{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T07:10:57Z","timestamp":1787987457111,"version":"build-2784847793"},"reference-count":50,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2022,12,8]],"date-time":"2022-12-08T00:00:00Z","timestamp":1670457600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004488","name":"Croatian Science Foundation","doi-asserted-by":"publisher","award":["IP-06-2016-2575"],"award-info":[{"award-number":["IP-06-2016-2575"]}],"id":[{"id":"10.13039\/501100004488","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>This study aimed to explore the spatio-temporal expression patterns of congenital anomalies of kidney and urinary tract (CAKUT) candidate genes, Fibroblast Growth Factor Receptor 1 (FGFR1), Fibroblast Growth Factor Receptor 2 (FGFR2) and Receptor-Interacting Protein Kinase 5 (RIP5), in human fetal kidney development (CTRL) and kidneys affected with CAKUT. Human fetal kidneys from the 22nd to 41st developmental week (duplex, hypoplastic, dysplastic, and controls) were stained with antibodies and analyzed by epifluorescence microscopy and RT\u2212qPCR. The effect of CAKUT candidate genes on kidney nephrogenesis and function is confirmed by statistically significant variations in the spatio-temporal expression patterns of the investigated markers. The nuclear localization of FGFR1, elevated expression score of FGFR1 mRNA, the increased area percentage of FGFR1-positive cells in the kidney cortex, and the overall decrease in the expression after the peak at the 27th developmental week in dysplastic kidneys (DYS), suggest an altered expression pattern and protein function in response to CAKUT pathophysiology. The RT\u2212qPCR analysis revealed a significantly higher FGFR2 mRNA expression score in the CAKUT kidneys compared to the CTRL. This increase could be due to the repair mechanism involving the downstream mediator, Extracellular Signal-Regulated Kinase 1\/2 (ERK1\/2). The expression of RIP5 during normal human kidney development was reduced temporarily, due to urine production and increased later since it undertakes additional functions in the maturation of the postnatal kidney and homeostasis, while the expression dynamics in CAKUT-affected kidneys exhibited a decrease in the percentage of RIP5-positive cells during the investigated developmental period. Our findings highlight the importance of FGFR1, FGFR2, and RIP5 as markers in normal and pathological kidney development.<\/jats:p>","DOI":"10.3390\/ijms232415537","type":"journal-article","created":{"date-parts":[[2022,12,8]],"date-time":"2022-12-08T04:05:29Z","timestamp":1670472329000},"page":"15537","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Aberrations in FGFR1, FGFR2, and RIP5 Expression in Human Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6529-5474","authenticated-orcid":false,"given":"Nela","family":"Kelam","sequence":"first","affiliation":[{"name":"Department of Anatomy, Histology and Embryology, University of Split School of Medicine, \u0160oltanska 2, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anita","family":"Racetin","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Histology and Embryology, University of Split School of Medicine, \u0160oltanska 2, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mirjana","family":"Polovi\u0107","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Histology and Embryology, University of Split School of Medicine, \u0160oltanska 2, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3681-8499","authenticated-orcid":false,"given":"Benjamin","family":"Benzon","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Histology and Embryology, University of Split School of Medicine, \u0160oltanska 2, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3801-1026","authenticated-orcid":false,"given":"Marin","family":"Ogorevc","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Histology and Embryology, University of Split School of Medicine, \u0160oltanska 2, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2182-2890","authenticated-orcid":false,"given":"Katarina","family":"Vukojevi\u0107","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Histology and Embryology, University of Split School of Medicine, \u0160oltanska 2, 21000 Split, Croatia"},{"name":"Department of Anatomy, University of Mostar School of Medicine, 88000 Mostar, Bosnia and Herzegovina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Merica","family":"Glavina Durdov","sequence":"additional","affiliation":[{"name":"Department of Pathology, University Hospital Center Split, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana","family":"Dunatov Huljev","sequence":"additional","affiliation":[{"name":"Department of Pathology, University Hospital Center Split, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5875-3839","authenticated-orcid":false,"given":"Ivana","family":"Kuzmi\u0107 Prusac","sequence":"additional","affiliation":[{"name":"Department of Pathology, University Hospital Center Split, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2999-4937","authenticated-orcid":false,"given":"Davor","family":"\u010cari\u0107","sequence":"additional","affiliation":[{"name":"Department of Orthopaedics and Traumatology, University Hospital in Split, Spinciceva 1, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fila","family":"Ragu\u017e","sequence":"additional","affiliation":[{"name":"Department of Nephrology, University Hospital Center Mostar, 88000 Mostar, Bosnia and Herzegovina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6389-5604","authenticated-orcid":false,"given":"Sandra","family":"Kosti\u0107","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Histology and Embryology, University of Split School of Medicine, \u0160oltanska 2, 21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"(1993). Birth Defects Monitoring Program (BDMP)\/Commission on Professional and Hospital Activities (CPHA) surveillance data, 1988-1991. Teratology, 48, 658\u2013675.","DOI":"10.1002\/tera.1420480608"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"S31","DOI":"10.1002\/bdra.20778","article-title":"Paper 4: EUROCAT statistical monitoring: Identification and investigation of ten year trends of congenital anomalies in Europe","volume":"91","author":"Loane","year":"2011","journal-title":"Birth Defects Res. Part A Clin. Mol. Teratol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e382","DOI":"10.1542\/peds.111.4.e382","article-title":"Epidemiology of chronic renal failure in children: Data from the ItalKid project","volume":"111","author":"Ardissino","year":"2003","journal-title":"Pediatrics"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1038\/ki.2009.220","article-title":"Renal outcome in patients with congenital anomalies of the kidney and urinary tract","volume":"76","author":"Ravani","year":"2009","journal-title":"Kidney Int."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2793","DOI":"10.1007\/s00467-022-05678-z","article-title":"The term CAKUT has outlived its usefulness: The case for the defense","volume":"37","author":"Knoers","year":"2022","journal-title":"Pediatr. Nephrol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.2215\/CJN.10221012","article-title":"Severe prenatal renal anomalies associated with mutations in HNF1B or PAX2 genes","volume":"8","author":"Madariaga","year":"2013","journal-title":"Clin. J. Am. Soc. Nephrol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1007\/s00467-013-2684-4","article-title":"Single-gene causes of congenital anomalies of the kidney and urinary tract (CAKUT) in humans","volume":"29","author":"Vivante","year":"2014","journal-title":"Pediatr. Nephrol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2348","DOI":"10.1681\/ASN.2017121265","article-title":"Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies of the Kidney and Urinary Tract","volume":"29","author":"Connaughton","year":"2018","journal-title":"J. Am. Soc. Nephrol. JASN"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1056\/NEJMoa1214479","article-title":"Mutations in DSTYK and dominant urinary tract malformations","volume":"369","author":"Sampogna","year":"2013","journal-title":"N. Engl. J. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1373","DOI":"10.1007\/s00467-010-1747-z","article-title":"Role of fibroblast growth factor receptor signaling in kidney development","volume":"26","author":"Bates","year":"2011","journal-title":"Pediatr. Nephrol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1016\/j.acthis.2018.01.008","article-title":"Growth factors FGF8 and FGF2 and their receptor FGFR1, transcriptional factors Msx-1 and MSX-2, and apoptotic factors p19 and RIP5 participate in the early human limb development","volume":"120","author":"Becic","year":"2018","journal-title":"Acta Histochem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1016\/j.acthis.2019.04.011","article-title":"Immunohistochemical expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the normal human kidney development","volume":"121","author":"Racetin","year":"2019","journal-title":"Acta Histochem."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Kelam, N., Racetin, A., Katsuyama, Y., Vukojevic, K., and Kostic, S. (2022). Immunohistochemical Expression Pattern of FGFR1, FGFR2, RIP5, and HIP2 in Developing and Postnatal Kidneys of Dab1(-\/-) (yotari) Mice. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23042025"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Capone, V.P., Morello, W., Taroni, F., and Montini, G. (2017). Genetics of Congenital Anomalies of the Kidney and Urinary Tract: The Current State of Play. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18040796"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1007\/s00467-015-3151-1","article-title":"Fibroblast growth factor receptor signaling in kidney and lower urinary tract development","volume":"31","author":"Walker","year":"2016","journal-title":"Pediatr. Nephrol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1002\/(SICI)1098-1004(1999)14:2<115::AID-HUMU3>3.0.CO;2-2","article-title":"Clinical spectrum of fibroblast growth factor receptor mutations","volume":"14","author":"Wilcox","year":"1999","journal-title":"Hum. Mutat."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1016\/S0344-0338(97)80018-0","article-title":"A 19-week-old fetus with craniosynostosis, renal agenesis and gastroschisis: Case report and differential diagnosis","volume":"193","author":"Sergi","year":"1997","journal-title":"Pathol. Res. Pract."},{"key":"ref_18","first-page":"200","article-title":"High-grade vesicoureteral reflux in Pfeiffer syndrome","volume":"5","author":"Seyedzadeh","year":"2008","journal-title":"Urol. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"758","DOI":"10.1002\/ajmg.1320450618","article-title":"Visceral anomalies in the Apert syndrome","volume":"45","author":"Cohen","year":"1993","journal-title":"Am. J. Med. Genet."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1203\/PDR.0b013e318187cc12","article-title":"Role of fibroblast growth factor receptor 2 in kidney mesenchyme","volume":"64","author":"Hains","year":"2008","journal-title":"Pediatr. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2077","DOI":"10.1016\/j.juro.2009.12.095","article-title":"High incidence of vesicoureteral reflux in mice with Fgfr2 deletion in kidney mesenchyma","volume":"183","author":"Hains","year":"2010","journal-title":"J. Urol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2525","DOI":"10.1681\/ASN.2009050532","article-title":"Three-dimensional imaging reveals ureteric and mesenchymal defects in Fgfr2-mutant kidneys","volume":"20","author":"Argyropoulos","year":"2009","journal-title":"J. Am. Soc. Nephrol. JASN"},{"key":"ref_23","first-page":"240","article-title":"Fgfr1 and the IIIc isoform of Fgfr2 play critical roles in the metanephric mesenchyme mediating early inductive events in kidney development","volume":"240","author":"Cusack","year":"2011","journal-title":"Dev. Dyn. Off. Publ. Am. Assoc. Anat."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/j.ydbio.2004.09.002","article-title":"Role of fibroblast growth factor receptors 1 and 2 in the ureteric bud","volume":"276","author":"Zhao","year":"2004","journal-title":"Dev. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5082","DOI":"10.1073\/pnas.95.9.5082","article-title":"Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development","volume":"95","author":"Arman","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3045","DOI":"10.1101\/gad.8.24.3045","article-title":"Murine FGFR-1 is required for early postimplantation growth and axial organization","volume":"8","author":"Deng","year":"1994","journal-title":"Genes Dev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1242\/dev.125.4.753","article-title":"Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction","volume":"125","author":"Xu","year":"1998","journal-title":"Development"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3032","DOI":"10.1101\/gad.8.24.3032","article-title":"fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation","volume":"8","author":"Yamaguchi","year":"1994","journal-title":"Genes Dev."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1203\/01.pdr.0000202764.02295.45","article-title":"Link between reduced nephron number and hypertension: Studies in a mutant mouse model","volume":"59","author":"Poladia","year":"2006","journal-title":"Pediatr. Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1642","DOI":"10.1093\/emboj\/17.6.1642","article-title":"Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning","volume":"17","author":"Celli","year":"1998","journal-title":"EMBO J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1007\/s00467-010-1629-4","article-title":"Genetics of congenital anomalies of the kidney and urinary tract","volume":"26","author":"Song","year":"2011","journal-title":"Pediatr. Nephrol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"F888","DOI":"10.1152\/ajprenal.00624.2014","article-title":"Fgfr2 is integral for bladder mesenchyme patterning and function","volume":"308","author":"Walker","year":"2015","journal-title":"Am. J. Physiol. Ren. Physiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1016\/j.ydbio.2005.12.034","article-title":"Role of fibroblast growth factor receptors 1 and 2 in the metanephric mesenchyme","volume":"291","author":"Poladia","year":"2006","journal-title":"Dev. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"298","DOI":"10.1016\/j.bbrc.2004.04.194","article-title":"RIP5 is a RIP-homologous inducer of cell death","volume":"319","author":"Zha","year":"2004","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1122","DOI":"10.1038\/s41419-021-04385-1","article-title":"Loss of DSTYK activates Wnt\/beta-catenin signaling and glycolysis in lung adenocarcinoma","volume":"12","author":"Zhong","year":"2021","journal-title":"Cell Death Dis."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e2965","DOI":"10.1038\/cddis.2017.353","article-title":"FGFR1 is critical for the anti-endothelial mesenchymal transition effect of N-acetyl-seryl-aspartyl-lysyl-proline via induction of the MAP4K4 pathway","volume":"8","author":"Li","year":"2017","journal-title":"Cell Death Dis."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Srivastava, S.P., Goodwin, J.E., Tripathi, P., Kanasaki, K., and Koya, D. (2021). Interactions among Long Non-Coding RNAs and microRNAs Influence Disease Phenotype in Diabetes and Diabetic Kidney Disease. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22116027"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1007\/s003830050750","article-title":"Increased expression of fibroblast growth factors in segmental renal dysplasia","volume":"16","author":"Shima","year":"2000","journal-title":"Pediatr. Surg. Int."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1038\/s41392-020-00222-7","article-title":"FGF\/FGFR signaling in health and disease","volume":"5","author":"Xie","year":"2020","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_40","first-page":"265","article-title":"FGFR2 overexpression predicts survival outcome in patients with metastatic papillary renal cell carcinoma","volume":"19","author":"Tsimafeyeu","year":"2017","journal-title":"Clin. Transl. Oncol. Off. Publ. Fed. Span. Oncol. Soc. Natl. Cancer Inst. Mex."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1159\/000505661","article-title":"FGF\/FGFR2 Protects against Tubular Cell Death and Acute Kidney Injury Involving Erk1\/2 Signaling Activation","volume":"6","author":"Xu","year":"2020","journal-title":"Kidney Dis."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1604","DOI":"10.1681\/ASN.2004090800","article-title":"Extracellular signal-regulated kinase inhibition slows disease progression in mice with polycystic kidney disease","volume":"17","author":"Omori","year":"2006","journal-title":"J. Am. Soc. Nephrol. JASN"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"492","DOI":"10.1016\/j.jpedsurg.2021.04.017","article-title":"Protein expression in vesicoureteral reflux: What about children?","volume":"57","author":"Tokat","year":"2022","journal-title":"J. Pediatr. Surg."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1265","DOI":"10.1007\/s00467-011-2053-0","article-title":"PAX2 in human kidney malformations and disease","volume":"27","author":"Harshman","year":"2012","journal-title":"Pediatr. Nephrol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"962","DOI":"10.1007\/s00467-007-0466-6","article-title":"Expression profiles of congenital renal dysplasia reveal new insights into renal development and disease","volume":"22","author":"Jain","year":"2007","journal-title":"Pediatr. Nephrol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"650","DOI":"10.2471\/BLT.08.050955","article-title":"The Declaration of Helsinki and public health","volume":"86","author":"Williams","year":"2008","journal-title":"Bull. World Health Organ."},{"key":"ref_47","first-page":"556","article-title":"Guide to the staging of human embryos","volume":"130","year":"1972","journal-title":"Anat. Anz."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Lozic, M., Filipovic, N., Juric, M., Kosovic, I., Benzon, B., Solic, I., Kelam, N., Racetin, A., Watanabe, K., and Katsuyama, Y. (2021). Alteration of Cx37, Cx40, Cx43, Cx45, Panx1, and Renin Expression Patterns in Postnatal Kidneys of Dab1-\/- (yotari) Mice. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22031284"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Pastar, V., Lozic, M., Kelam, N., Filipovic, N., Bernard, B., Katsuyama, Y., and Vukojevic, K. (2021). Connexin Expression Is Altered in Liver Development of Yotari (dab1 -\/-) Mice. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms221910712"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1037\/1040-3590.6.4.284","article-title":"Guidelines, Criteria, and Rules of Thumb for Evaluating Normed and Standardized Assessment Instrument in Psychology","volume":"6","author":"Cicchetti","year":"1994","journal-title":"Psychol. Assess."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/24\/15537\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:36:19Z","timestamp":1760146579000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/24\/15537"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,12,8]]},"references-count":50,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2022,12]]}},"alternative-id":["ijms232415537"],"URL":"https:\/\/doi.org\/10.3390\/ijms232415537","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,12,8]]}}}