Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK
Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids, including DNA, mRNA, and small interfering RNA. Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessit...
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| Veröffentlicht in: | Gene therapy 2013-02, Vol.20 (2), p.136-142 |
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| creator | Anderson, B R Karikó, K Weissman, D |
| description | Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids, including DNA, mRNA, and small interfering RNA. Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined the factors that are important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of the delivered nucleic acid. We studied the involvement of three ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies. |
| doi_str_mv | 10.1038/gt.2012.5 |
| format | Article |
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Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined the factors that are important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of the delivered nucleic acid. We studied the involvement of three ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.</description><identifier>ISSN: 0969-7128</identifier><identifier>EISSN: 1476-5462</identifier><identifier>DOI: 10.1038/gt.2012.5</identifier><identifier>PMID: 22301437</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/154/51/391 ; 631/1647/2300 ; 631/250/516 ; Animals ; Biomedical and Life Sciences ; Biomedicine ; Biotechnology ; Cell Biology ; Cell Line ; Deoxyribonucleic acid ; DNA ; DNA binding proteins ; Drug development ; eIF-2 kinase ; eIF-2 Kinase - genetics ; eIF-2 Kinase - metabolism ; Endoplasmic reticulum ; Eukaryotic Initiation Factor-2 - genetics ; Eukaryotic Initiation Factor-2 - metabolism ; Gene Expression ; Gene Therapy ; Human Genetics ; Humans ; Initiation factor eIF-2 ; Initiation factor eIF-2α ; Kinases ; Mammalian cells ; Messenger RNA ; Mice ; mRNA ; Nanotechnology ; nucleic acids ; original-article ; Phosphorylation ; Physiological aspects ; Protein kinase ; Protein-Serine-Threonine Kinases - genetics ; Protein-Serine-Threonine Kinases - metabolism ; siRNA ; Transfection ; Transfection - methods ; Translation</subject><ispartof>Gene therapy, 2013-02, Vol.20 (2), p.136-142</ispartof><rights>Macmillan Publishers Limited 2013</rights><rights>COPYRIGHT 2013 Nature Publishing Group</rights><rights>Macmillan Publishers Limited 2013.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c603t-e643e91320fb3b847f5d4a67f8b09c74915382d74fdb43d03ab9e2f997a949343</citedby><cites>FETCH-LOGICAL-c603t-e643e91320fb3b847f5d4a67f8b09c74915382d74fdb43d03ab9e2f997a949343</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22301437$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Anderson, B R</creatorcontrib><creatorcontrib>Karikó, K</creatorcontrib><creatorcontrib>Weissman, D</creatorcontrib><title>Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK</title><title>Gene therapy</title><addtitle>Gene Ther</addtitle><addtitle>Gene Ther</addtitle><description>Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids, including DNA, mRNA, and small interfering RNA. Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined the factors that are important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of the delivered nucleic acid. We studied the involvement of three ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.</description><subject>631/154/51/391</subject><subject>631/1647/2300</subject><subject>631/250/516</subject><subject>Animals</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>Biotechnology</subject><subject>Cell Biology</subject><subject>Cell Line</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA binding proteins</subject><subject>Drug development</subject><subject>eIF-2 kinase</subject><subject>eIF-2 Kinase - genetics</subject><subject>eIF-2 Kinase - metabolism</subject><subject>Endoplasmic reticulum</subject><subject>Eukaryotic Initiation Factor-2 - genetics</subject><subject>Eukaryotic Initiation Factor-2 - metabolism</subject><subject>Gene Expression</subject><subject>Gene Therapy</subject><subject>Human Genetics</subject><subject>Humans</subject><subject>Initiation factor eIF-2</subject><subject>Initiation factor eIF-2α</subject><subject>Kinases</subject><subject>Mammalian cells</subject><subject>Messenger RNA</subject><subject>Mice</subject><subject>mRNA</subject><subject>Nanotechnology</subject><subject>nucleic acids</subject><subject>original-article</subject><subject>Phosphorylation</subject><subject>Physiological aspects</subject><subject>Protein kinase</subject><subject>Protein-Serine-Threonine Kinases - genetics</subject><subject>Protein-Serine-Threonine Kinases - metabolism</subject><subject>siRNA</subject><subject>Transfection</subject><subject>Transfection - methods</subject><subject>Translation</subject><issn>0969-7128</issn><issn>1476-5462</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNqNkt9qFDEUxoModl298AVkQBAVds2_SSY3QlnadbFUqXodMjPJbMpssk0y4j6WL-IzmbG17movJIRAzu98nPOdA8BTBOcIkupNl-YYIjwv74EJopzNSsrwfTCBgokZR7g6Ao9ivIQQUl7hh-AIYwIRJXwCludD02tvdJOsd4V17dDoWKSgXLTapUKvTvGP78V27WO-YderX2C9K5aLc1wo1xYfTy7ePwYPjOqjfnLzTsGX05PPi3ezsw_L1eL4bNYwSNJMM0q0QARDU5O6otyULVWMm6qGouFUoJJUuOXUtDUlLSSqFhobIbgSVBBKpuDtte52qDe6bXKJQfVyG-xGhZ30ysrDiLNr2fmvkhBaYlFmgZc3AsFfDTomubGx0X2vnPZDlAgLxHKBJfwPtBqNxnRUff4XeumH4LITEjNKGasg36M61WtpnfG5xGYUlcfZklIgmpucgvkdVD6t3tjGO21s_j9IeHWQkJmkv6VODTHK1aeLQ_bFHrvWqk_r6PthHGq8U7QJPsagza3HCMpx62SX5Lh1cuzr2f5Qbsnfa5aB19dAzCHX6fDHnn_VfgImF9uh</recordid><startdate>20130201</startdate><enddate>20130201</enddate><creator>Anderson, B R</creator><creator>Karikó, K</creator><creator>Weissman, D</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</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>ISR</scope><scope>3V.</scope><scope>7QP</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</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>AFKRA</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>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</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>7X8</scope><scope>7QO</scope><scope>5PM</scope></search><sort><creationdate>20130201</creationdate><title>Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK</title><author>Anderson, B R ; Karikó, K ; Weissman, D</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c603t-e643e91320fb3b847f5d4a67f8b09c74915382d74fdb43d03ab9e2f997a949343</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>631/154/51/391</topic><topic>631/1647/2300</topic><topic>631/250/516</topic><topic>Animals</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedicine</topic><topic>Biotechnology</topic><topic>Cell Biology</topic><topic>Cell Line</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA binding proteins</topic><topic>Drug development</topic><topic>eIF-2 kinase</topic><topic>eIF-2 Kinase - genetics</topic><topic>eIF-2 Kinase - metabolism</topic><topic>Endoplasmic reticulum</topic><topic>Eukaryotic Initiation Factor-2 - genetics</topic><topic>Eukaryotic Initiation Factor-2 - metabolism</topic><topic>Gene Expression</topic><topic>Gene Therapy</topic><topic>Human Genetics</topic><topic>Humans</topic><topic>Initiation factor eIF-2</topic><topic>Initiation factor eIF-2α</topic><topic>Kinases</topic><topic>Mammalian cells</topic><topic>Messenger RNA</topic><topic>Mice</topic><topic>mRNA</topic><topic>Nanotechnology</topic><topic>nucleic acids</topic><topic>original-article</topic><topic>Phosphorylation</topic><topic>Physiological aspects</topic><topic>Protein kinase</topic><topic>Protein-Serine-Threonine Kinases - genetics</topic><topic>Protein-Serine-Threonine Kinases - metabolism</topic><topic>siRNA</topic><topic>Transfection</topic><topic>Transfection - methods</topic><topic>Translation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Anderson, B R</creatorcontrib><creatorcontrib>Karikó, K</creatorcontrib><creatorcontrib>Weissman, D</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: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</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 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 Central UK/Ireland</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>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>Research Library</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>MEDLINE - Academic</collection><collection>Biotechnology Research Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Gene therapy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Anderson, B R</au><au>Karikó, K</au><au>Weissman, D</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK</atitle><jtitle>Gene therapy</jtitle><stitle>Gene Ther</stitle><addtitle>Gene Ther</addtitle><date>2013-02-01</date><risdate>2013</risdate><volume>20</volume><issue>2</issue><spage>136</spage><epage>142</epage><pages>136-142</pages><issn>0969-7128</issn><eissn>1476-5462</eissn><abstract>Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids, including DNA, mRNA, and small interfering RNA. Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined the factors that are important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of the delivered nucleic acid. We studied the involvement of three ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>22301437</pmid><doi>10.1038/gt.2012.5</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 631/154/51/391 631/1647/2300 631/250/516 Animals Biomedical and Life Sciences Biomedicine Biotechnology Cell Biology Cell Line Deoxyribonucleic acid DNA DNA binding proteins Drug development eIF-2 kinase eIF-2 Kinase - genetics eIF-2 Kinase - metabolism Endoplasmic reticulum Eukaryotic Initiation Factor-2 - genetics Eukaryotic Initiation Factor-2 - metabolism Gene Expression Gene Therapy Human Genetics Humans Initiation factor eIF-2 Initiation factor eIF-2α Kinases Mammalian cells Messenger RNA Mice mRNA Nanotechnology nucleic acids original-article Phosphorylation Physiological aspects Protein kinase Protein-Serine-Threonine Kinases - genetics Protein-Serine-Threonine Kinases - metabolism siRNA Transfection Transfection - methods Translation |
| title | Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK |
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