{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T03:21:05Z","timestamp":1791429665527,"version":"4.3.3"},"reference-count":89,"publisher":"American Society for Cell Biology (ASCB)","issue":"2","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MBoC"],"published-print":{"date-parts":[[2019,1,15]]},"abstract":"<jats:p>Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used F\u00f6rster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca<jats:sup>2+<\/jats:sup>-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca<jats:sup>2+<\/jats:sup>and slows the kinetics of structural changes in troponin induced by Ca<jats:sup>2+<\/jats:sup>dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin\u2019s thin-filament pointed-end assembly but does not affect tropomodulin\u2019s assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin\u2019s affinity for F-actin and affects leiomodin\u2019s function. We suggest that the K15N mutation causes DCM by altering Ca<jats:sup>2+<\/jats:sup>-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin\u2019s function.<\/jats:p>","DOI":"10.1091\/mbc.e18-06-0406","type":"journal-article","created":{"date-parts":[[2018,11,21]],"date-time":"2018-11-21T17:08:24Z","timestamp":1542820104000},"page":"268-281","source":"Crossref","is-referenced-by-count":19,"title":["Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics"],"prefix":"10.1091","volume":"30","author":[{"given":"Thu","family":"Ly","sequence":"first","affiliation":[{"name":"Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher T.","family":"Pappas","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Medicine and Sarver Molecular Cardiovascular Research Program, University of Arizona, Tucson, AZ 85721"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dylan","family":"Johnson","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William","family":"Schlecht","sequence":"additional","affiliation":[{"name":"Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164"},{"name":"Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA 99164"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mert","family":"Colpan","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Medicine and Sarver Molecular Cardiovascular Research Program, University of Arizona, Tucson, AZ 85721"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vitold E.","family":"Galkin","sequence":"additional","affiliation":[{"name":"Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23507"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carol C.","family":"Gregorio","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Medicine and Sarver Molecular Cardiovascular Research Program, University of Arizona, Tucson, AZ 85721"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wen-Ji","family":"Dong","sequence":"additional","affiliation":[{"name":"Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164"},{"name":"Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA 99164"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alla S.","family":"Kostyukova","sequence":"additional","affiliation":[{"name":"Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1076","reference":[{"key":"B1","doi-asserted-by":"crossref","first-page":"915","DOI":"10.1093\/cvr\/cvx068","volume":"113","author":"Alves ML","year":"2017","journal-title":"Cardiovasc Res"},{"key":"B2","doi-asserted-by":"crossref","first-page":"10150","DOI":"10.1073\/pnas.1101221108","volume":"108","author":"Barua B","year":"2011","journal-title":"Proc Natl Acad Sci USA"},{"key":"B3","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/j.bbrc.2009.02.054","volume":"381","author":"Borovikov YS","year":"2009","journal-title":"Biochem Biophys Res Commun"},{"key":"B4","doi-asserted-by":"crossref","first-page":"2432","DOI":"10.1016\/S0021-9258(18)34942-1","volume":"257","author":"Chalovich JM","year":"1982","journal-title":"J Biol Chem"},{"key":"B5","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1126\/science.1155313","volume":"320","author":"Chereau D","year":"2008","journal-title":"Science"},{"key":"B6","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.abb.2017.07.006","volume":"630","author":"Colpan M","year":"2017","journal-title":"Arch Biochem Biophys"},{"key":"B7","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.abb.2016.04.006","volume":"600","author":"Colpan M","year":"2016","journal-title":"Arch Biochem Biophys"},{"key":"B8","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1016\/j.bbapap.2016.02.009","volume":"1864","author":"Colpan M","year":"2016","journal-title":"Biochim Biophys Acta"},{"key":"B9","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1006\/geno.2000.6501","volume":"73","author":"Conley CA","year":"2001","journal-title":"Genomics"},{"key":"B10","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1007\/BF00712034","volume":"4","author":"Cooper JA","year":"1983","journal-title":"J Muscle Res Cell Motil"},{"key":"B11","doi-asserted-by":"crossref","first-page":"6754","DOI":"10.1021\/bi9622276","volume":"36","author":"Dong WJ","year":"1997","journal-title":"Biochemistry"},{"key":"B12","doi-asserted-by":"crossref","first-page":"6745","DOI":"10.1021\/bi962226d","volume":"36","author":"Dong WJ","year":"1997","journal-title":"Biochemistry"},{"key":"B13","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1006\/jmbi.2001.5118","volume":"314","author":"Dong WJ","year":"2001","journal-title":"J Mol Biol"},{"key":"B14","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1161\/CIRCRESAHA.106.146670","volume":"101","author":"Du CK","year":"2007","journal-title":"Circ Res"},{"key":"B15","doi-asserted-by":"crossref","first-page":"1948","DOI":"10.1021\/bi00859a010","volume":"6","author":"Edelhoch H","year":"1967","journal-title":"Biochemistry"},{"key":"B16","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1016\/0076-6879(88)57093-3","volume":"157","author":"Fabiato A","year":"1988","journal-title":"Methods Enzymol"},{"key":"B17","volume-title":"Practical Handbook of Biochemistry and Molecular Biology","author":"Fasman GD","year":"1989"},{"key":"B18","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1136\/jmg.2010.083345","volume":"48","author":"Fokstuen S","year":"2011","journal-title":"J Med Genet"},{"key":"B19","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1038\/nrcardio.2011.159","volume":"9","author":"Frey N","year":"2011","journal-title":"Nat Rev Cardiol"},{"key":"B20","doi-asserted-by":"crossref","first-page":"1033","DOI":"10.1083\/jcb.200308164","volume":"163","author":"Fritz-Six KL","year":"2003","journal-title":"J Cell Biol"},{"key":"B21","doi-asserted-by":"crossref","first-page":"4908","DOI":"10.1021\/bi100349a","volume":"49","author":"Frye J","year":"2010","journal-title":"Biochemistry"},{"key":"B22","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/B978-0-12-374920-8.00416-1","volume-title":"Comprehensive Biophysics","author":"Geeves MA","year":"2012"},{"key":"B23","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1016\/j.jmb.2006.08.033","volume":"364","author":"Greenfield NJ","year":"2006","journal-title":"J Mol Biol"},{"key":"B24","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1083\/jcb.129.3.683","volume":"129","author":"Gregorio CC","year":"1995","journal-title":"J Cell Biol"},{"key":"B25","doi-asserted-by":"crossref","first-page":"2965","DOI":"10.1242\/jcs.172502","volume":"128","author":"Gunning PW","year":"2015","journal-title":"J Cell Sci"},{"key":"B26","doi-asserted-by":"crossref","first-page":"15210","DOI":"10.1016\/S0021-9258(17)36593-6","volume":"269","author":"Guo X","year":"1994","journal-title":"J Biol Chem"},{"key":"B27","doi-asserted-by":"crossref","first-page":"13627","DOI":"10.1016\/S0021-9258(19)76474-6","volume":"262","author":"Guth K","year":"1987","journal-title":"J Biol Chem"},{"issue":"3","key":"B28","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1242\/jcs.112.3.371","volume":"112","author":"Helfman DM","year":"1999","journal-title":"J Cell Sci"},{"key":"B29","doi-asserted-by":"crossref","first-page":"1125","DOI":"10.1152\/jappl.2001.90.3.1125","volume":"90","author":"Hernandez OM","year":"2001","journal-title":"J Appl Physiol"},{"key":"B30","volume-title":"GeneReviews((R))","author":"Hershberger RE","year":"1993"},{"key":"B31","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1161\/CIRCGENETICS.109.912345","volume":"3","author":"Hershberger RE","year":"2010","journal-title":"Circ Cardiovasc Genet"},{"key":"B32","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1161\/CIRCGENETICS.108.846733","volume":"2","author":"Hershberger RE","year":"2009","journal-title":"Circ Cardiovasc Genet"},{"key":"B33","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/j.abb.2010.06.001","volume":"500","author":"Jin JP","year":"2010","journal-title":"Arch Biochem Biophys"},{"key":"B34","doi-asserted-by":"crossref","first-page":"6470","DOI":"10.1046\/j.1432-1327.2000.01738.x","volume":"267","author":"Kostyukova A","year":"2000","journal-title":"Eur J Biochem"},{"key":"B35","doi-asserted-by":"crossref","first-page":"5066","DOI":"10.1074\/jbc.M311186200","volume":"279","author":"Kostyukova AS","year":"2004","journal-title":"J Biol Chem"},{"key":"B36","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1111\/j.1432-1033.1981.tb06167.x","volume":"114","author":"Kouyama T","year":"1981","journal-title":"Eur J Biochem"},{"key":"B37","doi-asserted-by":"crossref","first-page":"3208","DOI":"10.1016\/S0006-3495(99)77151-1","volume":"77","author":"Leinweber B","year":"1999","journal-title":"Biophys J"},{"key":"B38","doi-asserted-by":"crossref","first-page":"1005","DOI":"10.1016\/j.bpj.2010.12.3697","volume":"100","author":"Li XE","year":"2011","journal-title":"Biophys J"},{"key":"B39","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1186\/1472-6750-8-91","volume":"8","author":"Liu H","year":"2008","journal-title":"BMC Biotechnol"},{"key":"B40","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1002\/pro.3341","volume":"27","author":"Ly T","year":"2018","journal-title":"Protein Sci"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.e16-03-0200"},{"key":"B42","doi-asserted-by":"crossref","first-page":"2975","DOI":"10.1021\/bi00817a007","volume":"9","author":"Lymn RW","year":"1970","journal-title":"Biochemistry"},{"key":"B43","doi-asserted-by":"crossref","first-page":"4617","DOI":"10.1021\/bi00801a004","volume":"10","author":"Lymn RW","year":"1971","journal-title":"Biochemistry"},{"key":"B44","first-page":"1308","volume":"287","author":"Maron BJ","year":"2002","journal-title":"J Am Med Assoc"},{"key":"B45","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1161\/CIRCULATIONAHA.108.804617","volume":"119","author":"Maron BJ","year":"2009","journal-title":"Circulation"},{"key":"B46","doi-asserted-by":"crossref","first-page":"1807","DOI":"10.1161\/CIRCULATIONAHA.106.174287","volume":"113","author":"Maron BJ","year":"2006","journal-title":"Circulation"},{"key":"B47","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.bbrc.2011.01.112","volume":"406","author":"Mathur MC","year":"2011","journal-title":"Biochem Biophys Res Commun"},{"key":"B48","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1016\/0022-2836(74)90031-X","volume":"86","author":"McGhee JD","year":"1974","journal-title":"J Mol Biol"},{"key":"B49","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1016\/S0006-3495(93)81110-X","volume":"65","author":"McKillop DF","year":"1993","journal-title":"Biophys J"},{"key":"B50","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1016\/0022-2836(76)90313-2","volume":"103","author":"McLachlan AD","year":"1976","journal-title":"J Mol Biol"},{"key":"B51","doi-asserted-by":"crossref","first-page":"28498","DOI":"10.1074\/jbc.M412281200","volume":"280","author":"Mirza M","year":"2005","journal-title":"J Biol Chem"},{"key":"B52","doi-asserted-by":"crossref","first-page":"13487","DOI":"10.1074\/jbc.M701071200","volume":"282","author":"Mirza M","year":"2007","journal-title":"J Biol Chem"},{"key":"B53","doi-asserted-by":"crossref","first-page":"10461","DOI":"10.1016\/S0021-9258(17)34082-6","volume":"269","author":"Monteiro PB","year":"1994","journal-title":"J Biol Chem"},{"key":"B54","doi-asserted-by":"crossref","first-page":"7200","DOI":"10.1073\/pnas.0801950105","volume":"105","author":"Murakami K","year":"2008","journal-title":"Proc Natl Acad Sci USA"},{"key":"B55","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1093\/oxfordjournals.jbchem.a132549","volume":"86","author":"Ohtsuki I","year":"1979","journal-title":"J Biochem"},{"key":"B56","doi-asserted-by":"crossref","first-page":"1036","DOI":"10.1016\/j.jmb.2010.06.007","volume":"400","author":"Ouyang Y","year":"2010","journal-title":"J Mol Biol"},{"key":"B57","doi-asserted-by":"crossref","first-page":"2827","DOI":"10.1016\/S0006-3495(01)75924-3","volume":"81","author":"Palm T","year":"2001","journal-title":"Biophys J"},{"key":"B58","doi-asserted-by":"crossref","first-page":"13573","DOI":"10.1073\/pnas.1508273112","volume":"112","author":"Pappas CT","year":"2015","journal-title":"Proc Natl Acad Sci USA"},{"key":"B59","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0022-2836(73)90527-5","volume":"75","author":"Parry DA","year":"1973","journal-title":"J Mol Biol"},{"key":"B60","doi-asserted-by":"crossref","first-page":"15007","DOI":"10.1074\/jbc.M808615200","volume":"284","author":"Paul DM","year":"2009","journal-title":"J Biol Chem"},{"key":"B61","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/j.jsb.2017.01.004","volume":"197","author":"Paul DM","year":"2017","journal-title":"J Struct Biol"},{"key":"B62","doi-asserted-by":"crossref","first-page":"1032","DOI":"10.1139\/o77-154","volume":"55","author":"Pearlstone JR","year":"1977","journal-title":"Can J Biochem"},{"key":"B63","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1016\/j.jmb.2005.12.050","volume":"357","author":"Pirani A","year":"2006","journal-title":"J Mol Biol"},{"key":"B64","doi-asserted-by":"crossref","first-page":"761","DOI":"10.1016\/j.jmb.2004.12.013","volume":"346","author":"Pirani A","year":"2005","journal-title":"J Mol Biol"},{"key":"B65","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/j.jsb.2006.02.020","volume":"155","author":"Poole KJ","year":"2006","journal-title":"J Struct Biol"},{"key":"B66","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1007\/s10974-013-9358-5","volume":"34","author":"Redwood C","year":"2013","journal-title":"J Muscle Res Cell Motil"},{"key":"B67","first-page":"91","volume-title":"Troponin: Regulator of Muscle Contraction","author":"Rieck DC","year":"2014"},{"key":"B68","doi-asserted-by":"crossref","first-page":"6782","DOI":"10.1073\/pnas.1700868114","volume":"114","author":"Risi C","year":"2017","journal-title":"Proc Natl Acad Sci USA"},{"key":"B69","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1016\/j.jmb.2004.04.046","volume":"340","author":"Robinson JM","year":"2004","journal-title":"J Mol Biol"},{"key":"B70","doi-asserted-by":"crossref","first-page":"40710","DOI":"10.1074\/jbc.M203446200","volume":"277","author":"Robinson P","year":"2002","journal-title":"J Biol Chem"},{"key":"B71","doi-asserted-by":"crossref","DOI":"10.1161\/CIRCHEARTFAILURE.115.002637","volume":"10","author":"Rusconi P","year":"2017","journal-title":"Circ Heart Fail"},{"key":"B72","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.abb.2014.03.013","volume":"550","author":"Schlecht W","year":"2014","journal-title":"Arch Biochem Biophys"},{"key":"B73","doi-asserted-by":"crossref","first-page":"600","DOI":"10.1046\/j.1432-1327.1998.2520600.x","volume":"252","author":"She M","year":"1998","journal-title":"Eur J Biochem"},{"key":"B74","doi-asserted-by":"crossref","first-page":"14917","DOI":"10.1021\/bi701570b","volume":"46","author":"Singh A","year":"2007","journal-title":"Biochemistry"},{"key":"B75","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.e10-02-0109"},{"key":"B76","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1146\/annurev.biophys.050708.133751","volume":"39","author":"Sweeney HL","year":"2010","journal-title":"Annu Rev Biophys"},{"key":"B77","doi-asserted-by":"crossref","first-page":"869","DOI":"10.1517\/21678707.2015.1057498","volume":"3","author":"Sweet M","year":"2015","journal-title":"Expert Opin Orphan Drugs"},{"key":"B78","doi-asserted-by":"crossref","first-page":"e0186288","DOI":"10.1371\/journal.pone.0186288","volume":"12","author":"Szatmari D","year":"2017","journal-title":"PLoS One"},{"key":"B79","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1038\/nature01780","volume":"424","author":"Takeda S","year":"2003","journal-title":"Nature"},{"key":"B80","doi-asserted-by":"crossref","first-page":"3136","DOI":"10.1242\/jcs.071837","volume":"123","author":"Tsukada T","year":"2010","journal-title":"J Cell Sci"},{"key":"B81","doi-asserted-by":"crossref","first-page":"5038","DOI":"10.1073\/pnas.0408882102","volume":"102","author":"Vinogradova MV","year":"2005","journal-title":"Proc Natl Acad Sci USA"},{"key":"B82","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1023\/A:1005559613516","volume":"20","author":"Wang Y","year":"1999","journal-title":"J Muscle Res Cell Motil"},{"key":"B83","doi-asserted-by":"crossref","first-page":"1627","DOI":"10.1083\/jcb.127.6.1627","volume":"127","author":"Weber A","year":"1994","journal-title":"J Cell Biol"},{"key":"B84","doi-asserted-by":"crossref","first-page":"34637","DOI":"10.1074\/jbc.274.49.34637","volume":"274","author":"Weber A","year":"1999","journal-title":"J Biol Chem"},{"key":"B85","doi-asserted-by":"crossref","first-page":"E1844","DOI":"10.1073\/pnas.1516598113","volume":"113","author":"Wulf SF","year":"2016","journal-title":"Proc Natl Acad Sci USA"},{"key":"B86","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1006\/abbi.1994.1381","volume":"313","author":"Xing J","year":"1994","journal-title":"Arch Biochem Biophys"},{"key":"B87","doi-asserted-by":"crossref","first-page":"16432","DOI":"10.1074\/jbc.M808075200","volume":"284","author":"Xing J","year":"2009","journal-title":"J Biol Chem"},{"key":"B88","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1016\/j.bpj.2014.01.007","volume":"106","author":"Yang S","year":"2014","journal-title":"Biophys J"},{"key":"B89","doi-asserted-by":"crossref","first-page":"7661","DOI":"10.1074\/jbc.M111.281600","volume":"287","author":"Zhou Z","year":"2012","journal-title":"J Biol Chem"}],"container-title":["Molecular Biology of the Cell"],"original-title":[],"language":"en","deposited":{"date-parts":[[2022,9,6]],"date-time":"2022-09-06T17:31:56Z","timestamp":1662485516000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.molbiolcell.org\/doi\/10.1091\/mbc.E18-06-0406"}},"subtitle":[],"editor":[{"given":"Laurent","family":"Blanchoin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2019,1,15]]},"references-count":89,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2019,1,15]]}},"alternative-id":["10.1091\/mbc.E18-06-0406"],"URL":"https:\/\/doi.org\/10.1091\/mbc.e18-06-0406","relation":{},"ISSN":["1059-1524","1939-4586"],"issn-type":[{"value":"1059-1524","type":"print"},{"value":"1939-4586","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,1,15]]}}}