{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,31]],"date-time":"2026-10-31T04:08:24Z","timestamp":1793419704779,"version":"4.5.0"},"reference-count":41,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,3,4]],"date-time":"2021-03-04T00:00:00Z","timestamp":1614816000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,3,4]],"date-time":"2021-03-04T00:00:00Z","timestamp":1614816000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100003803","name":"University of Hong Kong","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100003803","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["CREATE-528202-2019"],"award-info":[{"award-number":["CREATE-528202-2019"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001805","name":"Canada Foundation for Innovation","doi-asserted-by":"publisher","award":["36138"],"award-info":[{"award-number":["36138"]}],"id":[{"id":"10.13039\/501100001805","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Sonoporation via microbubble-mediated ultrasound exposure has shown potential in drug and gene delivery. However, there is a general lack of mechanistic knowledge on sonoporation-induced cellular impact after membrane resealing, and this issue has made it challenging to apply sonoporation efficiently in practice. Here, we present new evidence on how sonoporation, without endangering immediate cell viability, may disrupt downstream cellular hemostasis in ways that are distinguished from the bioeffects observed in other sonicated and unsonoporated cells. Sonoporation was realized on HL-60 leukemia cells by delivering pulsed ultrasound (1\u00a0MHz frequency, 0.50\u00a0MPa peak negative pressure; 10% duty cycle; 30\u00a0s exposure period; 29.1\u00a0J\/cm<jats:sup>2<\/jats:sup> acoustic energy density) in the presence of lipid-shelled microbubbles (1:1 cell-to-bubble ratio). Results showed that 54.6% of sonoporated cells, despite remaining initially viable, underwent apoptosis or necrosis at 24\u00a0h after sonoporation. Anti-proliferation behavior was also observed in sonoporated cells as their subpopulation size was reduced by 43.8% over 24\u00a0h. Preceding these cytotoxic events, the percentages of sonoporated cells in different cell cycle phases were found to be altered by 12\u00a0h after exposure. As well, for sonoporated cells, their expressions of cytoprotective genes in the heat shock protein-70 (HSP-70) family were upregulated by at least 4.1 fold at 3\u00a0h after exposure. Taken altogether, these findings indicate that sonoporated cells attempted to restore homeostasis after membrane resealing, but many of them ultimately failed to recover. Such mechanistic knowledge should be taken into account to devise more efficient sonoporation-mediated therapeutic protocols.<\/jats:p>","DOI":"10.1038\/s41598-021-84341-3","type":"journal-article","created":{"date-parts":[[2021,3,4]],"date-time":"2021-03-04T11:04:29Z","timestamp":1614855869000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":36,"title":["Sonoporation generates downstream cellular impact after membrane resealing"],"prefix":"10.1038","volume":"11","author":[{"given":"Xinxing","family":"Duan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qian","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer M. F.","family":"Wan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alfred C. H.","family":"Yu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,3,4]]},"reference":[{"key":"84341_CR1","doi-asserted-by":"publisher","first-page":"375","DOI":"10.2147\/DDDT.S31564","volume":"7","author":"S Ibsen","year":"2013","unstructured":"Ibsen, S., Schutt, C. E. & Esener, S. Microbubble-mediated ultrasound therapy: A review of its potential in cancer treatment. Drug Des. Dev. Ther. 7, 375 (2013).","journal-title":"Drug Des. Dev. Ther."},{"key":"84341_CR2","doi-asserted-by":"crossref","unstructured":"Qin, J., Wang, T.-Y., & Willmann, J. K. Sonoporation: Applications for cancer therapy. In: Therapeutic Ultrasound 263\u2013291 (Springer, 2016).","DOI":"10.1007\/978-3-319-22536-4_15"},{"key":"84341_CR3","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1016\/j.gene.2013.03.095","volume":"525","author":"A Delalande","year":"2013","unstructured":"Delalande, A., Kotopoulis, S., Postema, M., Midoux, P. & Pichon, C. Sonoporation: Mechanistic insights and ongoing challenges for gene transfer. Gene 525, 191\u2013199 (2013).","journal-title":"Gene"},{"key":"84341_CR4","doi-asserted-by":"crossref","unstructured":"Bouakaz, A., Zeghimi, A., & Doinikov, A. A. Sonoporation: Concept and mechanisms. in Therapeutic Ultrasound 175\u2013189. (Springer, 2016).","DOI":"10.1007\/978-3-319-22536-4_10"},{"key":"84341_CR5","doi-asserted-by":"crossref","unstructured":"Mignet, N., Marie, C., Delalande, A., Manta, S., Bureau, M.-F., Renault, G., Scherman, D., & Pichon, C. Microbubbles for nucleic acid delivery in liver using mild sonoporation. in Nanotechnology for Nucleic Acid Delivery 377\u2013387 (Springer, 2019).","DOI":"10.1007\/978-1-4939-9092-4_25"},{"key":"84341_CR6","doi-asserted-by":"publisher","first-page":"446","DOI":"10.7150\/thno.13518","volume":"6","author":"SM Nejad","year":"2016","unstructured":"Nejad, S. M., Hosseini, H., Akiyama, H. & Tachibana, K. Reparable cell sonoporation in suspension: Theranostic potential of microbubble. Theranostics 6, 446 (2016).","journal-title":"Theranostics"},{"key":"84341_CR7","doi-asserted-by":"publisher","first-page":"4866","DOI":"10.1016\/j.bpj.2009.02.072","volume":"96","author":"N Kudo","year":"2009","unstructured":"Kudo, N., Okada, K. & Yamamoto, K. Sonoporation by single-shot pulsed ultrasound with microbubbles adjacent to cells. Biophys. J . 96, 4866\u20134876 (2009).","journal-title":"Biophys. J ."},{"key":"84341_CR8","doi-asserted-by":"publisher","first-page":"876","DOI":"10.1016\/j.ultrasmedbio.2012.01.006","volume":"38","author":"Y Liu","year":"2012","unstructured":"Liu, Y., Yan, J. & Prausnitz, M. R. Can ultrasound enable efficient intracellular uptake of molecules? A retrospective literature review and analysis. Ultrasound Med. Biol. 38, 876\u2013888 (2012).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR9","doi-asserted-by":"publisher","first-page":"657","DOI":"10.1016\/j.ultrasmedbio.2006.11.001","volume":"33","author":"P Campbell","year":"2007","unstructured":"Campbell, P. & Prausnitz, M. R. Future directions for therapeutic ultrasound. Ultrasound Med. Biol. 33, 657 (2007).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR10","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1016\/j.addr.2013.11.008","volume":"72","author":"I Lentacker","year":"2014","unstructured":"Lentacker, I., de Cock, I., Deckers, R., de Smedt, S. C. & Moonen, C. T. W. Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms. Adv. Drug Deliv. Rev. 72, 49\u201364 (2014).","journal-title":"Adv. Drug Deliv. Rev."},{"key":"84341_CR11","doi-asserted-by":"publisher","first-page":"2393","DOI":"10.1016\/j.ultrasmedbio.2013.08.003","volume":"39","author":"Y Hu","year":"2013","unstructured":"Hu, Y., Wan, J. M. F. & Yu, A. C. H. Membrane perforation and recovery dynamics in microbubble-mediated sonoporation. Ultrasound Med. Biol. 39, 2393\u20132405 (2013).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR12","doi-asserted-by":"publisher","first-page":"9983","DOI":"10.1073\/pnas.1606915113","volume":"113","author":"B Helfield","year":"2016","unstructured":"Helfield, B., Chen, X., Watkins, S. C. & Villaneuva, F. S. Biophysical insight into mechanisms of sonoporation. Proc. Natl. Acad. Sci. 113, 9983\u20139988 (2016).","journal-title":"Proc. Natl. Acad. Sci."},{"key":"84341_CR13","doi-asserted-by":"publisher","first-page":"20140071","DOI":"10.1098\/rsif.2014.0071","volume":"11","author":"X Chen","year":"2014","unstructured":"Chen, X., Leow, R. S., Hu, Y., Wan, J. M. F. & Yu, A. C. H. Single-site sonoporation disrupts actin cytoskeleton organization. J. R. Soc. Interface 11, 20140071 (2014).","journal-title":"J. R. Soc. Interface"},{"key":"84341_CR14","doi-asserted-by":"publisher","first-page":"2017","DOI":"10.1016\/j.ultrasmedbio.2020.03.029","volume":"46","author":"I Beekers","year":"2020","unstructured":"Beekers, I. et al. High-resolution imaging of intracellular calcium fluctuations by oscillating microbubbles. Ultrasound Med. Biol. 46, 2017\u20132029 (2020).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR15","doi-asserted-by":"publisher","first-page":"20150029","DOI":"10.1098\/rsif.2015.0029","volume":"12","author":"RS Leow","year":"2015","unstructured":"Leow, R. S., Wan, J. M. F. & Yu, A. C. H. Membrane blebbing as a recovery manoeuvre in site-specific sonoporation mediated by targeted microbubbles. J. R. Soc. Interface 12, 20150029 (2015).","journal-title":"J. R. Soc. Interface"},{"key":"84341_CR16","doi-asserted-by":"publisher","first-page":"169","DOI":"10.1016\/j.jconrel.2018.01.001","volume":"272","author":"P Qin","year":"2018","unstructured":"Qin, P., Han, T., Yu, A. C. H. & Xu, L. Mechanistic understanding the bioeffects of ultrasound-driven microbubbles to enhance macromolecule delivery. J. Control. Release 272, 169\u2013181 (2018).","journal-title":"J. Control. Release"},{"key":"84341_CR17","doi-asserted-by":"crossref","unstructured":"Guzman, H. R., Nguyen, D. X., Khan, S., & Prausnitz, M. R. Ultrasound-mediated disruption of cell membranes. II. Heterogeneous effects on cells. J. Acoust. Soc. Am. 110, 597\u2013606 (2001).","DOI":"10.1121\/1.1376130"},{"key":"84341_CR18","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1016\/j.ultsonch.2015.12.001","volume":"31","author":"P Qin","year":"2016","unstructured":"Qin, P., Xu, L., Han, T., Du, L. & Yu, A. C. H. Effect of non-acoustic parameters on heterogeneous sonoporation mediated by single-pulse ultrasound and microbubbles. Ultrason. Sonochem. 31, 107\u2013115 (2016).","journal-title":"Ultrason. Sonochem."},{"key":"84341_CR19","doi-asserted-by":"publisher","first-page":"1008","DOI":"10.1016\/j.ultrasmedbio.2010.03.011","volume":"36","author":"JD Hutcheson","year":"2010","unstructured":"Hutcheson, J. D., Schlicher, R. K., Hicks, H. K. & Prausnitz, M. R. Saving cells from ultrasound-induced apoptosis: Quantification of cell death and uptake following sonication and effects of targeted calcium chelation. Ultrasound Med. Biol. 36, 1008\u20131021 (2010).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR20","doi-asserted-by":"publisher","first-page":"1211","DOI":"10.1016\/S0301-5629(03)00899-8","volume":"29","author":"HR Guzm\u00e1n","year":"2003","unstructured":"Guzm\u00e1n, H. R., McNamara, A. J., Nguyen, D. X. & Prausnitz, M. R. Bioeffects caused by changes in acoustic cavitation bubble density and cell concentration: A unified explanation based on cell-to-bubble ratio and blast radius. Ultrasound Med. Biol. 29, 1211\u20131222 (2003).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR21","doi-asserted-by":"publisher","first-page":"479","DOI":"10.1269\/jrr.45.479","volume":"45","author":"LB Feril","year":"2004","unstructured":"Feril, L. B. & Kondo, T. Biological effects of low intensity ultrasound: the mechanism involved, and its implications on therapy and on biosafety of ultrasound. J. Radiat. Res. 45, 479\u2013489 (2004).","journal-title":"J. Radiat. Res."},{"key":"84341_CR22","doi-asserted-by":"publisher","first-page":"144","DOI":"10.1016\/j.ultrasmedbio.2008.06.007","volume":"35","author":"DL Miller","year":"2009","unstructured":"Miller, D. L. & Dou, C. Induction of apoptosis in sonoporation and ultrasonic gene transfer. Ultrasound Med. Biol. 35, 144\u2013154 (2009).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR23","doi-asserted-by":"publisher","first-page":"2149","DOI":"10.1016\/j.ultrasmedbio.2011.09.012","volume":"37","author":"W Zhong","year":"2011","unstructured":"Zhong, W., Sit, W. H., Wan, J. M. F. & Yu, A. C. H. Sonoporation induces apoptosis and cell cycle arrest in human promyelocytic leukemia cells. Ultrasound Med. Biol. 37, 2149\u20132159 (2011).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR24","doi-asserted-by":"publisher","first-page":"64","DOI":"10.17305\/bjbms.2012.2497","volume":"12","author":"J-Z Zhang","year":"2012","unstructured":"Zhang, J.-Z., Saggar, J. K., Zhou, Z.-L. & Hu, B. Different effects of sonoporation on cell morphology and viability. Bosnian J. Basic Med. Sci. 12, 64\u201368 (2012).","journal-title":"Bosnian J. Basic Med. Sci."},{"key":"84341_CR25","doi-asserted-by":"publisher","first-page":"1075","DOI":"10.1016\/j.ultrasmedbio.2013.01.008","volume":"39","author":"X Chen","year":"2013","unstructured":"Chen, X., Wan, J. M. F. & Yu, A. C. H. Sonoporation as a cellular stress: Induction of morphological repression and developmental delays. Ultrasound Med. Biol. 39, 1075\u20131086 (2013).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR26","doi-asserted-by":"publisher","first-page":"670","DOI":"10.1007\/s00018-004-4464-6","volume":"62","author":"MP Mayer","year":"2005","unstructured":"Mayer, M. P. & Bukau, B. Hsp70 chaperones: Cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62, 670\u2013684 (2005).","journal-title":"Cell. Mol. Life Sci."},{"key":"84341_CR27","doi-asserted-by":"publisher","first-page":"1422","DOI":"10.1109\/TUFFC.2019.2923443","volume":"66","author":"X Duan","year":"2019","unstructured":"Duan, X., Yu, A. C. H. & Wan, J. M. F. Cellular bioeffect investigations on low-intensity pulsed ultrasound and sonoporation: Platform design and flow cytometry protocol. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 66, 1422\u20131434 (2019).","journal-title":"IEEE Trans. Ultrason. Ferroelectr. Freq. Control"},{"key":"84341_CR28","doi-asserted-by":"publisher","first-page":"790","DOI":"10.1073\/pnas.82.3.790","volume":"82","author":"D Wolf","year":"1985","unstructured":"Wolf, D. & Rotter, V. Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells. Proc. Natl. Acad. Sci. 82, 790\u2013794 (1985).","journal-title":"Proc. Natl. Acad. Sci."},{"key":"84341_CR29","doi-asserted-by":"publisher","first-page":"2382","DOI":"10.1016\/j.ultrasmedbio.2013.08.005","volume":"39","author":"W Zhong","year":"2013","unstructured":"Zhong, W. et al. Induction of endoplasmic reticulum stress by sonoporation: Linkage to mitochondria-mediated apoptosis initiation. Ultrasound Med. Biol. 39, 2382\u20132392 (2013).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR30","doi-asserted-by":"publisher","first-page":"379","DOI":"10.1007\/s12192-016-0676-6","volume":"21","author":"J Radons","year":"2016","unstructured":"Radons, J. The human HSP70 family of chaperones: Where do we stand?. Cell Stress Chaperones 21, 379\u2013404 (2016).","journal-title":"Cell Stress Chaperones"},{"key":"84341_CR31","doi-asserted-by":"publisher","first-page":"1587","DOI":"10.1016\/j.ultrasmedbio.2014.01.003","volume":"40","author":"Y Hu","year":"2014","unstructured":"Hu, Y., Wan, J. M. F. & Yu, A. C. H. Cytomechanical perturbations during low-intensity ultrasound pulsing. Ultrasound Med. Biol. 40, 1587\u20131598 (2014).","journal-title":"Ultrasound Med. Biol."},{"key":"84341_CR32","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1158\/1535-7163.537.3.5","volume":"3","author":"E Crescenzi","year":"2004","unstructured":"Crescenzi, E. et al. Photodynamic therapy with indocyanine green complements and enhances low-dose cisplatin cytotoxicity in MCF-7 breast cancer cells. Mol. Cancer Ther. 3, 537\u2013544 (2004).","journal-title":"Mol. Cancer Ther."},{"key":"84341_CR33","doi-asserted-by":"publisher","first-page":"8697","DOI":"10.1158\/1078-0432.CCR-04-1166","volume":"10","author":"TO Kirby","year":"2004","unstructured":"Kirby, T. O. et al. A novel ex vivo model system for evaluation of conditionally replicative adenoviruses therapeutic efficacy and toxicity. Clin. Cancer Res. 10, 8697\u20138703 (2004).","journal-title":"Clin. Cancer Res."},{"key":"84341_CR34","doi-asserted-by":"publisher","first-page":"423","DOI":"10.1593\/neo.04169","volume":"6","author":"M Naidu","year":"2004","unstructured":"Naidu, M. et al. Chemotherapy-induced and\/or radiation therapy-induced oral mucositis\u2014Complicating the treatment of cancer. Neoplasia 6, 423\u2013431 (2004).","journal-title":"Neoplasia"},{"key":"84341_CR35","doi-asserted-by":"crossref","unstructured":"Chugh, R. et al. A preclinical evaluation of Minnelide as a therapeutic agent against pancreatic cancer. Sci. Transl. Med. 4, 156ra39 (2012).","DOI":"10.1126\/scitranslmed.3004334"},{"key":"84341_CR36","doi-asserted-by":"publisher","first-page":"8441","DOI":"10.1158\/1078-0432.CCR-05-1646","volume":"11","author":"L Varghese","year":"2005","unstructured":"Varghese, L., Agarwal, C., Tyagi, A., Singh, R. P. & Agarwal, R. Silibinin efficacy against human hepatocellular carcinoma. Clin. Cancer Res. 11, 8441\u20138448 (2005).","journal-title":"Clin. Cancer Res."},{"key":"84341_CR37","doi-asserted-by":"publisher","first-page":"512","DOI":"10.1016\/j.ultsonch.2018.12.031","volume":"52","author":"M Cheng","year":"2019","unstructured":"Cheng, M., Li, F., Han, T., Yu, A. C. H. & Qin, P. Effects of ultrasound pulse parameters on cavitation properties of flowing microbubbles under physiologically relevant conditions. Ultrason. Sonochem. 52, 512\u2013521 (2019).","journal-title":"Ultrason. Sonochem."},{"key":"84341_CR38","doi-asserted-by":"publisher","first-page":"57","DOI":"10.1007\/s10616-005-2106-y","volume":"46","author":"L Moc\u00e9-Llivina","year":"2004","unstructured":"Moc\u00e9-Llivina, L. & Jofre, J. A method to maintain mammalian cells for days alive at 4 C. Cytotechnology 46, 57\u201361 (2004).","journal-title":"Cytotechnology"},{"key":"84341_CR39","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1016\/0022-1759(95)00072-I","volume":"184","author":"I Vermes","year":"1995","unstructured":"Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutellingsperger, C. A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J. Immunol. Methods 184, 39\u201351 (1995).","journal-title":"J. Immunol. Methods"},{"key":"84341_CR40","first-page":"67","volume":"3","author":"R Nunez","year":"2001","unstructured":"Nunez, R. DNA measurement and cell cycle analysis by flow cytometry. Curr. Issues Mol. Biol. 3, 67\u201370 (2001).","journal-title":"Curr. Issues Mol. Biol."},{"key":"84341_CR41","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1006\/meth.2001.1262","volume":"25","author":"KJ Livak","year":"2001","unstructured":"Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2\u2212\u0394\u0394CT method. Methods 25, 402\u2013408 (2001).","journal-title":"Methods"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-84341-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-84341-3","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-84341-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,3]],"date-time":"2022-12-03T04:49:52Z","timestamp":1670042992000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-84341-3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,4]]},"references-count":41,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2021,12]]}},"alternative-id":["84341"],"URL":"https:\/\/doi.org\/10.1038\/s41598-021-84341-3","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,4]]},"assertion":[{"value":"27 September 2020","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"15 February 2021","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"4 March 2021","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"5161"}}