{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T16:46:30Z","timestamp":1781282790956,"version":"3.54.1"},"reference-count":51,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2024,6,17]],"date-time":"2024-06-17T00:00:00Z","timestamp":1718582400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["21K11594"],"award-info":[{"award-number":["21K11594"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Urakami Foundation for Food and Food Culture Promotion","award":["21K11594"],"award-info":[{"award-number":["21K11594"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Vitamin D hydroxylation in the liver\/kidney results in conversion to its physiologically active form of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 controls gene expression through the nuclear vitamin D receptor (VDR) mainly expressed in intestinal epithelial cells. Cytochrome P450 (CYP) 24A1 is a catabolic enzyme expressed in the kidneys. Interestingly, a recently identified mutation in another CYP enzyme, CYP3A4 (gain-of-function), caused type III vitamin D-dependent rickets. CYP3A are also expressed in the intestine, but their hydroxylation activities towards vitamin D substrates are unknown. We evaluated CYP3A or CYP24A1 activities on vitamin D action in cultured cells. In addition, we examined the expression level and regulation of CYP enzymes in intestines from mice. The expression of CYP3A or CYP24A1 significantly reduced 1,25(OH)2D3-VDRE activity. Moreover, in mice, Cyp24a1 mRNA was significantly induced by 1,25(OH)2D3 in the intestine, but a mature form (approximately 55 kDa protein) was also expressed in mitochondria and induced by 1,25(OH)2D3, and this mitochondrial enzyme appears to hydroxylate 25OHD3 to 24,25(OH)2D3. Thus, CYP3A or CYP24A1 could locally attenuate 25OHD3 or 1,25(OH)2D3 action, and we suggest the small intestine is both a vitamin D target tissue, as well as a newly recognized vitamin D-metabolizing tissue.<\/jats:p>","DOI":"10.3390\/biom14060717","type":"journal-article","created":{"date-parts":[[2024,6,17]],"date-time":"2024-06-17T11:14:16Z","timestamp":1718622856000},"page":"717","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["The Role of Intestinal Cytochrome P450s in Vitamin D Metabolism"],"prefix":"10.3390","volume":"14","author":[{"given":"Minori","family":"Uga","sequence":"first","affiliation":[{"name":"Department of Applied Nutrition, Tokushima University Graduate School of Biomedical Sciences, Tokushima 770-8503, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2710-9188","authenticated-orcid":false,"given":"Ichiro","family":"Kaneko","sequence":"additional","affiliation":[{"name":"Research Institute for Food and Nutritional Sciences, School of Human Science and Environment, University of Hyogo, Hyogo 670-0092, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuji","family":"Shiozaki","sequence":"additional","affiliation":[{"name":"Department of Applied Nutrition, Tokushima University Graduate School of Biomedical Sciences, Tokushima 770-8503, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megumi","family":"Koike","sequence":"additional","affiliation":[{"name":"Department of Applied Nutrition, Tokushima University Graduate School of Biomedical Sciences, Tokushima 770-8503, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naoko","family":"Tsugawa","sequence":"additional","affiliation":[{"name":"Faculty of Nutrition, Kobe Gakuin University, Hyogo 651-2180, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4950-9161","authenticated-orcid":false,"given":"Peter W.","family":"Jurutka","sequence":"additional","affiliation":[{"name":"Mathematical and Natural Sciences, Arizona State University, Glendale, AZ 85306, USA"},{"name":"College of Medicine, The University of Arizona, Phoenix, AZ 85004, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ken-Ichi","family":"Miyamoto","sequence":"additional","affiliation":[{"name":"Department of Applied Nutrition, Tokushima University Graduate School of Biomedical Sciences, Tokushima 770-8503, Japan"},{"name":"Graduate School of Agriculture, Ryukoku University, Shiga 520-2194, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0905-246X","authenticated-orcid":false,"given":"Hiroko","family":"Segawa","sequence":"additional","affiliation":[{"name":"Department of Applied Nutrition, Tokushima University Graduate School of Biomedical Sciences, Tokushima 770-8503, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,6,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1080\/003655202753416876","article-title":"Vitamin D status, parathyroid hormone and bone mineral density in patients with inflammatory bowel disease","volume":"37","author":"Jahnsen","year":"2002","journal-title":"Scand. J. Gastroenterol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Ratajczak, A.E., Rychter, A.M., Zawada, A., Dobrowolska, A., and Krela-Ka\u017amierczak, I. (2020). Nutrients in the Prevention of Osteoporosis in Patients with Inflammatory Bowel Diseases. Nutrients, 12.","DOI":"10.3390\/nu12061702"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Grozi\u0107, A., Coker, K., Dussik, C.M., Sabir, M.S., Sabir, Z., Bradley, A., Zhang, L., Park, J., Yale, S., and Kaneko, I. (2022). Identification of putative transcriptomic biomarkers in irritable bowel syndrome (IBS): Differential gene expression and regulation of TPH1 and SERT by vitamin D. PLoS ONE, 17.","DOI":"10.1371\/journal.pone.0275683"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Aspelund, T., Grubler, M.R., Smith, A.V., Gudmundsson, E.F., Keppel, M., Cotch, M.F., Harris, T.B., Jorde, R., Grimnes, G., and Joakimsen, R. (2019). Effect of Genetically Low 25-Hydroxyvitamin D on Mortality Risk: Mendelian Randomization Analysis in 3 Large European Cohorts. Nutrients, 11.","DOI":"10.3390\/nu11010074"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"782","DOI":"10.3945\/ajcn.112.047712","article-title":"Strong associations of 25-hydroxyvitamin D concentrations with all-cause, cardiovascular, cancer, and respiratory disease mortality in a large cohort study","volume":"97","author":"Schottker","year":"2013","journal-title":"Am. J. Clin. Nutr."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1210\/edrv.22.4.0437","article-title":"Vitamin D deficiency and secondary hyperparathyroidism in the elderly: Consequences for bone loss and fractures and therapeutic implications","volume":"22","author":"Lips","year":"2001","journal-title":"Endocr. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"17541","DOI":"10.1074\/jbc.M117.806901","article-title":"A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D(3) activation","volume":"292","author":"Meyer","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"14467","DOI":"10.1074\/jbc.RA119.010173","article-title":"A chromatin-based mechanism controls differential regulation of the cytochrome P450 gene Cyp24a1 in renal and non-renal tissues","volume":"294","author":"Meyer","year":"2019","journal-title":"J. Biol. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1007\/s00223-012-9619-0","article-title":"Molecular mechanisms of vitamin D action","volume":"92","author":"Haussler","year":"2013","journal-title":"Calcif. Tissue Int."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1111\/j.1582-4934.2007.00209.x","article-title":"Molecular mechanism of regulation of the calcium-binding protein calbindin-D9k, and its physiological role(s) in mammals: A review of current research","volume":"12","author":"Choi","year":"2008","journal-title":"J. Cell. Mol. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1007\/s00210-005-1021-2","article-title":"The epithelial calcium channels TRPV5 and TRPV6: Regulation and implications for disease","volume":"371","author":"Hoenderop","year":"2005","journal-title":"Naunyn Schmiedebergs Arch. Pharmacol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1194\/jlr.R031534","article-title":"Cytochrome P450-mediated metabolism of vitamin D","volume":"55","author":"Jones","year":"2014","journal-title":"J. Lipid Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1","DOI":"10.4062\/biomolther.2021.102","article-title":"Inhibition of Cytochrome P450 Enzymes by Drugs-Molecular Basis and Practical Applications","volume":"30","author":"Guengerich","year":"2022","journal-title":"Biomol. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.pharmthera.2012.12.007","article-title":"Cytochrome P450 enzymes in drug metabolism: Regulation of gene expression, enzyme activities, and impact of genetic variation","volume":"138","author":"Zanger","year":"2013","journal-title":"Pharmacol. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4543","DOI":"10.1073\/pnas.90.10.4543","article-title":"Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA","volume":"90","author":"Chen","year":"1993","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0167-4781(95)00060-T","article-title":"Cloning of the human 1 alpha,25-dihydroxyvitamin D-3 24-hydroxylase gene promoter and identification of two vitamin D-responsive elements","volume":"1263","author":"Chen","year":"1995","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"825","DOI":"10.1210\/en.2004-1116","article-title":"Altered pharmacokinetics of 1alpha,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 in the blood and tissues of the 25-hydroxyvitamin D-24-hydroxylase (Cyp24a1) null mouse","volume":"146","author":"Masuda","year":"2005","journal-title":"Endocrinology"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3498","DOI":"10.1016\/S0021-9258(19)50758-X","article-title":"Immunopurified 25-hydroxyvitamin D 1 alpha-hydroxylase and 1,25-dihydroxyvitamin D 24-hydroxylase are closely related but distinct enzymes","volume":"267","author":"Ettinger","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.abb.2012.04.001","article-title":"Where is the vitamin D receptor?","volume":"523","author":"Wang","year":"2012","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"13324","DOI":"10.1073\/pnas.231474698","article-title":"Duodenal calcium absorption in vitamin D receptor-knockout mice: Functional and molecular aspects","volume":"98","author":"Dewerchin","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1146\/annurev.pharmtox.39.1.1","article-title":"Cytochrome P-450 3A4: Regulation and role in drug metabolism","volume":"39","author":"Guengerich","year":"1999","journal-title":"Annu. Rev. Pharmacol. Toxicol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1124\/mol.105.017392","article-title":"Intestinal and hepatic CYP3A4 catalyze hydroxylation of 1alpha,25-dihydroxyvitamin D(3): Implications for drug-induced osteomalacia","volume":"69","author":"Xu","year":"2006","journal-title":"Mol. Pharmacol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1124\/mol.111.076356","article-title":"An inducible cytochrome P450 3A4-dependent vitamin D catabolic pathway","volume":"81","author":"Wang","year":"2012","journal-title":"Mol. Pharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Yasuda, K., Nishikawa, M., Okamoto, K., Horibe, K., Mano, H., Yamaguchi, M., Okon, R., Nakagawa, K., Tsugawa, N., and Okano, T. (2021). Elucidation of metabolic pathways of 25-hydroxyvitamin D3 mediated by CYP24A1 and CYP3A using Cyp24a1 knockout rats generated by CRISPR\/Cas9 system. J. Biol. Chem., 296.","DOI":"10.1016\/j.jbc.2021.100668"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1074\/mcp.M113.035600","article-title":"Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics","volume":"13","author":"Fagerberg","year":"2014","journal-title":"Mol. Cell. Proteom."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1124\/jpet.103.054841","article-title":"Developmental expression of the major human hepatic CYP3A enzymes","volume":"307","author":"Stevens","year":"2003","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1913","DOI":"10.1172\/JCI98680","article-title":"CYP3A4 mutation causes vitamin D-dependent rickets type 3","volume":"128","author":"Roizen","year":"2018","journal-title":"J. Clin. Investig."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1007\/s00223-022-01051-2","article-title":"Vitamin D-Dependent Rickets Type 3: A Case Report and Systematic Review","volume":"112","author":"Mantoanelli","year":"2023","journal-title":"Calcif. Tissue Int."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1213361","DOI":"10.3389\/fendo.2023.1213361","article-title":"Molecular insights into mineralotropic hormone inter-regulation","volume":"14","author":"Pike","year":"2023","journal-title":"Front. Endocrinol."},{"key":"ref_30","first-page":"e10433","article-title":"Genomic Mechanisms Governing Mineral Homeostasis and the Regulation and Maintenance of Vitamin D Metabolism","volume":"5","author":"Pike","year":"2021","journal-title":"J. Bone Miner. Res. Plus"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2403","DOI":"10.1124\/dmd.111.041541","article-title":"Proluciferin acetals as bioluminogenic substrates for cytochrome P450 activity and probes for CYP3A inhibition","volume":"39","author":"Meisenheimer","year":"2011","journal-title":"Drug Metab. Dispos."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1803","DOI":"10.1172\/JCI45890","article-title":"Normocalcemia is maintained in mice under conditions of calcium malabsorption by vitamin D-induced inhibition of bone mineralization","volume":"122","author":"Lieben","year":"2012","journal-title":"J. Clin. Investig."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1008","DOI":"10.1210\/en.2012-1542","article-title":"Vitamin D receptor in osteoblasts is a negative regulator of bone mass control","volume":"154","author":"Yamamoto","year":"2013","journal-title":"Endocrinology"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"22344","DOI":"10.1074\/jbc.M703475200","article-title":"Characterizing early events associated with the activation of target genes by 1,25-dihydroxyvitamin D3 in mouse kidney and intestine in vivo","volume":"282","author":"Meyer","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3001","DOI":"10.1021\/ac048249c","article-title":"Determination of 25-hydroxyvitamin D in human plasma using high-performance liquid chromatography--tandem mass spectrometry","volume":"77","author":"Tsugawa","year":"2005","journal-title":"Anal. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1359\/JBMR.0301257","article-title":"CYP3A4 is a human microsomal vitamin D 25-hydroxylase","volume":"19","author":"Gupta","year":"2004","journal-title":"J. Bone Miner. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1210","DOI":"10.1210\/jc.2004-0966","article-title":"CYP3A4 is a vitamin D-24- and 25-hydroxylase: Analysis of structure function by site-directed mutagenesis","volume":"90","author":"Gupta","year":"2005","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1002\/jbmr.1839","article-title":"Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: Implications for drug-induced osteomalacia","volume":"28","author":"Wang","year":"2013","journal-title":"J. Bone Miner. Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1016\/S0006-291X(02)02742-0","article-title":"Liganded VDR induces CYP3A4 in small intestinal and colon cancer cells via DR3 and ER6 vitamin D responsive elements","volume":"299","author":"Thompson","year":"2002","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"18199","DOI":"10.1074\/jbc.M115.665794","article-title":"1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium-regulating Components","volume":"290","author":"Lee","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1313","DOI":"10.1126\/science.1070477","article-title":"Vitamin D Receptor As an Intestinal Bile Acid Sensor","volume":"296","author":"Makishima","year":"2002","journal-title":"Science"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1111\/j.1432-1033.1994.00335.x","article-title":"Further oxidation of hydroxycalcidiol by calcidiol 24-hydroxylase. A study with the mature enzyme expressed in Escherichia coli","volume":"224","author":"Sakaki","year":"1994","journal-title":"Eur. J. Biochem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1080\/15216540152845894","article-title":"Protein import into mitochondria","volume":"52","author":"Paschen","year":"2001","journal-title":"IUBMB Life"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"20604","DOI":"10.1074\/jbc.M414522200","article-title":"Alternative splicing of vitamin D-24-hydroxylase: A novel mechanism for the regulation of extrarenal 1,25-dihydroxyvitamin D synthesis","volume":"280","author":"Ren","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"49","DOI":"10.3164\/jcbn.19-79","article-title":"Association of increased renal Cyp24a1 gene expression with low plasma 1,25-dihydroxyvitamin D levels in rats with streptozotocin-induced diabetes","volume":"66","author":"Tajiri","year":"2020","journal-title":"J. Clin. Biochem. Nutr."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1016","DOI":"10.1172\/JCI3703","article-title":"The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions","volume":"102","author":"Lehmann","year":"1998","journal-title":"J. Clin. Investig."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"15122","DOI":"10.1074\/jbc.M001215200","article-title":"Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands","volume":"275","author":"Moore","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1029","DOI":"10.1172\/JCI113156","article-title":"Identification of glucocorticoid-inducible cytochromes P-450 in the intestinal mucosa of rats and man","volume":"80","author":"Watkins","year":"1987","journal-title":"J. Clin. Investig."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1089","DOI":"10.1007\/s11095-006-0277-7","article-title":"CYP induction-mediated drug interactions: In vitro assessment and clinical implications","volume":"23","author":"Lin","year":"2006","journal-title":"Pharm. Res."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1440","DOI":"10.1210\/jc.2016-4048","article-title":"CYP3A4 Induction by Rifampin: An Alternative Pathway for Vitamin D Inactivation in Patients with CYP24A1 Mutations","volume":"102","author":"Hawkes","year":"2017","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Su, Y.W., Lee, A.M.C., Xu, X., Hua, B., Tapp, H., Wen, X.S., and Xian, C.J. (2023). Methotrexate Chemotherapy Causes Growth Impairments, Vitamin D Deficiency, Bone Loss, and Altered Intestinal Metabolism-Effects of Calcitriol Supplementation. Cancers, 15.","DOI":"10.3390\/cancers15174367"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/14\/6\/717\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:00:05Z","timestamp":1760108405000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/14\/6\/717"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,6,17]]},"references-count":51,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["biom14060717"],"URL":"https:\/\/doi.org\/10.3390\/biom14060717","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,6,17]]}}}