{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T08:41:02Z","timestamp":1780044062432,"version":"3.53.1"},"reference-count":39,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,6,11]],"date-time":"2021-06-11T00:00:00Z","timestamp":1623369600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,6,11]],"date-time":"2021-06-11T00:00:00Z","timestamp":1623369600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Biol Proced Online"],"published-print":{"date-parts":[[2021,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec>\n                <jats:title>Background<\/jats:title>\n                <jats:p>Expression of stemness factors, such as octamer-binding transcription factor 3\/4 (<jats:italic>OCT3\/4<\/jats:italic>), sex determining region Y-box 2 (<jats:italic>SOX2<\/jats:italic>), and alkaline phosphatase (<jats:italic>ALP<\/jats:italic>) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>Two or more colored cells (~\u200910), after staining with a chromogen such a 3,3\u2032-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as <jats:italic>OCT3\/4<\/jats:italic>, <jats:italic>SOX2<\/jats:italic>, and <jats:italic>ALP<\/jats:italic>) was confined to positively stained cells.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusion<\/jats:title>\n                <jats:p>These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12575-021-00149-5","type":"journal-article","created":{"date-parts":[[2021,6,11]],"date-time":"2021-06-11T07:03:58Z","timestamp":1623395038000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["RNA analysis based on a small number of manually isolated fixed cells (RNA-snMIFxC) to profile stem cells from human deciduous tooth-derived dental pulp cells"],"prefix":"10.1186","volume":"23","author":[{"given":"Emi","family":"Inada","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Issei","family":"Saitoh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naoko","family":"Kubota","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoko","family":"Iwase","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuki","family":"Kiyokawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hirofumi","family":"Noguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Youichi","family":"Yamasaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masahiro","family":"Sato","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,6,11]]},"reference":[{"key":"149_CR1","doi-asserted-by":"publisher","first-page":"305","DOI":"10.1007\/978-4-431-56847-6_17","volume":"1103","author":"M Dezawa","year":"2018","unstructured":"Dezawa M. Clinical Trials of Muse Cells. Adv Exp Med Biol. 2018;1103:305\u20137.","journal-title":"Adv Exp Med Biol"},{"key":"149_CR2","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1016\/j.jjcc.2018.10.002","volume":"73","author":"M Isomi","year":"2019","unstructured":"Isomi M, Sadahiro T, Ieda M. Progress and Challenge of Cardiac Regeneration to Treat Heart Failure. J Cardiol. 2019;73:97\u2013101.","journal-title":"J Cardiol"},{"key":"149_CR3","first-page":"80","volume":"122","author":"K Sakai","year":"2012","unstructured":"Sakai K, Yamamoto A, Matsubara K, Nakamura S, Naruse M, Yamagata M, et al. Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms. J Clin Invest. 2012;122:80\u201390.","journal-title":"J Clin Invest"},{"key":"149_CR4","doi-asserted-by":"publisher","first-page":"5807","DOI":"10.1073\/pnas.0937635100","volume":"100","author":"M Miura","year":"2003","unstructured":"Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100:5807\u201312.","journal-title":"Proc Natl Acad Sci U S A"},{"key":"149_CR5","doi-asserted-by":"publisher","first-page":"166","DOI":"10.1016\/j.joen.2007.11.021","volume":"34","author":"W Sonoyama","year":"2008","unstructured":"Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod. 2008;34:166\u201371.","journal-title":"J Endod"},{"key":"149_CR6","doi-asserted-by":"publisher","first-page":"433","DOI":"10.1007\/s00784-009-0310-4","volume":"14","author":"C Morsczeck","year":"2010","unstructured":"Morsczeck C, Vollner F, Saugspier M, Brandl C, Reichert TE, Driemel O, et al. Comparison of human dental follicle cells (DFCs) and stem cells from human exfoliated deciduous teeth (SHED) after neural differentiation in vitro. Clin Oral Investig. 2010;14:433\u201340.","journal-title":"Clin Oral Investig"},{"key":"149_CR7","doi-asserted-by":"publisher","first-page":"13625","DOI":"10.1073\/pnas.240309797","volume":"97","author":"S Gronthos","year":"2000","unstructured":"Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acd Sci USA. 2000;97:13625\u201330.","journal-title":"Proc Natl Acd Sci USA"},{"key":"149_CR8","doi-asserted-by":"publisher","first-page":"501","DOI":"10.1016\/j.joms.2008.09.011","volume":"67","author":"N Koyama","year":"2009","unstructured":"Koyama N, Okubo Y, Nakao K, Bessho K. Evaluation of pluripotency in human dental pulp cells. J Oral Maxillofac Surg. 2009;67:501\u20136.","journal-title":"J Oral Maxillofac Surg"},{"key":"149_CR9","doi-asserted-by":"publisher","first-page":"1536","DOI":"10.1016\/j.joen.2009.07.024","volume":"35","author":"S Nakamura","year":"2009","unstructured":"Nakamura S, Yamada Y, Katagiri W, Sugito T, Ito K, Ueda M. Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. J Endod. 2009;35:1536\u201342.","journal-title":"J Endod"},{"key":"149_CR10","doi-asserted-by":"publisher","first-page":"95","DOI":"10.3892\/mmr.2016.5214","volume":"14","author":"N Zhang","year":"2016","unstructured":"Zhang N, Chen B, Wang W, Chen C, Kang J, Deng SQ, et al. Isolation, characterization and multi-lineage differentiation of stem cells from human exfoliated deciduous teeth. Mol Med Rep. 2016;14:95\u2013102.","journal-title":"Mol Med Rep"},{"key":"149_CR11","doi-asserted-by":"publisher","first-page":"295","DOI":"10.1007\/s13577-020-00323-z","volume":"33","author":"F Xie","year":"2020","unstructured":"Xie F, He J, Chen Y, Hu Z, Qin M, Hui T. Multi-lineage differentiation and clinical application of stem cells from exfoliated deciduous teeth. Hum Cell. 2020;33:295\u2013302.","journal-title":"Hum Cell"},{"key":"149_CR12","doi-asserted-by":"publisher","first-page":"445","DOI":"10.1002\/sctm.19-0398","volume":"9","author":"X Shi","year":"2020","unstructured":"Shi X, Mao J, Liu Y. Pulp stem cells derived from human permanent and deciduous teeth: Biological characteristics and therapeutic applications. Stem Cells Transl Med. 2020;9:445\u201364.","journal-title":"Stem Cells Transl Med"},{"key":"149_CR13","doi-asserted-by":"publisher","first-page":"4904","DOI":"10.3390\/ijms20194904","volume":"20","author":"E Inada","year":"2019","unstructured":"Inada E, Saitoh I, Kubota N, Iwase Y, Kiyokawa Y, Shibasaki S, et al. piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential. Int J Mol Sci. 2019;20:4904.","journal-title":"Int J Mol Sci"},{"key":"149_CR14","doi-asserted-by":"crossref","unstructured":"Inada E, Saitoh I, Kubota N, Soda M, Matsueda K, Murakami T, et al. Alkaline phosphatase and OCT-3\/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells. J Investig Clin Dent. 2017; 8(4).","DOI":"10.1111\/jicd.12236"},{"key":"149_CR15","doi-asserted-by":"publisher","first-page":"4395","DOI":"10.1007\/s12035-018-1367-3","volume":"56","author":"K Ganapathy","year":"2019","unstructured":"Ganapathy K, Datta I, Bhonde R. Astrocyte-Like Cells Differentiated from Dental Pulp Stem Cells Protect Dopaminergic Neurons Against 6-Hydroxydopamine Toxicity. Mol Neurobiol. 2019;56:4395\u2013413.","journal-title":"Mol Neurobiol"},{"key":"149_CR16","doi-asserted-by":"publisher","first-page":"899","DOI":"10.1089\/ten.tea.2011.0311","volume":"18","author":"K Schriebl","year":"2012","unstructured":"Schriebl K, Satianegara G, Hwang A, Tan HL, Fong WJ, Yang HH, et al. Selective removal of undifferentiated human embryonic stem cells using magnetic activated cell sorting followed by a cytotoxic antibody. Tissue Eng Part A. 2012;18:899\u2013909.","journal-title":"Tissue Eng Part A"},{"key":"149_CR17","doi-asserted-by":"publisher","first-page":"7959","DOI":"10.1038\/srep07959","volume":"5","author":"G Welzel","year":"2015","unstructured":"Welzel G, Seitz D, Schuster S. Magnetic-activated cell sorting (MACS) can be used as a large-scale method for establishing zebrafish neuronal cell cultures. Sci Rep. 2015;5:7959.","journal-title":"Sci Rep"},{"key":"149_CR18","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1016\/S0958-1669(02)00009-5","volume":"14","author":"SF Ibrahim","year":"2003","unstructured":"Ibrahim SF, van den Engh G. High-speed cell sorting: fundamentals and recent advances. Curr Opin Biotechnol. 2003;14:5\u201312.","journal-title":"Curr Opin Biotechnol"},{"key":"149_CR19","doi-asserted-by":"crossref","unstructured":"Sato M, Kubota N, Inada E, Saitoh I, Ohtsuka, M, Nakamura S, et al. Hela Cells Consist of Two Cell Types, as Evidenced by Cytochemical Staining for Alkaline Phosphatase Activity: A Possible Model for Cancer Stem Cell Study. Advance in Sten Cells. 2013:15. Article ID 208514.","DOI":"10.5171\/2013.208514"},{"key":"149_CR20","doi-asserted-by":"publisher","first-page":"618","DOI":"10.1038\/nrg3542","volume":"14","author":"E Shapiro","year":"2013","unstructured":"Shapiro E, Biezuner T, Linnarsson S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet. 2013;14:618\u201330.","journal-title":"Nat Rev Genet"},{"key":"149_CR21","doi-asserted-by":"publisher","first-page":"407","DOI":"10.1111\/1574-6976.12015","volume":"37","author":"PC Blainey","year":"2013","unstructured":"Blainey PC. The future is now: single-cell genomics of bacteria and archaea. FEMS Microbiol Rev. 2013;37:407\u201327.","journal-title":"FEMS Microbiol Rev"},{"key":"149_CR22","doi-asserted-by":"publisher","first-page":"8845","DOI":"10.1093\/nar\/gku555","volume":"42","author":"AE Saliba","year":"2014","unstructured":"Saliba AE, Westermann AJ, Gorski SA, Vogel J. Single-cell RNA-seq: advances and future challenges. Nucleic Acids Res. 2014;42:8845\u201360.","journal-title":"Nucleic Acids Res"},{"key":"149_CR23","doi-asserted-by":"publisher","first-page":"1191","DOI":"10.1038\/srep01191","volume":"3","author":"N Yoshimoto","year":"2013","unstructured":"Yoshimoto N, Kida A, Jie X, Kurokawa M, Iijima M, Niimi T, et al. An automated system for high-throughput single cell-based breeding. Sci Rep. 2013;3:1191.","journal-title":"Sci Rep"},{"key":"149_CR24","first-page":"77590W_1","volume":"7759","author":"A P Hsiao","year":"2010","unstructured":"Hsiao A P, Barbee K D, Huang X. Microfluidic Device for Capture and Isolation of Single Cells. Proc SPIE Int Soc Opt Eng. 2010;7759:77590W_1.","journal-title":"Proc SPIE Int Soc Opt Eng"},{"key":"149_CR25","doi-asserted-by":"publisher","first-page":"4955","DOI":"10.1002\/j.1460-2075.1993.tb06189.x","volume":"12","author":"R Kuppers","year":"1993","unstructured":"Kuppers R, Zhao M, Hansmann ML, Rajewsky K. Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J. 1993;12:4955\u201367.","journal-title":"EMBO J"},{"key":"149_CR26","volume-title":"Manipulating the mouse embryo: A Laboratory Manual","author":"B Hogan","year":"1994","unstructured":"Hogan B, Beddington R, Costantini F, Lacy E. Manipulating the mouse embryo: A Laboratory Manual. 2nd ed. NewYork: Cold Spring Harbor Laboratory Press; 1994.","edition":"2"},{"key":"149_CR27","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1016\/j.jim.2004.08.010","volume":"294","author":"HR Bohnenkamp","year":"2004","unstructured":"Bohnenkamp HR, Burchell JM, Taylor-Papadimitriou J, Noll T. Apoptosis of monocytes and the influence on yield of monocyte-derived dendritic cells. J Immunol Methods. 2004;294:67\u201380.","journal-title":"J Immunol Methods"},{"key":"149_CR28","doi-asserted-by":"publisher","first-page":"333","DOI":"10.1002\/(SICI)1097-0320(19990801)36:4<333::AID-CYTO8>3.0.CO;2-F","volume":"36","author":"YW Chu","year":"1999","unstructured":"Chu YW, Wang R, Schmid I, Sakamoto KM. Analysis with flow cytometry of green fluorescent protein expression in leukemic cells. Cytometry. 1999;36:333\u20139.","journal-title":"Cytometry"},{"key":"149_CR29","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1002\/cyto.b.20367","volume":"74","author":"R Bekkema","year":"2008","unstructured":"Bekkema R, Tadema A, Daenen SM, Kluin-Nelemans HC, Mulder AB. An improved flow cytometric method using FACS Lysing Solution for measurement of ZAP-70 expression in B-cell chronic lymphocytic leukemia. Cytometry B Clin Cytom. 2008;74:40\u20134.","journal-title":"Cytometry B Clin Cytom"},{"key":"149_CR30","doi-asserted-by":"publisher","first-page":"e73849","DOI":"10.1371\/journal.pone.0073849","volume":"8","author":"JN Russell","year":"2013","unstructured":"Russell JN, Clements JE, Gama L. Quantitation of gene expression in formaldehyde-fixed and fluorescence-activated sorted cells. PLoS ONE. 2013;8:e73849.","journal-title":"PLoS ONE"},{"key":"149_CR31","doi-asserted-by":"publisher","first-page":"1391","DOI":"10.1016\/S0140-6736(89)92837-7","volume":"1","author":"DP Jackson","year":"1989","unstructured":"Jackson DP, Quirke P, Lewis F, Boylston AW, Sloan JM, Robertson D, et al. Detection of measles virus RNA in paraffin-embedded tissue. Lancet. 1989;1:1391.","journal-title":"Lancet"},{"key":"149_CR32","doi-asserted-by":"publisher","first-page":"176","DOI":"10.1016\/0006-291X(91)90502-X","volume":"174","author":"F von Weizsacker","year":"1991","unstructured":"von Weizsacker F, Labeit S, Koch HK, Oehlert W, Gerok W, Blum HE. A simple and rapid method for the detection of RNA in formalin-fixed, paraffin-embedded tissues by PCR amplification. Biochem Biophys Res Commun. 1991;174:176\u201380.","journal-title":"Biochem Biophys Res Commun"},{"key":"149_CR33","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1016\/S1525-1578(10)60621-6","volume":"2","author":"TE Godfrey","year":"2000","unstructured":"Godfrey TE, Kim SH, Chavira M, Ruff DW, Warren RS, Gray JW, et al. Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5\u2019 nuclease quantitative reverse transcription-polymerase chain reaction. J Mol Diagn. 2000;2:84\u201391.","journal-title":"J Mol Diagn"},{"key":"149_CR34","doi-asserted-by":"publisher","first-page":"277","DOI":"10.1097\/00019606-199710000-00005","volume":"6","author":"RB Guerrero","year":"1997","unstructured":"Guerrero RB, Batts KP, Brandhagen DJ, Germer JJ, Perez RG, Persing DH. Effects of formalin fixation and prolonged block storage on detection of hepatitis C virus RNA in liver tissue. Diagn Mol Pathol. 1997;6:277\u201381.","journal-title":"Diagn Mol Pathol"},{"key":"149_CR35","doi-asserted-by":"publisher","first-page":"2198","DOI":"10.1098\/rstb.2011.0016","volume":"366","author":"K Okita","year":"2011","unstructured":"Okita K, Yamanaka S. Induced pluripotent stem cells: opportunities and challenges. Philos Trans R Soc Lond B Biol Sci. 2011;366:2198\u2013207.","journal-title":"Philos Trans R Soc Lond B Biol Sci"},{"key":"149_CR36","doi-asserted-by":"publisher","first-page":"1546","DOI":"10.1089\/scd.2014.0552","volume":"24","author":"G Xu","year":"2015","unstructured":"Xu G, Yang L, Zhang W, Wei X. All the Tested Human Somatic Cells Express Both Oct4A and Its Pseudogenes but Express Oct4A at Much Lower Levels Compared with Its Pseudogenes and Human Embryonic Stem Cells. Stem Cells Dev. 2015;24:1546\u201357.","journal-title":"Stem Cells Dev"},{"key":"149_CR37","doi-asserted-by":"publisher","first-page":"364","DOI":"10.1016\/j.stem.2007.09.003","volume":"1","author":"S Liedtke","year":"2007","unstructured":"Liedtke S, Enczmann J, Waclawczyk S, Wernet P, Kogler G. Oct4 and its pseudogenes confuse stem cell research. Cell Stem Cell. 2007;1:364\u20136.","journal-title":"Cell Stem Cell"},{"key":"149_CR38","doi-asserted-by":"publisher","first-page":"1651","DOI":"10.3390\/ijms20071651","volume":"20","author":"E Inada","year":"2019","unstructured":"Inada E, Saitoh I, Kubota N, Iwase Y, Murakami T, Sawami T, et al. Increased Expression of Cell Surface SSEA-1 is Closely Associated with Naive-Like Conversion from Human Deciduous Teeth Dental Pulp Cells-Derived iPS Cells. Int J Mol Sci. 2019;20:1651.","journal-title":"Int J Mol Sci"},{"key":"149_CR39","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1016\/j.jim.2005.12.004","volume":"310","author":"MD Chapman","year":"2006","unstructured":"Chapman MD, Keir G, Petzold A, Thompson EJ. Measurement of high affinity antibodies on antigen-immunoblots. J Immunol Methods. 2006;310:62\u20136.","journal-title":"J Immunol Methods"}],"container-title":["Biological Procedures Online"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12575-021-00149-5.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12575-021-00149-5\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12575-021-00149-5.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,6,11]],"date-time":"2021-06-11T07:04:10Z","timestamp":1623395050000},"score":1,"resource":{"primary":{"URL":"https:\/\/biologicalproceduresonline.biomedcentral.com\/articles\/10.1186\/s12575-021-00149-5"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,11]]},"references-count":39,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2021,12]]}},"alternative-id":["149"],"URL":"https:\/\/doi.org\/10.1186\/s12575-021-00149-5","relation":{},"ISSN":["1480-9222"],"issn-type":[{"value":"1480-9222","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,11]]},"assertion":[{"value":"21 February 2021","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"17 May 2021","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"11 June 2021","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"This study was approved guidelines set by the ethical committee for the use and experimentation of the Kagoshima University Graduate School of Medical and Dental Science (permission no. 27\u201311). Informed consent was obtained from the subjects or their parents prior to their entry into the study.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"12"}}