye.
Short-term gene expression responses to dietary interventions as measured in the in vitro bioassay have a low predictability for long-term responses as measured in the intestinal mucosa in vivo. The short-term responses of a set regulatory and effector genes, as measured in this bioassay, however, provided additional insight into how specific processes in piglets and broilers may be modulated by "early" signalling molecules produced by enterocytes. The relevance of this set of regulatory/effector genes and cognate biological processes for zinc deficiency and supplementation, gluten allergy (rye), and amoxicillin administration in humans is discussed.</abstract><cop>Germany</cop><pub>BioMed Central</pub><pmid>28413565</pmid><doi>10.1186/s12263-017-0559-1</doi><tpages>1</tpages><orcidid>https://orcid.org/0000-0001-8020-8853</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Additives Allergies Amoxicillin Animal Breeding & Genomics Animal Breeding and Genetics Animal Nutrition Animal welfare Antibiotics Bioassays Bioinformatics Broilers CVI Infectiebiologie CVI Infection Biology Cytokines Cytoskeleton Datasets Diarrhea Diervoeding Diet Enterocytes Epithelial cells Fokkerij & Genomica Fokkerij en Genetica Gene expression Gene regulation Gluten Immune system Infectiebiologie Infection Biology Intervention Intestinal mucosa Intestine Laboratory animals Livestock LR - Animal Breeding & Genomics LR - Animal Nutrition LR - Diervoeding LR - Genomica Microbiota Mucosa Mucus Nutrient deficiency Pathogens Penicillin Pigs Rye Signal transduction Small intestine Transcription WIAS Zinc Zinc oxides |
title | Enrichment of in vivo transcription data from dietary intervention studies with in vitro data provides improved insight into gene regulation mechanisms in the intestinal mucosa |
url | http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-02T20%3A07%3A09IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_doaj_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Enrichment%20of%20in%20vivo%20transcription%20data%20from%20dietary%20intervention%20studies%20with%20in%20vitro%20data%20provides%20improved%20insight%20into%20gene%20regulation%20mechanisms%20in%20the%20intestinal%20mucosa&rft.jtitle=Genes%20&%20nutrition&rft.au=Hulst,%20Marcel&rft.date=2017-04-13&rft.volume=12&rft.issue=1&rft.spage=11&rft.epage=11&rft.pages=11-11&rft.artnum=11&rft.issn=1555-8932&rft.eissn=1865-3499&rft_id=info:doi/10.1186/s12263-017-0559-1&rft_dat=%3Cproquest_doaj_%3E1888965767%3C/proquest_doaj_%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-c544t-b614a92c46ab89b8560deb82e219e6cf3f116283e996b6eec8ed9425860d839f3%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_pqid=2575275373&rft_id=info:pmid/28413565&rfr_iscdi=true |