{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,3]],"date-time":"2026-11-03T06:13:11Z","timestamp":1793686391692,"version":"4.5.1"},"reference-count":43,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2020,4,30]],"date-time":"2020-04-30T00:00:00Z","timestamp":1588204800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004587","name":"Instituto de Salud Carlos III","doi-asserted-by":"publisher","award":["PI13\/00556; PI15\/00484;  PI16\/00789; PI18\/00151; CP16\/00046"],"award-info":[{"award-number":["PI13\/00556; PI15\/00484;  PI16\/00789; PI18\/00151; CP16\/00046"]}],"id":[{"id":"10.13039\/501100004587","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008530","name":"European Regional Development Fund","doi-asserted-by":"publisher","award":["PI13\/00556; PI15\/00484;  PI16\/00789; PI18\/00151 and CP16\/00046"],"award-info":[{"award-number":["PI13\/00556; PI15\/00484;  PI16\/00789; PI18\/00151 and CP16\/00046"]}],"id":[{"id":"10.13039\/501100008530","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003176","name":"Ministerio de Educaci\u00f3n, Cultura y Deporte","doi-asserted-by":"publisher","award":["FPU13\/00544"],"award-info":[{"award-number":["FPU13\/00544"]}],"id":[{"id":"10.13039\/501100003176","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characterized by neurodegeneration and astrogliosis in the basal ganglia or the brain stem. At present, there is no cure or treatment for this disease, partly due to scarcity of LS models. Current models generally fail to recapitulate important traits of the disease. Therefore, there is an urgent need to develop new human in vitro models. Establishment of induced pluripotent stem cells (iPSCs) followed by differentiation into neurons is a powerful tool to obtain an in vitro model for LS. Here, we describe the generation and characterization of iPSCs, neural stem cells (NSCs) and iPSC-derived neurons harboring the mtDNA mutation m.13513G&gt;A in heteroplasmy. We have performed mitochondrial characterization, analysis of electrophysiological properties and calcium imaging of LS neurons. Here, we show a clearly compromised oxidative phosphorylation (OXPHOS) function in LS patient neurons. This is also the first report of electrophysiological studies performed on iPSC-derived neurons harboring an mtDNA mutation, which revealed that, in spite of having identical electrical properties, diseased neurons manifested mitochondrial dysfunction together with a diminished calcium buffering capacity. This could lead to an overload of cytoplasmic calcium concentration and the consequent cell death observed in patients. Importantly, our results highlight the importance of calcium homeostasis in LS pathology.<\/jats:p>","DOI":"10.3390\/ijms21093191","type":"journal-article","created":{"date-parts":[[2020,5,4]],"date-time":"2020-05-04T14:00:43Z","timestamp":1588600843000},"page":"3191","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":42,"title":["Mitochondrial Dysfunction and Calcium Dysregulation in Leigh Syndrome Induced Pluripotent Stem Cell Derived Neurons"],"prefix":"10.3390","volume":"21","author":[{"given":"Teresa","family":"Galera-Monge","sequence":"first","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Facultad de Medicina, Universidad Aut\u00f3noma de Madrid, 28029 Madrid, Spain"},{"name":"Departamento de Modelos Experimentales de Enfermedades Humanas, Instituto de Investigaciones Biom\u00e9dicas \u201cAlberto Sols\u201d UAM-CSIC, 28029 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red (CIBERER), 28029 Madrid, Spain"},{"name":"Instituto de Investigaci\u00f3n Sanitaria Hospital 12 de Octubre (i + 12), 28041 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco","family":"Zurita-D\u00edaz","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Facultad de Medicina, Universidad Aut\u00f3noma de Madrid, 28029 Madrid, Spain"},{"name":"Departamento de Modelos Experimentales de Enfermedades Humanas, Instituto de Investigaciones Biom\u00e9dicas \u201cAlberto Sols\u201d UAM-CSIC, 28029 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red (CIBERER), 28029 Madrid, Spain"},{"name":"Instituto de Investigaci\u00f3n Sanitaria Hospital 12 de Octubre (i + 12), 28041 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2689-268X","authenticated-orcid":false,"given":"Isaac","family":"Canals","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Neurology, Lund Stem Cell Center, Lund University, 221 00 Lund, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marita","family":"Gr\u00f8nning Hansen","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Neurology, Lund Stem Cell Center, Lund University, 221 00 Lund, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Rufi\u00e1n-V\u00e1zquez","sequence":"additional","affiliation":[{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red (CIBERER), 28029 Madrid, Spain"},{"name":"Instituto de Investigaci\u00f3n Sanitaria Hospital 12 de Octubre (i + 12), 28041 Madrid, Spain"},{"name":"Laboratorio de enfermedades mitocondriales y Neurometab\u00f3licas, Hospital 12 de Octubre, 28041 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2417-5767","authenticated-orcid":false,"given":"Johannes K.","family":"Ehinger","sequence":"additional","affiliation":[{"name":"Mitochondrial Medicine, Department of Clinical Sciences Lund, Faculty of Medicine, Lund University, BMC A13, 221 84 Lund, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7901-1826","authenticated-orcid":false,"given":"Eskil","family":"Elm\u00e9r","sequence":"additional","affiliation":[{"name":"Mitochondrial Medicine, Department of Clinical Sciences Lund, Faculty of Medicine, Lund University, BMC A13, 221 84 Lund, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4741-772X","authenticated-orcid":false,"given":"Miguel A.","family":"Martin","sequence":"additional","affiliation":[{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red (CIBERER), 28029 Madrid, Spain"},{"name":"Instituto de Investigaci\u00f3n Sanitaria Hospital 12 de Octubre (i + 12), 28041 Madrid, Spain"},{"name":"Laboratorio de enfermedades mitocondriales y Neurometab\u00f3licas, Hospital 12 de Octubre, 28041 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafael","family":"Garesse","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Facultad de Medicina, Universidad Aut\u00f3noma de Madrid, 28029 Madrid, Spain"},{"name":"Departamento de Modelos Experimentales de Enfermedades Humanas, Instituto de Investigaciones Biom\u00e9dicas \u201cAlberto Sols\u201d UAM-CSIC, 28029 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red (CIBERER), 28029 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8958-6148","authenticated-orcid":false,"given":"Henrik","family":"Ahlenius","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Neurology, Lund Stem Cell Center, Lund University, 221 00 Lund, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8699-8734","authenticated-orcid":false,"given":"M. Esther","family":"Gallardo","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Facultad de Medicina, Universidad Aut\u00f3noma de Madrid, 28029 Madrid, Spain"},{"name":"Departamento de Modelos Experimentales de Enfermedades Humanas, Instituto de Investigaciones Biom\u00e9dicas \u201cAlberto Sols\u201d UAM-CSIC, 28029 Madrid, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red (CIBERER), 28029 Madrid, Spain"},{"name":"Instituto de Investigaci\u00f3n Sanitaria Hospital 12 de Octubre (i + 12), 28041 Madrid, Spain"},{"name":"Grupo de Investigaci\u00f3n Traslacional con c\u00e9lulas iPS. Instituto de Investigaci\u00f3n Sanitaria Hospital 12 de Octubre (i + 12), 28041 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1524","DOI":"10.1126\/science.1244360","article-title":"mTOR inhibition alleviates mitochondrial disease in a mouse model of Leigh syndrome","volume":"342","author":"Johnson","year":"2013","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2359","DOI":"10.1172\/JCI62923","article-title":"Fatal breathing dysfunction in a mouse model of Leigh syndrome","volume":"122","author":"Quintana","year":"2012","journal-title":"J. Clin. Investig."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1002\/ana.24551","article-title":"Leigh syndrome: One disorder, more than 75 monogenic causes","volume":"79","author":"Lake","year":"2016","journal-title":"Ann. Neurol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"227s","DOI":"10.1083\/jcb.91.3.227s","article-title":"Mitochondria: A historical review","volume":"91","author":"Ernster","year":"1981","journal-title":"J. Cell Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"544","DOI":"10.1016\/j.bbabio.2015.03.001","article-title":"Emerging concepts in the therapy of mitochondrial disease","volume":"1847","author":"Viscomi","year":"2015","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.semcdb.2016.02.018","article-title":"Energy metabolism in neuronal\/glial induction and in iPSC models of brain disorders","volume":"52","author":"Mlody","year":"2016","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1038\/290457a0","article-title":"Sequence and organization of the human mitochondrial genome","volume":"290","author":"Anderson","year":"1981","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"530","DOI":"10.1038\/nrg3966","article-title":"The dynamics of mitochondrial DNA heteroplasmy: Implications for human health and disease","volume":"16","author":"Stewart","year":"2015","journal-title":"Nat. Rev. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1531","DOI":"10.1177\/0883073812460580","article-title":"Leigh Syndrome and the Mitochondrial m.13513G>A Mutation: Expanding the Clinical Spectrum","volume":"28","author":"Toro","year":"2013","journal-title":"J. Child Neurol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2317","DOI":"10.1002\/jcp.25386","article-title":"iPSCs, a Future Tool for Therapeutic Intervention in Mitochondrial Disorders: Pros and Cons","volume":"231","author":"Galera","year":"2016","journal-title":"J. Cell. Physiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1038\/nature14546","article-title":"Metabolic rescue in pluripotent cells from patients with mtDNA disease","volume":"524","author":"Ma","year":"2015","journal-title":"Nature"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e13378","DOI":"10.7554\/eLife.13378","article-title":"Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration","volume":"5","author":"Zheng","year":"2016","journal-title":"eLife"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.scr.2015.12.005","article-title":"Generation of a human iPSC line from a patient with Leigh syndrome","volume":"16","author":"Galera","year":"2016","journal-title":"Stem Cell Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"785","DOI":"10.1016\/j.neuron.2013.05.029","article-title":"Rapid single-step induction of functional neurons from human pluripotent stem cells","volume":"78","author":"Zhang","year":"2013","journal-title":"Neuron"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"138","DOI":"10.15252\/embj.201490446","article-title":"Pluripotent stem cell energy metabolism: An update","volume":"34","author":"Teslaa","year":"2015","journal-title":"EMBO J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1068","DOI":"10.1038\/nprot.2012.048","article-title":"Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells","volume":"7","author":"Zhang","year":"2012","journal-title":"Nat. Protoc."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"12317","DOI":"10.1038\/ncomms12317","article-title":"Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency","volume":"7","author":"Ehinger","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2784","DOI":"10.1016\/j.bbamcr.2015.03.013","article-title":"Selective removal of mitochondria via mitophagy: Distinct pathways for different mitochondrial stresses","volume":"1853","author":"Wei","year":"2015","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"19","DOI":"10.4161\/auto.5.1.7174","article-title":"Coenzyme Q deficiency triggers mitochondria degradation by mitophagy","volume":"5","author":"Cordero","year":"2009","journal-title":"Autophagy"},{"key":"ref_20","first-page":"2669","article-title":"Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts","volume":"25","author":"Cotan","year":"2011","journal-title":"FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol."},{"key":"ref_21","first-page":"446","article-title":"Recovery of MERRF fibroblasts and cybrids pathophysiology by coenzyme Q10","volume":"9","author":"Cotan","year":"2012","journal-title":"Neurother. J. Am. Soc. Exp. NeuroTher."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1038\/cdd.2015.84","article-title":"Reduced mitochondrial Ca(2+) transients stimulate autophagy in human fibroblasts carrying the 13514A>G mutation of the ND5 subunit of NADH dehydrogenase","volume":"23","author":"Granatiero","year":"2016","journal-title":"Cell Death Differ."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1016\/j.bbadis.2014.03.013","article-title":"Bulk autophagy, but not mitophagy, is increased in cellular model of mitochondrial disease","volume":"1842","author":"Moran","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1002\/stem.307","article-title":"Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells","volume":"28","author":"Armstrong","year":"2010","journal-title":"Stem Cells"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Prigione, A., Hossini, A.M., Lichtner, B., Serin, A., Fauler, B., Megges, M., Lurz, R., Lehrach, H., Makrantonaki, E., and Zouboulis, C.C. (2011). Mitochondrial-associated cell death mechanisms are reset to an embryonic-like state in aged donor-derived iPS cells harboring chromosomal aberrations. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0027352"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4860","DOI":"10.1038\/emboj.2011.401","article-title":"UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells","volume":"30","author":"Zhang","year":"2011","journal-title":"EMBO J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1038\/35036035","article-title":"The versatility and universality of calcium signalling","volume":"1","author":"Berridge","year":"2000","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"862","DOI":"10.1016\/j.neuron.2012.02.011","article-title":"Imaging calcium in neurons","volume":"73","author":"Grienberger","year":"2012","journal-title":"Neuron"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1038\/nrm1150","article-title":"Regulation of cell death: The calcium-apoptosis link","volume":"4","author":"Orrenius","year":"2003","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_30","first-page":"1835","article-title":"Intracellular ATP levels determine cell death fate by apoptosis or necrosis","volume":"57","author":"Eguchi","year":"1997","journal-title":"Cancer Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1481","DOI":"10.1084\/jem.185.8.1481","article-title":"Intracellular adenosine triphosphate (ATP) concentration: A switch in the decision between apoptosis and necrosis","volume":"185","author":"Leist","year":"1997","journal-title":"J. Exp. Med."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Cameron, M., Kekesi, O., Morley, J.W., Tapson, J., Breen, P.P., van Schaik, A., and Buskila, Y. (2016). Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0155468"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/j.1469-7793.1999.001aa.x","article-title":"Contributions of mitochondria to animal physiology: From homeostatic sensor to calcium signalling and cell death","volume":"516","author":"Duchen","year":"1999","journal-title":"J. Physiol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"593","DOI":"10.1042\/bj3470593","article-title":"Inositol 1,4,5-trisphosphate-induced Ca2+ release is inhibited by mitochondrial depolarization","volume":"347","author":"Collins","year":"2000","journal-title":"Biochem. J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"659","DOI":"10.1016\/j.stem.2016.12.013","article-title":"Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders","volume":"20","author":"Lorenz","year":"2017","journal-title":"Cell Stem Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4028","DOI":"10.1038\/ncomms5028","article-title":"iPSC-derived neurons from GBA1-associated Parkinson\u2019s disease patients show autophagic defects and impaired calcium homeostasis","volume":"5","author":"Schondorf","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"34904","DOI":"10.1038\/srep34904","article-title":"Calcium dysregulation contributes to neurodegeneration in FTLD patient iPSC-derived neurons","volume":"6","author":"Imamura","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_38","first-page":"563","article-title":"Myocardial fiber necrosis due to intracellular Ca overload-a new principle in cardiac pathophysiology","volume":"4","author":"Fleckenstein","year":"1974","journal-title":"Recent Adv. Stud. Card. Struct. Metab."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1317","DOI":"10.2174\/156802609789869646","article-title":"Neuron protection as a therapeutic target in acute ischemic stroke","volume":"9","author":"Tuttolomondo","year":"2009","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"McKenzie, M., and Duchen, M.R. (2016). Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0154371"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"19511","DOI":"10.1002\/jcp.28549","article-title":"The mutation m.13513G>A impairs cardiac function, favoring a neuroectoderm commitment, in a mutant-load dependent way","volume":"234","author":"Garesse","year":"2019","journal-title":"J. Cell. Physiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.mito.2009.05.001","article-title":"Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis","volume":"9","author":"Medja","year":"2009","journal-title":"Mitochondrion"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1007\/978-1-4939-9007-8_6","article-title":"In Vitro Functional Characterization of Human Neurons and Astrocytes Using Calcium Imaging and Electrophysiology","volume":"1919","author":"Hansen","year":"2019","journal-title":"Methods Mol. Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/9\/3191\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:52:13Z","timestamp":1760363533000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/9\/3191"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,4,30]]},"references-count":43,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2020,5]]}},"alternative-id":["ijms21093191"],"URL":"https:\/\/doi.org\/10.3390\/ijms21093191","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,4,30]]}}}