{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T13:22:20Z","timestamp":1774876940180,"version":"3.50.1"},"reference-count":59,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2018,3,19]],"date-time":"2018-03-19T00:00:00Z","timestamp":1521417600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Osteoporosis is characterized by a reduction of the bone mineral density (BMD) and microarchitectural deterioration of the bone, which lead to bone fragility and susceptibility to fracture. Astaxanthin (AST) has a variety of biological activities, such as a protective effect against asthma or neuroinflammation, antioxidant effect, and decrease of the osteoclast number in the right mandibles in the periodontitis model. Although treatment with AST is known to have an effect on inflammation, no studies on the effect of AST exposure on bone loss have been performed. Thus, in the present study, we examined the antiosteoporotic effect of AST on bone mass in ovariectomized (OVX) mice and its possible mechanism of action. The administration of AST (5, 10 mg\/kg) for 6 weeks suppressed the enhancement of serum calcium, inorganic phosphorus, alkaline phosphatase, total cholesterol, and tartrate-resistant acid phosphatase (TRAP) activity. The bone mineral density (BMD) and bone microarchitecture of the trabecular bone in the tibia and femur were recovered by AST exposure. Moreover, in the in vitro experiment, we demonstrated that AST inhibits osteoclast formation through the expression of the nuclear factor of activated T cells (NFAT) c1, dendritic cell-specific transmembrane protein (DC-STAMP), TRAP, and cathepsin K without any cytotoxic effects on bone marrow-derived macrophages (BMMs). Therefore, we suggest that AST may have therapeutic potential for the treatment of postmenopausal osteoporosis.<\/jats:p>","DOI":"10.3390\/ijms19030912","type":"journal-article","created":{"date-parts":[[2018,3,20]],"date-time":"2018-03-20T06:57:11Z","timestamp":1521529031000},"page":"912","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":48,"title":["Suppression Effect of Astaxanthin on Osteoclast Formation In Vitro and Bone Loss In Vivo"],"prefix":"10.3390","volume":"19","author":[{"given":"Yun-Ho","family":"Hwang","sequence":"first","affiliation":[{"name":"Department of Pharmacy, Sunchon National University, 255 Jungangno, Suncheon 540-950, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kwang-Jin","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Sunchon National University, 255 Jungangno, Suncheon 540-950, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Su-Jin","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Sunchon National University, 255 Jungangno, Suncheon 540-950, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Seul-Ki","family":"Mun","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Sunchon National University, 255 Jungangno, Suncheon 540-950, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Seong-Gyeol","family":"Hong","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Sunchon National University, 255 Jungangno, Suncheon 540-950, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Young-Jin","family":"Son","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Sunchon National University, 255 Jungangno, Suncheon 540-950, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sung-Tae","family":"Yee","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Sunchon National University, 255 Jungangno, Suncheon 540-950, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2018,3,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1007\/BF01622200","article-title":"Assessment of fracture risk and its application to screening for postmenopausal osteoporosis: Synopsis of a WHO report","volume":"4","author":"Kanis","year":"1994","journal-title":"Osteoporos. Int."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Weycker (2016). D.; Li, X.; Barron, R.; Bornheimer, R.; Chandler, D. Hospitalizations for osteoporosis-related fractures: Economic costs and clinical outcomes. Bone Rep., 5, 186\u2013191.","DOI":"10.1016\/j.bonr.2016.07.005"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1590\/S0004-27302006000400016","article-title":"Estrogen therapy for postmenopausal osteoporosis","volume":"50","author":"Fitzpatrick","year":"2006","journal-title":"Arq. Bras. Endocrinol. Metab."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"825","DOI":"10.7150\/ijms.5180","article-title":"Immune regulation of osteoclast function in postmenopausal osteoporosis: A critical interdisciplinary perspective","volume":"9","author":"Zhao","year":"2012","journal-title":"Int. J. Med. Sci."},{"key":"ref_5","unstructured":"Torres, A.Y.P., Flores, M.V., Orozco, L., and Cruz, R.V. (2013). Molecular aspects of bone remodeling. Top. Osteoporos."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1186","DOI":"10.1172\/JCI28550","article-title":"Estrogen deficiency and bone loss: An inflammatory tale","volume":"116","author":"Weitzmann","year":"2006","journal-title":"J. Clin. Investig."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"889","DOI":"10.1016\/S1534-5807(02)00369-6","article-title":"Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts","volume":"3","author":"Takayanagi","year":"2002","journal-title":"Dev. Cell"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1196\/annals.1402.071","article-title":"The role of NFAT in osteoclast formation","volume":"1116","author":"Takayanagi","year":"2007","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1016\/j.bone.2015.07.023","article-title":"Decursin inhibits osteoclastogenesis by downregulating NFATc1 and blocking fusion of pre-osteoclasts","volume":"81","author":"Kim","year":"2015","journal-title":"Bone"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1001\/jama.288.3.321","article-title":"Writing Group for the Women\u2019s Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: Principal results From the Women\u2019s Health Initiative randomized controlled trial","volume":"288","author":"Rossouw","year":"2002","journal-title":"JAMA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"982","DOI":"10.1016\/0002-9378(95)90247-3","article-title":"Benefits and risks of estrogen replacement therapy","volume":"173","author":"Lobo","year":"1995","journal-title":"Am. J. Obstet. Gynecol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.lfs.2016.01.024","article-title":"Natural products for treatment of osteoporosis: The effects and mechanisms on promoting osteoblast-mediated bone formation","volume":"147","author":"An","year":"2016","journal-title":"Life Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1769","DOI":"10.3892\/etm.2017.4695","article-title":"Combination of alendronate and genistein synergistically suppresses osteoclastic differentiation of RAW267.4 cells in vitro","volume":"14","author":"Yamaguchi","year":"2017","journal-title":"Exp. Ther. Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1893","DOI":"10.1210\/endo.140.4.6663","article-title":"Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen deficiency","volume":"140","author":"Ishimi","year":"1999","journal-title":"Endocrinology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1123","DOI":"10.1007\/s00198-013-2512-5","article-title":"Genistein effects on quality of life and depression symptoms in osteopenic postmenopausal women: A 2-year randomized, double-blind, controlled study","volume":"25","author":"Atteritano","year":"2014","journal-title":"Osteoporos. Int."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Arcoraci, V., Atteritano, M., Squadrito, F., D\u2019Anna, R., Marini, H., Santoro, D., Minutoli, L., Messina, S., Altavilla, D., and Bitto, A. (2017). Antiosteoporotic Activity of Genistein Aglycone in Postmenopausal Women: Evidence from a Post-Hoc Analysis of a Multicenter Randomized Controlled Trial. Nutrients, 9.","DOI":"10.3390\/nu9020179"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1016\/S0167-7799(00)01433-5","article-title":"Commercial potential for Haematococcus microalgae as a natural source of astaxanthin","volume":"18","author":"Lorenz","year":"2000","journal-title":"Trends Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1016\/S0167-7799(03)00078-7","article-title":"Haematococcus astaxanthin: Applications for human health and nutrition","volume":"21","author":"Guerin","year":"2003","journal-title":"Trends Biotechnol."},{"key":"ref_19","first-page":"27","article-title":"Inhibition of oxidative injury of biological membranes by astaxanthin","volume":"22","author":"Kurashige","year":"1990","journal-title":"Physiol. Chem. Phys. Med. NMR"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Hwang, Y.H., Hong, S.G., Mun, S.K., Kim, S.J., Lee, S.J., Kim, J.J., Kang, K.Y., and Yee, S.T. (2017). The Protective Effects of Astaxanthin on the OVA-Induced Asthma Mice Model. Molecules.","DOI":"10.3390\/molecules22112019"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Li, S., Takahara, T., Fujino, M., Fukuhara, Y., Sugiyama, T., Li, X.K., and Takahara, S. (2017). Astaxanthin prevents ischemia-reperfusion injury of the steatotic liver in mice. PLoS ONE.","DOI":"10.1371\/journal.pone.0187810"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.intimp.2017.01.028","article-title":"Astaxanthin pretreatment attenuates acetaminophen-induced liver injury in mice","volume":"45","author":"Zhang","year":"2017","journal-title":"Int. Immunopharmacol."},{"key":"ref_23","first-page":"784","article-title":"Astaxanthin inhibits proliferation and promotes apoptosis of A549 lung cancer cells via blocking JAK1\/STAT3 pathway","volume":"32","author":"Wu","year":"2016","journal-title":"Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"69370","DOI":"10.18632\/oncotarget.20628","article-title":"Astaxanthin acts via LRP-1 to inhibit inflammation and reverse lipopolysaccharide-induced M1\/M2 polarization of microglial cells","volume":"8","author":"Wen","year":"2017","journal-title":"Oncotarget"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1111\/jre.12497","article-title":"Investigation of the effect of astaxanthin on alveolar bone loss in experimental periodontitis","volume":"53","author":"Gevrek","year":"2018","journal-title":"J. Periodontal Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1835","DOI":"10.1007\/s00198-010-1419-7","article-title":"Medical costs of osteoporosis in the elderly Medicare population","volume":"22","author":"Blume","year":"2011","journal-title":"Osteoporos. Int."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1038\/nature01658","article-title":"Osteoclast differentiation and activation","volume":"423","author":"Boyle","year":"2003","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1210\/me.2007-0237","article-title":"NFATc1 induces osteoclast fusion via up-regulation of Atp6v0d2 and the dendritic cell-specific transmembrane protein (DC-STAMP)","volume":"22","author":"Kim","year":"2008","journal-title":"Mol. Endocrinol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1016\/j.ydbio.2006.01.017","article-title":"NFATc1 expression in the developing heart valves is responsive to the RANKL pathway and is required for endocardial expression of cathepsin K","volume":"292","author":"Lange","year":"2006","journal-title":"Dev. Biol."},{"key":"ref_30","first-page":"424","article-title":"The laboratory rat as an animal model for osteoporosis research","volume":"58","author":"Lelovas","year":"2008","journal-title":"Comp. Med."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"156","DOI":"10.18632\/aging.101145","article-title":"Salvia miltiorrhiza bunge increases estrogen level without side effects on reproductive tissues in immature\/ovariectomized mice","volume":"9","author":"Xu","year":"2016","journal-title":"Aging"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Wang, X., He, Y., Guo, B., Tsang, M.C., Tu, F., Dai, Y., Yao, Z., Zheng, L., Xie, X., and Wang, N. (2015). In Vivo Screening for Anti-Osteoporotic Fraction from Extract of Herbal Formula Xianlinggubao in Ovariectomized Mice. PLoS ONE.","DOI":"10.1371\/journal.pone.0118184"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Kanaya, N., Kubo, M., Liu, Z., Chu, P., Wang, C., Yuan, Y.C., and Chen, S. (2011). Protective Effects of White Button Mushroom (Agaricus bisporus) against Hepatic Steatosis in Ovariectomized Mice as a Model of Postmenopausal Women. PLoS ONE.","DOI":"10.1371\/journal.pone.0026654"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Hwang, Y.H., Kang, K.Y., Kim, J.J., Lee, S.J., Son, Y.J., Paik, S.H., and Yee, S.T. (2016). Effects of Hot Water Extracts from Polygonum multiflorum on Ovariectomy Induced Osteopenia in Mice. Evid Based Complement. Altern. Med.","DOI":"10.1155\/2016\/8970585"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"649","DOI":"10.3892\/ijmm.2016.2477","article-title":"The effects of Lycii Radicis Cortex on RANKL-induced osteoclast differentiation and activation in RAW 264.7 cells","volume":"37","author":"Kim","year":"2016","journal-title":"Int. J. Mol. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1359\/jbmr.2002.17.3.394","article-title":"Exposure of KS483 cells to estrogen enhances osteogenesis and inhibits adipogenesis","volume":"17","author":"Dang","year":"2002","journal-title":"J. Bone Miner. Res."},{"key":"ref_37","first-page":"93","article-title":"The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice","volume":"8","author":"Wehrle","year":"2015","journal-title":"Dis. Model. Mech."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Chung, H.J., Cho, L., Shin, J.S., Lee, J., Ha, I.H., Park, H.J., and Lee, S.K. (2014). Effects of JSOG-6 on protection against bone loss in ovariectomized mice through regulation of osteoblast differentiation and osteoclast formation. BMC Complement. Altern. Med., 14.","DOI":"10.1186\/1472-6882-14-184"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"5019","DOI":"10.2147\/DDDT.S89505","article-title":"Bu-Shen-Ning-Xin Decoction ameliorated the osteoporotic phenotype of ovariectomized mice without affecting the serum estrogen concentration or uterus","volume":"9","author":"Wang","year":"2015","journal-title":"Drug. Des. Develop. Ther."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Han, J., and Wang, W. (2017). Effects of tanshinol on markers of bone turnover in ovariectomized rats and osteoblast cultures. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0181175"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"664","DOI":"10.1016\/S0899-9007(97)83011-0","article-title":"Calcium and osteoporosis","volume":"13","author":"Nordin","year":"1997","journal-title":"Nutrition"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1359","DOI":"10.1210\/jcem-51-6-1359","article-title":"Effect of estrogen on calcium absorption and serum vitamin D metabolites in postmenopausal osteoporosis","volume":"51","author":"Gallagher","year":"1980","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2039","DOI":"10.1007\/s00467-012-2175-z","article-title":"Phosphate homeostasis and its role in bone health","volume":"27","author":"Penido","year":"2012","journal-title":"Pediatr. Nephrol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1865","DOI":"10.3892\/mmr.2016.5432","article-title":"Polydatin possesses notable anti\u2011osteoporotic activity via regulation of OPG, RANKL and \u03b2-catenin","volume":"14","author":"Zhou","year":"2016","journal-title":"Mol. Med. Rep."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"5077","DOI":"10.1038\/s41598-017-05392-z","article-title":"Evaluation of Osteogenesis and Angiogenesis of Icariin in Local Controlled Release and Systemic Delivery for Calvarial Defect in Ovariectomized Rats","volume":"7","author":"Wu","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1046\/j.1365-2796.1997.96114000.x","article-title":"Influence of menopause on blood cholesterol levels in women: The role of body composition, fat distribution and hormonal milieu","volume":"241","author":"Pasquali","year":"1997","journal-title":"J. Int. Med."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1615","DOI":"10.1007\/s00198-010-1367-2","article-title":"High serum total cholesterol is a long-term cause of osteoporotic fracture","volume":"22","author":"Trimpou","year":"2011","journal-title":"Osteoporos. Int."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1016\/S0022-3565(24)37618-9","article-title":"An investigation of the relationship between estrogen, estrogen metabolites and blood cholesterol levels in ovariectomized rats","volume":"286","author":"Liu","year":"1998","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"5224","DOI":"10.1210\/endo.139.12.6343","article-title":"Idoxifene: A novel selective estrogen receptor modulator prevents bone loss and lowers cholesterol levels in ovariectomized rats and decreases uterine weight in intact rats","volume":"139","author":"Nuttall","year":"1998","journal-title":"Endocrinology"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2734","DOI":"10.3390\/nu5072734","article-title":"Effects of dietary isoflavones from Puerariae radix on lipid and bone metabolism in ovariectomized rats","volume":"5","author":"Kim","year":"2013","journal-title":"Nutrients"},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Brandi, M.L. (2009). Microarchitecture, the key to bone quality. Rheumatology.","DOI":"10.1093\/rheumatology\/kep273"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1590\/S0004-27302010000200002","article-title":"Bone histomorphometry: A concise review for endocrinologists and clinicians","volume":"54","author":"Kulak","year":"2010","journal-title":"Arq. Bras. Endocrinol. Metabol."},{"key":"ref_53","first-page":"294","article-title":"Bone histomorphometry revisited","volume":"37","author":"Vidal","year":"2012","journal-title":"Acta Reumatol. Port."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"506","DOI":"10.1007\/s00774-008-0856-2","article-title":"Interindividual and intraspecimen variability of 3-D bone microarchitectural parameters in iliac crest biopsies imaged by conventional micro-computed tomography","volume":"26","author":"Chappard","year":"2008","journal-title":"J. Bone Miner. Metab."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1016\/j.bone.2007.01.010","article-title":"Changes in micro-CT 3D bone parameters reflect effects of a potent cathepsin K inhibitor (SB-553484) on bone resorption and cortical bone formation in ovariectomized mice","volume":"40","author":"Xiang","year":"2007","journal-title":"Bone"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"1840","DOI":"10.1016\/j.jnutbio.2013.04.005","article-title":"Pomegranate seed oil prevents bone loss in a mice model of osteoporosis, through osteoblastic stimulation, osteoclastic inhibition and decreased inflammatory status","volume":"24","author":"Spilmont","year":"2013","journal-title":"J. Nutr. Biochem."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/S0001-2998(97)80024-4","article-title":"Overview of role of BMD measurements in managing osteoporosis","volume":"27","author":"Ryan","year":"1997","journal-title":"Semin. Nucl. Med."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_59","first-page":"365","article-title":"Primer3 on the WWW for general users and for biologist programmers","volume":"132","author":"Rozen","year":"2000","journal-title":"Methods Mol. Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/3\/912\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T14:57:42Z","timestamp":1760194662000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/19\/3\/912"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,3,19]]},"references-count":59,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2018,3]]}},"alternative-id":["ijms19030912"],"URL":"https:\/\/doi.org\/10.3390\/ijms19030912","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,3,19]]}}}