{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T01:23:23Z","timestamp":1771464203265,"version":"3.50.1"},"reference-count":88,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2023,2,5]],"date-time":"2023-02-05T00:00:00Z","timestamp":1675555200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2023,2,5]],"date-time":"2023-02-05T00:00:00Z","timestamp":1675555200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["UIDB\/50026\/2020;"],"award-info":[{"award-number":["UIDB\/50026\/2020;"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Norte Portugal Regional Operational Programme","award":["NORTE-01-0145-FEDER-000013; NORTE-01-0145-FEDER-000023"],"award-info":[{"award-number":["NORTE-01-0145-FEDER-000013; NORTE-01-0145-FEDER-000023"]}]},{"name":"ICVS Scientific Microscopy Platform; PPBI - Portuguese Platform of Bioimaging","award":["PPBI-POCI-01-0145-FEDER-022122"],"award-info":[{"award-number":["PPBI-POCI-01-0145-FEDER-022122"]}]},{"name":"FCT; Jos\u00e9 de Melo Sa\u00fade-2CA","award":["PD\/BDE\/127829\/2016"],"award-info":[{"award-number":["PD\/BDE\/127829\/2016"]}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Respir Res"],"abstract":"<jats:title>Abstract<\/jats:title><jats:sec>\n                <jats:title>Background<\/jats:title>\n                <jats:p>Clinical and experimental evidence shows lung fluid volume as a modulator of fetal lung growth with important value in treating fetal lung hypoplasia. Thus, understanding the mechanisms underlying these morphological dynamics has been the topic of multiple investigations with, however, limited results, partially due to the difficulty of capturing or recapitulating these movements in the lab. In this sense, this study aims to establish an ex vivo model allowing the study of lung fluid function in branching morphogenesis and identify the subsequent molecular\/ cellular mechanisms.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>Ex vivo lung explant culture was selected as a model to study branching morphogenesis, and intraluminal injections were performed to change the composition of lung fluid. Distinct chloride (Cl<jats:sup>\u2212<\/jats:sup>) concentrations (5.8, 29, 143, and 715\u00a0mM) or Cl<jats:sup>\u2212<\/jats:sup> channels inhibitors [antracene-9-carboxylic acid (A9C), cystic fibrosis transmembrane conductance regulator inhibitor172 (CFTRinh), and calcium-dependent Cl<jats:sup>\u2212<\/jats:sup> channel inhibitorA01 (CaCCinh)] were injected into lung lumen at two timepoints, day0 (D0) and D2. At D4, morphological and molecular analyses were performed in terms of branching morphogenesis, spatial distribution (immunofluorescence), and protein quantification (western blot) of mechanoreceptors (PIEZO1 and PIEZO2), neuroendocrine (bombesin, ghrelin, and PGP9.5) and smooth muscle [alpha-smooth muscle actin (\u03b1-SMA) and myosin light chain 2 (MLC2)] markers.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>For the first time, we described effective intraluminal injections at D0 and D2 and demonstrated intraluminal movements at D4 in ex vivo lung explant cultures. Through immunofluorescence assay in in vivo and ex vivo branching morphogenesis, we show that PGP9.5 colocalizes with PIEZO1 and PIEZO2 receptors. Fetal lung growth is increased at higher [Cl<jats:sup>\u2212<\/jats:sup>], 715\u00a0mM Cl<jats:sup>\u2212<\/jats:sup>, through the overexpression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and \u03b1-SMA. In contrast, intraluminal injection of CFTRinh or CaCCinh decreases fetal lung growth and the expression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and \u03b1-SMA. Finally, the inhibition of PIEZO1\/PIEZO2 by GsMTx4 decreases branching morphogenesis and ghrelin, bombesin, MLC2, and \u03b1-SMA expression in an intraluminal injection-independent manner.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>Our results identify PIEZO1\/PIEZO2 expressed in neuroendocrine cells as a regulator of fetal lung growth induced by lung fluid.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12931-023-02328-2","type":"journal-article","created":{"date-parts":[[2023,2,5]],"date-time":"2023-02-05T14:02:51Z","timestamp":1675605771000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Intraluminal chloride regulates lung branching morphogenesis: involvement of PIEZO1\/PIEZO2"],"prefix":"10.1186","volume":"24","author":[{"given":"Ana N.","family":"Gon\u00e7alves","sequence":"first","affiliation":[]},{"given":"Rute S.","family":"Moura","sequence":"additional","affiliation":[]},{"given":"Jorge","family":"Correia-Pinto","sequence":"additional","affiliation":[]},{"given":"Cristina","family":"Nogueira-Silva","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2023,2,5]]},"reference":[{"key":"2328_CR1","doi-asserted-by":"publisher","first-page":"951","DOI":"10.1203\/00006450-198610000-00012","volume":"20","author":"AC Moessinger","year":"1986","unstructured":"Moessinger AC, Collins MH, Blanc WA, Rey HR, James LS. Oligohydramnios-induced lung hypoplasia: the influence of timing and duration in gestation. Pediatr Res. 1986;20:951\u20134.","journal-title":"Pediatr Res"},{"key":"2328_CR2","doi-asserted-by":"publisher","first-page":"1904","DOI":"10.1016\/0002-9378(90)90772-Y","volume":"163","author":"R Harding","year":"1990","unstructured":"Harding R, Hooper SB, Dickson KA. A mechanism leading to reduced lung expansion and lung hypoplasia in fetal sheep during oligohydramnios. Am J Obstet Gynecol. 1990;163:1904\u201313.","journal-title":"Am J Obstet Gynecol"},{"issue":"Suppl A","key":"2328_CR3","doi-asserted-by":"publisher","first-page":"S259","DOI":"10.1016\/S1526-0542(04)90049-8","volume":"5","author":"I Copland","year":"2004","unstructured":"Copland I, Post M. Lung development and fetal lung growth. Paediatr Respir Rev. 2004;5(Suppl A):S259-264.","journal-title":"Paediatr Respir Rev"},{"key":"2328_CR4","doi-asserted-by":"publisher","first-page":"651","DOI":"10.1378\/chest.06-2663","volume":"132","author":"W Shi","year":"2007","unstructured":"Shi W, Bellusci S, Warburton D. Lung development and adult lung diseases. Chest. 2007;132:651\u20136.","journal-title":"Chest"},{"key":"2328_CR5","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1016\/j.resp.2007.10.004","volume":"159","author":"SM Wilson","year":"2007","unstructured":"Wilson SM, Olver RE, Walters DV. Developmental regulation of lumenal lung fluid and electrolyte transport. Respir Physiol Neurobiol. 2007;159:247\u201355.","journal-title":"Respir Physiol Neurobiol"},{"key":"2328_CR6","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1002\/uog.11064","volume":"39","author":"JC Jani","year":"2012","unstructured":"Jani JC, Nicolaides KH. Fetal surgery for severe congenital diaphragmatic hernia? Ultrasound Obstet Gynecol. 2012;39:7\u20139.","journal-title":"Ultrasound Obstet Gynecol"},{"key":"2328_CR7","doi-asserted-by":"publisher","first-page":"64","DOI":"10.1002\/uog.6141","volume":"33","author":"JC Jani","year":"2009","unstructured":"Jani JC, Benachi A, Nicolaides KH, Allegaert K, Gratac\u00f3s E, Mazkereth R, Matis J, Tibboel D, Van Heijst A, Storme L, et al. Prenatal prediction of neonatal morbidity in survivors with congenital diaphragmatic hernia: a multicenter study. Ultrasound Obstet Gynecol. 2009;33:64\u20139.","journal-title":"Ultrasound Obstet Gynecol"},{"key":"2328_CR8","doi-asserted-by":"publisher","first-page":"1459","DOI":"10.1080\/14767058.2019.1636029","volume":"34","author":"AN Gon\u00e7alves","year":"2021","unstructured":"Gon\u00e7alves AN, Correia-Pinto J, Nogueira-Silva C. Imagiological methods for prediction of fetal pulmonary hypoplasia: a systematic review. J Matern Fetal Neonatal Med. 2021;34:1459\u201368.","journal-title":"J Matern Fetal Neonatal Med"},{"key":"2328_CR9","doi-asserted-by":"publisher","first-page":"531","DOI":"10.1113\/jphysiol.1987.sp016506","volume":"385","author":"KA Dickson","year":"1987","unstructured":"Dickson KA, Harding R. Restoration of lung liquid volume following its acute alteration in fetal sheep. J Physiol. 1987;385:531\u201343.","journal-title":"J Physiol"},{"key":"2328_CR10","doi-asserted-by":"publisher","first-page":"3976","DOI":"10.1210\/endo.141.11.7770","volume":"141","author":"AJ Fletcher","year":"2000","unstructured":"Fletcher AJ, Edwards CM, Gardner DS, Fowden AL, Giussani DA. Neuropeptide Y in the sheep fetus: effects of acute hypoxemia and dexamethasone during late gestation. Endocrinology. 2000;141:3976\u201382.","journal-title":"Endocrinology"},{"key":"2328_CR11","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1002\/ajmg.c.30127","volume":"145","author":"PA Khan","year":"2007","unstructured":"Khan PA, Cloutier M, Piedboeuf B. Tracheal occlusion: a review of obstructing fetal lungs to make them grow and mature. Am J Med Genet C Semin Med Genet. 2007;145:125\u201338.","journal-title":"Am J Med Genet C Semin Med Genet"},{"key":"2328_CR12","doi-asserted-by":"publisher","first-page":"314","DOI":"10.1016\/j.mod.2007.10.013","volume":"125","author":"M Unbekandt","year":"2008","unstructured":"Unbekandt M, del Moral PM, Sala FG, Bellusci S, Warburton D, Fleury V. Tracheal occlusion increases the rate of epithelial branching of embryonic mouse lung via the FGF10-FGFR2b-Sprouty2 pathway. Mech Dev. 2008;125:314\u201324.","journal-title":"Mech Dev"},{"key":"2328_CR13","doi-asserted-by":"publisher","first-page":"78.e71","DOI":"10.1016\/j.ajog.2017.02.041","volume":"217","author":"JA Jim\u00e9nez","year":"2017","unstructured":"Jim\u00e9nez JA, Eixarch E, DeKoninck P, Bennini JR, Devlieger R, Peralta CF, Gratacos E, Deprest J. Balloon removal after fetoscopic endoluminal tracheal occlusion for congenital diaphragmatic hernia. Am J Obstet Gynecol. 2017;217:78.e71-78.e11.","journal-title":"Am J Obstet Gynecol"},{"key":"2328_CR14","doi-asserted-by":"publisher","first-page":"70","DOI":"10.1002\/uog.12414","volume":"42","author":"R Ruano","year":"2013","unstructured":"Ruano R, Peiro JL, da Silva MM, Campos JA, Carreras E, Tannuri U, Zugaib M. Early fetoscopic tracheal occlusion for extremely severe pulmonary hypoplasia in isolated congenital diaphragmatic hernia: preliminary results. Ultrasound Obstet Gynecol. 2013;42:70\u20136.","journal-title":"Ultrasound Obstet Gynecol"},{"key":"2328_CR15","doi-asserted-by":"publisher","first-page":"1071","DOI":"10.1007\/s00431-016-2742-6","volume":"175","author":"K Ali","year":"2016","unstructured":"Ali K, Bendapudi P, Polubothu S, Andradi G, Ofuya M, Peacock J, Hickey A, Davenport M, Nicolaides K, Greenough A. Congenital diaphragmatic hernia-influence of fetoscopic tracheal occlusion on outcomes and predictors of survival. Eur J Pediatr. 2016;175:1071\u20136.","journal-title":"Eur J Pediatr"},{"key":"2328_CR16","doi-asserted-by":"publisher","first-page":"979","DOI":"10.1097\/SLA.0000000000002595","volume":"269","author":"N Khoshgoo","year":"2019","unstructured":"Khoshgoo N, Kholdebarin R, Pereira-Terra P, Mahood TH, Falk L, Day CA, Iwasiow BM, Zhu F, Mulhall D, Fraser C, et al. Prenatal microRNA miR-200b therapy improves nitrofen-induced pulmonary hypoplasia associated with congenital diaphragmatic hernia. Ann Surg. 2019;269:979\u201387.","journal-title":"Ann Surg"},{"key":"2328_CR17","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1165\/ajrcmb.25.1.4500","volume":"25","author":"DJ Gillie","year":"2001","unstructured":"Gillie DJ, Pace AJ, Coakley RJ, Koller BH, Barker PM. Liquid and ion transport by fetal airway and lung epithelia of mice deficient in sodium-potassium-2-chloride transporter. Am J Respir Cell Mol Biol. 2001;25:14\u201320.","journal-title":"Am J Respir Cell Mol Biol"},{"key":"2328_CR18","doi-asserted-by":"publisher","first-page":"6007","DOI":"10.1113\/jphysiol.2008.161687","volume":"586","author":"BA Finney","year":"2008","unstructured":"Finney BA, del Moral PM, Wilkinson WJ, Cayzac S, Cole M, Warburton D, Kemp PJ, Riccardi D. Regulation of mouse lung development by the extracellular calcium-sensing receptor. CaR J Physiol. 2008;586:6007\u201319.","journal-title":"CaR J Physiol"},{"key":"2328_CR19","doi-asserted-by":"publisher","first-page":"L145","DOI":"10.1152\/ajplung.90525.2008","volume":"296","author":"O Bardou","year":"2009","unstructured":"Bardou O, Trinh NT, Brochiero E. Molecular diversity and function of K+ channels in airway and alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2009;296:L145-155.","journal-title":"Am J Physiol Lung Cell Mol Physiol"},{"key":"2328_CR20","doi-asserted-by":"publisher","first-page":"21975","DOI":"10.1038\/srep21975","volume":"6","author":"SC Brennan","year":"2016","unstructured":"Brennan SC, Wilkinson WJ, Tseng HE, Finney B, Monk B, Dibble H, Quilliam S, Warburton D, Galietta LJ, Kemp PJ, Riccardi D. The extracellular calcium-sensing receptor regulates human fetal lung development via CFTR. Sci Rep. 2016;6:21975.","journal-title":"Sci Rep"},{"key":"2328_CR21","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0080294","volume":"8","author":"SC Brennan","year":"2013","unstructured":"Brennan SC, Finney BA, Lazarou M, Rosser AE, Scherf C, Adriaensen D, Kemp PJ, Riccardi D. Fetal calcium regulates branching morphogenesis in the developing human and mouse lung: involvement of voltage-gated calcium channels. PLoS ONE. 2013;8: e80294.","journal-title":"PLoS ONE"},{"key":"2328_CR22","doi-asserted-by":"publisher","first-page":"327","DOI":"10.1113\/jphysiol.1974.sp010659","volume":"241","author":"RE Olver","year":"1974","unstructured":"Olver RE, Strang LB. Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the foetal lamb. J Physiol. 1974;241:327\u201357.","journal-title":"J Physiol"},{"key":"2328_CR23","doi-asserted-by":"publisher","first-page":"395","DOI":"10.1113\/jphysiol.1981.sp013754","volume":"315","author":"RE Olver","year":"1981","unstructured":"Olver RE, Schneeberger EE, Walters DV. Epithelial solute permeability, ion transport and tight junction morphology in the developing lung of the fetal lamb. J Physiol. 1981;315:395\u2013412.","journal-title":"J Physiol"},{"key":"2328_CR24","doi-asserted-by":"publisher","first-page":"227","DOI":"10.1007\/BF01870565","volume":"70","author":"MJ Welsh","year":"1982","unstructured":"Welsh MJ, Smith PL, Frizzell RA. Chloride secretion by canine tracheal epithelium: II. The cellular electrical potential profile. J Membr Biol. 1982;70:227\u201338.","journal-title":"J Membr Biol"},{"key":"2328_CR25","doi-asserted-by":"publisher","first-page":"C377","DOI":"10.1152\/ajpcell.1983.244.5.C377","volume":"244","author":"MJ Welsh","year":"1983","unstructured":"Welsh MJ. Evidence for basolateral membrane potassium conductance in canine tracheal epithelium. Am J Physiol. 1983;244:C377-384.","journal-title":"Am J Physiol"},{"key":"2328_CR26","doi-asserted-by":"publisher","first-page":"L420","DOI":"10.1152\/ajplung.00113.2003","volume":"286","author":"CJ Blaisdell","year":"2004","unstructured":"Blaisdell CJ, Morales MM, Andrade AC, Bamford P, Wasicko M, Welling P. Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts. Am J Physiol Lung Cell Mol Physiol. 2004;286:L420-426.","journal-title":"Am J Physiol Lung Cell Mol Physiol"},{"key":"2328_CR27","doi-asserted-by":"publisher","first-page":"L4","DOI":"10.1152\/ajplung.00372.2005","volume":"291","author":"JE Larson","year":"2006","unstructured":"Larson JE, Cohen JC. Improvement of pulmonary hypoplasia associated with congenital diaphragmatic hernia by in utero CFTR gene therapy. Am J Physiol Lung Cell Mol Physiol. 2006;291:L4-10.","journal-title":"Am J Physiol Lung Cell Mol Physiol"},{"key":"2328_CR28","doi-asserted-by":"publisher","first-page":"28698","DOI":"10.1074\/jbc.M109.012120","volume":"284","author":"J Ousingsawat","year":"2009","unstructured":"Ousingsawat J, Martins JR, Schreiber R, Rock JR, Harfe BD, Kunzelmann K. Loss of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport. J Biol Chem. 2009;284:28698\u2013703.","journal-title":"J Biol Chem"},{"key":"2328_CR29","doi-asserted-by":"publisher","first-page":"12771","DOI":"10.1073\/pnas.0906850106","volume":"106","author":"JR Rock","year":"2009","unstructured":"Rock JR, Onaitis MW, Rawlins EL, Lu Y, Clark CP, Xue Y, Randell SH, Hogan BL. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci USA. 2009;106:12771\u20135.","journal-title":"Proc Natl Acad Sci USA"},{"key":"2328_CR30","doi-asserted-by":"publisher","first-page":"825","DOI":"10.1038\/s41374-018-0026-7","volume":"98","author":"DK Meyerholz","year":"2018","unstructured":"Meyerholz DK, Stoltz DA, Gansemer ND, Ernst SE, Cook DP, Strub MD, LeClair EN, Barker CK, Adam RJ, Leidinger MR, et al. Lack of cystic fibrosis transmembrane conductance regulator disrupts fetal airway development in pigs. Lab Invest. 2018;98:825\u201338.","journal-title":"Lab Invest"},{"key":"2328_CR31","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1165\/ajrcmb.23.1.3926","volume":"23","author":"JC Schittny","year":"2000","unstructured":"Schittny JC, Miserocchi G, Sparrow MP. Spontaneous peristaltic airway contractions propel lung liquid through the bronchial tree of intact and fetal lung explants. Am J Respir Cell Mol Biol. 2000;23:11\u20138.","journal-title":"Am J Respir Cell Mol Biol"},{"key":"2328_CR32","doi-asserted-by":"publisher","first-page":"330","DOI":"10.1165\/rcmb.2006-0349OC","volume":"37","author":"M Santos","year":"2007","unstructured":"Santos M, Moura RS, Gonzaga S, Nogueira-Silva C, Ohlmeier S, Correia-Pinto J. Embryonic essential myosin light chain regulates fetal lung development in rats. Am J Respir Cell Mol Biol. 2007;37:330\u20138.","journal-title":"Am J Respir Cell Mol Biol"},{"key":"2328_CR33","doi-asserted-by":"publisher","first-page":"719","DOI":"10.1016\/j.devcel.2015.08.012","volume":"34","author":"HY Kim","year":"2015","unstructured":"Kim HY, Pang MF, Varner VD, Kojima L, Miller E, Radisky DC, Nelson CM. Localized smooth muscle differentiation is essential for epithelial bifurcation during branching morphogenesis of the mammalian lung. Dev Cell. 2015;34:719\u201326.","journal-title":"Dev Cell"},{"key":"2328_CR34","doi-asserted-by":"crossref","unstructured":"Goodwin K, Mao S, Guyomar T, Miller E, Radisky DC, Ko\u0161mrlj A, Nelson CM. Smooth muscle differentiation shapes domain branches during mouse lung development. Development. 2019; 146.","DOI":"10.1242\/dev.181172"},{"key":"2328_CR35","doi-asserted-by":"publisher","first-page":"199","DOI":"10.1165\/ajrcmb\/3.3.199","volume":"3","author":"ME Sunday","year":"1990","unstructured":"Sunday ME, Hua J, Dai HB, Nusrat A, Torday JS. Bombesin increases fetal lung growth and maturation in utero and in organ culture. Am J Respir Cell Mol Biol. 1990;3:199\u2013205.","journal-title":"Am J Respir Cell Mol Biol"},{"key":"2328_CR36","doi-asserted-by":"publisher","first-page":"531","DOI":"10.1203\/01.pdr.0000202748.66359.a9","volume":"59","author":"M Santos","year":"2006","unstructured":"Santos M, Bastos P, Gonzaga S, Roriz JM, Baptista MJ, Nogueira-Silva C, Melo-Rocha G, Henriques-Coelho T, Roncon-Albuquerque R Jr, Leite-Moreira AF, et al. Ghrelin expression in human and rat fetal lungs and the effect of ghrelin administration in nitrofen-induced congenital diaphragmatic hernia. Pediatr Res. 2006;59:531\u20137.","journal-title":"Pediatr Res"},{"key":"2328_CR37","doi-asserted-by":"publisher","first-page":"2150","DOI":"10.1016\/j.peptides.2008.08.012","volume":"29","author":"S Nunes","year":"2008","unstructured":"Nunes S, Nogueira-Silva C, Dias E, Moura RS, Correia-Pinto J. Ghrelin and obestatin: different role in fetal lung development? Peptides. 2008;29:2150\u20138.","journal-title":"Peptides"},{"key":"2328_CR38","doi-asserted-by":"publisher","first-page":"1749","DOI":"10.1016\/j.jpedsurg.2014.09.015","volume":"49","author":"K Sakai","year":"2014","unstructured":"Sakai K, Kimura O, Furukawa T, Fumino S, Higuchi K, Wakao J, Kimura K, Aoi S, Masumoto K, Tajiri T. Prenatal administration of neuropeptide bombesin promotes lung development in a rat model of nitrofen-induced congenital diaphragmatic hernia. J Pediatr Surg. 2014;49:1749\u201352.","journal-title":"J Pediatr Surg"},{"key":"2328_CR39","doi-asserted-by":"publisher","first-page":"3301","DOI":"10.1113\/JP270477","volume":"593","author":"P Pereira-Terra","year":"2015","unstructured":"Pereira-Terra P, Moura RS, Nogueira-Silva C, Correia-Pinto J. Neuroendocrine factors regulate retinoic acid receptors in normal and hypoplastic lung development. J Physiol. 2015;593:3301\u201311.","journal-title":"J Physiol"},{"key":"2328_CR40","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1016\/bs.ctdb.2018.12.002","volume":"132","author":"A Garg","year":"2019","unstructured":"Garg A, Sui P, Verheyden JM, Young LR, Sun X. Consider the lung as a sensory organ: a tip from pulmonary neuroendocrine cells. Curr Top Dev Biol. 2019;132:67\u201389.","journal-title":"Curr Top Dev Biol"},{"key":"2328_CR41","doi-asserted-by":"publisher","first-page":"176","DOI":"10.1038\/nature20793","volume":"541","author":"K Nonomura","year":"2017","unstructured":"Nonomura K, Woo SH, Chang RB, Gillich A, Qiu Z, Francisco AG, Ranade SS, Liberles SD, Patapoutian A. Piezo2 senses airway stretch and mediates lung inflation-induced apnoea. Nature. 2017;541:176\u201381.","journal-title":"Nature"},{"key":"2328_CR42","doi-asserted-by":"publisher","first-page":"79","DOI":"10.1113\/JP272718","volume":"595","author":"F Wang","year":"2017","unstructured":"Wang F, Knutson K, Alcaino C, Linden DR, Gibbons SJ, Kashyap P, Grover M, Oeckler R, Gottlieb PA, Li HJ, et al. Mechanosensitive ion channel Piezo2 is important for enterochromaffin cell response to mechanical forces. J Physiol. 2017;595:79\u201391.","journal-title":"J Physiol"},{"key":"2328_CR43","doi-asserted-by":"publisher","first-page":"464","DOI":"10.1126\/science.aau6324","volume":"362","author":"WZ Zeng","year":"2018","unstructured":"Zeng WZ, Marshall KL, Min S, Daou I, Chapleau MW, Abboud FM, Liberles SD, Patapoutian A. PIEZOs mediate neuronal sensing of blood pressure and the baroreceptor reflex. Science. 2018;362:464\u20137.","journal-title":"Science"},{"key":"2328_CR44","doi-asserted-by":"publisher","first-page":"176","DOI":"10.1038\/nature10812","volume":"483","author":"B Coste","year":"2012","unstructured":"Coste B, Xiao B, Santos JS, Syeda R, Grandl J, Spencer KS, Kim SE, Schmidt M, Mathur J, Dubin AE, et al. Piezo proteins are pore-forming subunits of mechanically activated channels. Nature. 2012;483:176\u201381.","journal-title":"Nature"},{"key":"2328_CR45","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1126\/science.1193270","volume":"330","author":"B Coste","year":"2010","unstructured":"Coste B, Mathur J, Schmidt M, Earley TJ, Ranade S, Petrus MJ, Dubin AE, Patapoutian A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science. 2010;330:55\u201360.","journal-title":"Science"},{"key":"2328_CR46","doi-asserted-by":"publisher","first-page":"64","DOI":"10.1038\/nature15247","volume":"527","author":"J Ge","year":"2015","unstructured":"Ge J, Li W, Zhao Q, Li N, Chen M, Zhi P, Li R, Gao N, Xiao B, Yang M. Architecture of the mammalian mechanosensitive Piezo1 channel. Nature. 2015;527:64\u20139.","journal-title":"Nature"},{"key":"2328_CR47","doi-asserted-by":"crossref","unstructured":"Cahalan SM, Lukacs V, Ranade SS, Chien S, Bandell M, Patapoutian A. Piezo1 links mechanical forces to red blood cell volume. Elife. 2015; 4.","DOI":"10.7554\/eLife.07370"},{"key":"2328_CR48","doi-asserted-by":"publisher","first-page":"622","DOI":"10.1038\/nature13251","volume":"509","author":"SH Woo","year":"2014","unstructured":"Woo SH, Ranade S, Weyer AD, Dubin AE, Baba Y, Qiu Z, Petrus M, Miyamoto T, Reddy K, Lumpkin EA, et al. Piezo2 is required for Merkel-cell mechanotransduction. Nature. 2014;509:622\u20136.","journal-title":"Nature"},{"key":"2328_CR49","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1038\/nature13980","volume":"516","author":"SS Ranade","year":"2014","unstructured":"Ranade SS, Woo SH, Dubin AE, Moshourab RA, Wetzel C, Petrus M, Mathur J, B\u00e9gay V, Coste B, Mainquist J, et al. Piezo2 is the major transducer of mechanical forces for touch sensation in mice. Nature. 2014;516:121\u20135.","journal-title":"Nature"},{"key":"2328_CR50","doi-asserted-by":"publisher","first-page":"530","DOI":"10.1126\/science.aar5703","volume":"360","author":"J Feng","year":"2018","unstructured":"Feng J, Luo J, Yang P, Du J, Kim BS, Hu H. Piezo2 channel-Merkel cell signaling modulates the conversion of touch to itch. Science. 2018;360:530\u20133.","journal-title":"Science"},{"key":"2328_CR51","doi-asserted-by":"publisher","first-page":"302","DOI":"10.1186\/s12931-020-01568-w","volume":"21","author":"AN Gon\u00e7alves","year":"2020","unstructured":"Gon\u00e7alves AN, Correia-Pinto J, Nogueira-Silva C. ROBO2 signaling in lung development regulates SOX2\/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia. Respir Res. 2020;21:302.","journal-title":"Respir Res"},{"key":"2328_CR52","doi-asserted-by":"publisher","first-page":"4040","DOI":"10.1073\/pnas.95.7.4040","volume":"95","author":"EF Nemeth","year":"1998","unstructured":"Nemeth EF, Steffey ME, Hammerland LG, Hung BC, Van Wagenen BC, DelMar EG, Balandrin MF. Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proc Natl Acad Sci USA. 1998;95:4040\u20135.","journal-title":"Proc Natl Acad Sci USA"},{"key":"2328_CR53","doi-asserted-by":"publisher","first-page":"1651","DOI":"10.1172\/JCI0216112","volume":"110","author":"T Ma","year":"2002","unstructured":"Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, Galietta LJ, Verkman AS. Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J Clin Invest. 2002;110:1651\u20138.","journal-title":"J Clin Invest"},{"key":"2328_CR54","doi-asserted-by":"publisher","first-page":"202","DOI":"10.3389\/fncel.2013.00202","volume":"7","author":"JV Raimondo","year":"2013","unstructured":"Raimondo JV, Joyce B, Kay L, Schlagheck T, Newey SE, Srinivas S, Akerman CJ. A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system. Front Cell Neurosci. 2013;7:202.","journal-title":"Front Cell Neurosci"},{"key":"2328_CR55","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1016\/j.bpj.2016.11.013","volume":"112","author":"R Gnanasambandam","year":"2017","unstructured":"Gnanasambandam R, Ghatak C, Yasmann A, Nishizawa K, Sachs F, Ladokhin AS, Sukharev SI, Suchyna TM. GsMTx4: mechanism of inhibiting mechanosensitive ion channels. Biophys J. 2017;112:31\u201345.","journal-title":"Biophys J"},{"key":"2328_CR56","doi-asserted-by":"publisher","first-page":"14267","DOI":"10.1038\/s41598-018-32572-2","volume":"8","author":"MM Maneshi","year":"2018","unstructured":"Maneshi MM, Ziegler L, Sachs F, Hua SZ, Gottlieb PA. Enantiomeric A\u03b2 peptides inhibit the fluid shear stress response of PIEZO1. Sci Rep. 2018;8:14267.","journal-title":"Sci Rep"},{"key":"2328_CR57","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1002\/ppul.1950150205","volume":"15","author":"EA Massoud","year":"1993","unstructured":"Massoud EA, Sekhon HS, Rotschild A, Puterman ML, Matsui R, Thurlbeck WM. In vitro branching morphogenesis of the fetal rat lung. Pediatr Pulmonol. 1993;15:89\u201397.","journal-title":"Pediatr Pulmonol"},{"key":"2328_CR58","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1002\/dvdy.20136","volume":"231","author":"F Bajanca","year":"2004","unstructured":"Bajanca F, Luz M, Duxson MJ, Thorsteinsdottir S. Integrins in the mouse myotome: developmental changes and differences between the epaxial and hypaxial lineage. Dev Dyn. 2004;231:402\u201315.","journal-title":"Dev Dyn"},{"key":"2328_CR59","doi-asserted-by":"publisher","first-page":"2359","DOI":"10.1242\/dev.060723","volume":"138","author":"A Lazarus","year":"2011","unstructured":"Lazarus A, Del-Moral PM, Ilovich O, Mishani E, Warburton D, Keshet E. A perfusion-independent role of blood vessels in determining branching stereotypy of lung airways. Development. 2011;138:2359\u201368.","journal-title":"Development"},{"key":"2328_CR60","first-page":"649","volume":"123","author":"D Alcorn","year":"1977","unstructured":"Alcorn D, Adamson TM, Lambert TF, Maloney JE, Ritchie BC, Robinson PM. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat. 1977;123:649\u201360.","journal-title":"J Anat"},{"issue":"81","key":"2328_CR61","first-page":"209","volume":"1996","author":"R Harding","year":"1985","unstructured":"Harding R, Hooper SB. Regulation of lung expansion and lung growth before birth. J Appl Physiol. 1985;1996(81):209\u201324.","journal-title":"J Appl Physiol"},{"key":"2328_CR62","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1111\/j.1440-1681.1995.tb01988.x","volume":"22","author":"SB Hooper","year":"1995","unstructured":"Hooper SB, Harding R. Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clin Exp Pharmacol Physiol. 1995;22:235\u201347.","journal-title":"Clin Exp Pharmacol Physiol"},{"key":"2328_CR63","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1016\/0034-5687(84)90032-X","volume":"57","author":"R Harding","year":"1984","unstructured":"Harding R, Sigger JN, Wickham PJ, Bocking AD. The regulation of flow of pulmonary fluid in fetal sheep. Respir Physiol. 1984;57:47\u201359.","journal-title":"Respir Physiol"},{"key":"2328_CR64","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1007\/978-1-4939-7714-7_8","volume":"1752","author":"B Yeganeh","year":"2018","unstructured":"Yeganeh B, Bilodeau C, Post M. Explant culture for studying lung development. Methods Mol Biol. 2018;1752:81\u201390.","journal-title":"Methods Mol Biol"},{"key":"2328_CR65","doi-asserted-by":"publisher","first-page":"L859","DOI":"10.1152\/ajplung.00285.2017","volume":"313","author":"R Bartoszewski","year":"2017","unstructured":"Bartoszewski R, Matalon S, Collawn JF. Ion channels of the lung and their role in disease pathogenesis. Am J Physiol Lung Cell Mol Physiol. 2017;313:L859-l872.","journal-title":"Am J Physiol Lung Cell Mol Physiol"},{"key":"2328_CR66","doi-asserted-by":"publisher","first-page":"E2308","DOI":"10.1073\/pnas.1216382110","volume":"110","author":"EA Solymosi","year":"2013","unstructured":"Solymosi EA, Kaestle-Gembardt SM, Vad\u00e1sz I, Wang L, Neye N, Chupin CJ, Rozowsky S, Ruehl R, Tabuchi A, Schulz H, et al. Chloride transport-driven alveolar fluid secretion is a major contributor to cardiogenic lung edema. Proc Natl Acad Sci USA. 2013;110:E2308-2316.","journal-title":"Proc Natl Acad Sci USA"},{"key":"2328_CR67","doi-asserted-by":"publisher","first-page":"L1229","DOI":"10.1152\/ajplung.00319.2015","volume":"309","author":"S Matalon","year":"2015","unstructured":"Matalon S, Bartoszewski R, Collawn JF. Role of epithelial sodium channels in the regulation of lung fluid homeostasis. Am J Physiol Lung Cell Mol Physiol. 2015;309:L1229-1238.","journal-title":"Am J Physiol Lung Cell Mol Physiol"},{"issue":"19","key":"2328_CR68","doi-asserted-by":"publisher","first-page":"4099","DOI":"10.1113\/jphysiol.2014.275529","volume":"592","author":"E Delpire","year":"2014","unstructured":"Delpire E, Staley KJ. Novel determinants of the neuronal Cl(\u2212) concentration. J Physiol. 2014;592(19):4099\u2013114.","journal-title":"J Physiol"},{"issue":"6171","key":"2328_CR69","doi-asserted-by":"publisher","first-page":"670","DOI":"10.1126\/science.1245423","volume":"343","author":"J Glykys","year":"2014","unstructured":"Glykys J, Dzhala V, Egawa K, Balena T, Saponjian Y, Kuchibhotla KV, et al. Local impermeant anions establish the neuronal chloride concentration. Science. 2014;343(6171):670\u20135.","journal-title":"Science"},{"key":"2328_CR70","doi-asserted-by":"crossref","unstructured":"D\u00fcsterwald KM, Currin CB, Burman RJ, Akerman CJ, Kay AR, Raimondo JV. Biophysical models reveal the relative importance of transporter proteins and impermeant anions in chloride homeostasis. Elife. 2018; 7.","DOI":"10.7554\/eLife.39575"},{"key":"2328_CR71","doi-asserted-by":"publisher","first-page":"184","DOI":"10.1203\/00006450-199802000-00005","volume":"43","author":"L Nardo","year":"1998","unstructured":"Nardo L, Hooper SB, Harding R. Stimulation of lung growth by tracheal obstruction in fetal sheep: relation to luminal pressure and lung liquid volume. Pediatr Res. 1998;43:184\u201390.","journal-title":"Pediatr Res"},{"key":"2328_CR72","doi-asserted-by":"publisher","first-page":"4","DOI":"10.1513\/pats.200704-049VS","volume":"5","author":"KR Badri","year":"2008","unstructured":"Badri KR, Zhou Y, Schuger L. Embryological origin of airway smooth muscle. Proc Am Thorac Soc. 2008;5:4\u201310.","journal-title":"Proc Am Thorac Soc"},{"issue":"4 Pt 1","key":"2328_CR73","first-page":"L472","volume":"262","author":"PB McCray Jr","year":"1992","unstructured":"McCray PB Jr, Reenstra WW, Louie E, Johnson J, Bettencourt JD, Bastacky J. Expression of CFTR and presence of cAMP-mediated fluid secretion in human fetal lung. Am J Physiol. 1992;262(4 Pt 1):L472\u201381.","journal-title":"Am J Physiol"},{"issue":"10","key":"2328_CR74","doi-asserted-by":"publisher","first-page":"1251","DOI":"10.1164\/rccm.201004-0643OC","volume":"182","author":"DK Meyerholz","year":"2010","unstructured":"Meyerholz DK, Stoltz DA, Namati E, Ramachandran S, Pezzulo AA, Smith AR, et al. Loss of cystic fibrosis transmembrane conductance regulator function produces abnormalities in tracheal development in neonatal pigs and young children. Am J Respir Crit Care Med. 2010;182(10):1251\u201361.","journal-title":"Am J Respir Crit Care Med"},{"issue":"6","key":"2328_CR75","doi-asserted-by":"publisher","first-page":"715","DOI":"10.1055\/s-0039-1694021","volume":"40","author":"C Bergeron","year":"2019","unstructured":"Bergeron C, Cantin AM. Cystic fibrosis: pathophysiology of lung disease. Semin Respir Crit Care Med. 2019;40(6):715\u201326.","journal-title":"Semin Respir Crit Care Med"},{"issue":"1","key":"2328_CR76","doi-asserted-by":"publisher","first-page":"12397","DOI":"10.1038\/s41598-017-10910-0","volume":"7","author":"R Benedetto","year":"2017","unstructured":"Benedetto R, Ousingsawat J, Wanitchakool P, Zhang Y, Holtzman MJ, Amaral M, et al. Epithelial chloride transport by CFTR requires TMEM16A. Sci Rep. 2017;7(1):12397.","journal-title":"Sci Rep"},{"key":"2328_CR77","doi-asserted-by":"crossref","unstructured":"Dinsdale RL, Pipatpolkai T, Agostinelli E, Russell AJ, Stansfeld PJ, Tammaro P. An outer-pore gate modulates the pharmacology of the TMEM16A channel. Proc Natl Acad Sci USA. 2021; 118(34)","DOI":"10.1073\/pnas.2023572118"},{"key":"2328_CR78","doi-asserted-by":"crossref","unstructured":"He M, Wu B, Ye W, Le DD, Sinclair AW, Padovano V, Chen Y, Li KX, Sit R, Tan M, et al: Chloride channels regulate differentiation and barrier functions of the mammalian airway. Elife. 2020; 9.","DOI":"10.7554\/eLife.53085"},{"key":"2328_CR79","doi-asserted-by":"publisher","first-page":"728","DOI":"10.1038\/s41588-019-0346-6","volume":"51","author":"Q Liu","year":"2019","unstructured":"Liu Q, Liu K, Cui G, Huang X, Yao S, Guo W, Qin Z, Li Y, Yang R, Pu W, et al. Lung regeneration by multipotent stem cells residing at the bronchioalveolar-duct junction. Nat Genet. 2019;51:728\u201338.","journal-title":"Nat Genet"},{"key":"2328_CR80","doi-asserted-by":"publisher","first-page":"143","DOI":"10.1111\/j.1432-0436.1990.tb00547.x","volume":"44","author":"KO Leslie","year":"1990","unstructured":"Leslie KO, Mitchell JJ, Woodcock-Mitchell JL, Low RB. Alpha smooth muscle actin expression in developing and adult human lung. Differentiation. 1990;44:143\u20139.","journal-title":"Differentiation"},{"key":"2328_CR81","doi-asserted-by":"publisher","first-page":"2157","DOI":"10.1242\/dev.01795","volume":"132","author":"AA Mailleux","year":"2005","unstructured":"Mailleux AA, Kelly R, Veltmaat JM, De Langhe SP, Zaffran S, Thiery JP, Bellusci S. Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage. Development. 2005;132:2157\u201366.","journal-title":"Development"},{"key":"2328_CR82","doi-asserted-by":"publisher","first-page":"750","DOI":"10.1002\/dvdy.21462","volume":"237","author":"L Shan","year":"2008","unstructured":"Shan L, Subramaniam M, Emanuel RL, Degan S, Johnston P, Tefft D, Warburton D, Sunday ME. Centrifugal migration of mesenchymal cells in embryonic lung. Dev Dyn. 2008;237:750\u20137.","journal-title":"Dev Dyn"},{"key":"2328_CR83","doi-asserted-by":"crossref","first-page":"120S","DOI":"10.1016\/S0006-3495(95)80237-7","volume":"68","author":"S Lowey","year":"1995","unstructured":"Lowey S, Trybus KM. Role of skeletal and smooth muscle myosin light chains. Biophys J. 1995;68:120S-126S (discussion 126S\u2013127S).","journal-title":"Biophys J"},{"key":"2328_CR84","doi-asserted-by":"publisher","first-page":"701","DOI":"10.3389\/fphys.2020.00701","volume":"11","author":"MD \u00c1lvarez-Santos","year":"2020","unstructured":"\u00c1lvarez-Santos MD, \u00c1lvarez-Gonz\u00e1lez M, Estrada-Soto S, Baz\u00e1n-Perkins B. Regulation of myosin light-chain phosphatase activity to generate airway smooth muscle hypercontractility. Front Physiol. 2020;11:701.","journal-title":"Front Physiol"},{"key":"2328_CR85","doi-asserted-by":"publisher","first-page":"779","DOI":"10.1007\/s00249-016-1128-z","volume":"45","author":"H Yu","year":"2016","unstructured":"Yu H, Chakravorty S, Song W, Ferenczi MA. Phosphorylation of the regulatory light chain of myosin in striated muscle: methodological perspectives. Eur Biophys J. 2016;45:779\u2013805.","journal-title":"Eur Biophys J"},{"key":"2328_CR86","doi-asserted-by":"publisher","first-page":"118","DOI":"10.1165\/rcmb.2004-0304OC","volume":"32","author":"EC Jesudason","year":"2005","unstructured":"Jesudason EC, Smith NP, Connell MG, Spiller DG, White MR, Fernig DG, Losty PD. Developing rat lung has a sided pacemaker region for morphogenesis-related airway peristalsis. Am J Respir Cell Mol Biol. 2005;32:118\u201327.","journal-title":"Am J Respir Cell Mol Biol"},{"key":"2328_CR87","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1203\/PDR.0b013e31815eba47","volume":"63","author":"T Seaborn","year":"2008","unstructured":"Seaborn T, St-Amand J, Cloutier M, Tremblay MG, Maltais F, Dinel S, Moulin V, Khan PA, Piedboeuf B. Identification of cellular processes that are rapidly modulated in response to tracheal occlusion within mice lungs. Pediatr Res. 2008;63:124\u201330.","journal-title":"Pediatr Res"},{"key":"2328_CR88","doi-asserted-by":"crossref","unstructured":"Benavides F, Rulicke T, Prins JB, Bussell J, Scavizzi F, Cinelli P, Herault Y, Wedekind D. Genetic quality assurance and genetic monitoring of laboratory mice and rats: FELASA Working Group Report. Lab Anim 2019:23677219867719.","DOI":"10.1177\/0023677219867719"}],"container-title":["Respiratory Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12931-023-02328-2.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12931-023-02328-2\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12931-023-02328-2.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,2,6]],"date-time":"2023-02-06T18:08:01Z","timestamp":1675706881000},"score":1,"resource":{"primary":{"URL":"https:\/\/respiratory-research.biomedcentral.com\/articles\/10.1186\/s12931-023-02328-2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,2,5]]},"references-count":88,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2023,12]]}},"alternative-id":["2328"],"URL":"https:\/\/doi.org\/10.1186\/s12931-023-02328-2","relation":{},"ISSN":["1465-993X"],"issn-type":[{"value":"1465-993X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,2,5]]},"assertion":[{"value":"30 April 2022","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"13 January 2023","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"5 February 2023","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 carried out in strict accordance with FELASA guidelines [] and European regulations (European Union Directive 86\/609\/EEC). All animal experiments were approved by the Life and Health Sciences Research Institute (ICVS), University of Minho, and by the Dire\u00e7\u00e3o Geral de Alimenta\u00e7\u00e3o e Veterin\u00e1ria (approval No. DGAV 021328).","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 interest.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"42"}}