{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T12:28:38Z","timestamp":1790080118663,"version":"4.0.1"},"reference-count":54,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2020,8,14]],"date-time":"2020-08-14T00:00:00Z","timestamp":1597363200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["GM104412 and HL132024"],"award-info":[{"award-number":["GM104412 and HL132024"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010554","name":"Clinical and Translational Science Institute, University of Pittsburgh","doi-asserted-by":"publisher","award":["PinCh 2018"],"award-info":[{"award-number":["PinCh 2018"]}],"id":[{"id":"10.13039\/100010554","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Background: Cilia are actin based cellular protrusions conserved from algae to complex multicellular organisms like Homo sapiens. Respiratory motile cilia line epithelial cells of the tracheobronchial tree, beat in a synchronous, metachronal wave, moving inhaled pollutants and pathogens cephalad. Their role in both congenital disorders like primary ciliary dyskinesia (PCD) to acquired disorders like chronic obstructive pulmonary disease (COPD) continues to evolve. In this current body of work we outline a protocol optimized to reciliate human nasal epithelial cells and mouse tracheal cells in vitro. Using this protocol, we knocked down known cilia genes, as well as use a small molecule inhibitor of Notch, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT), to assess the effect of these on ciliogenesis in order to show the validity of our protocol. Methods: Tracheas were harvested from wild-type, adult C57B6 mice, pronase digested and sloughed off epithelial cells grown to confluence in stationary culture on rat-tail collagen coated wells. Upon reaching confluence, collagen was digested and cells placed suspension culture protocol to reciliate the cells. Using this suspension culture protocol, we employed siRNA gene knockdown to assay gene functions required for airway ciliogenesis. Knock down of Dynein axonemal heavy chain 5 (Dnah5), a ciliary structural protein, was confirmed using immunostaining. Mouse tracheal cells were treated in suspension with varying doses of DAPT, an inhibitor of Notch, with the purpose of evaluating its effect and dose response on ciliogenesis. The optimum dose was then used on reciliating human nasal epithelial cells. Results: siRNA knockdown of Foxj1 prevented ciliation, consistent with its role as a master regulator of motile cilia. Knockdown of Dnai1 and Dnah5 resulted in immotile cilia, and Cand1 knockdown, a centrosome protein known to regulate centrosome amplification, inhibited airway ciliogenesis. Dnah5 knockdown was confirmed with significantly decreased immunostaining of cilia for this protein. Inhibiting Notch signaling by inhibiting gamma secretase with DAPT enhanced the percentage of ciliation, and resulted in longer cilia that beat with higher frequency in both mouse and human airway epithelia. Conclusions: Modifying existing reciliation protocols to suit both human nasal epithelial and mouse tracheal tissue, we have shown that knockdown of known cilia-related genes have the expected effects. Additionally, we have demonstrated the optimal dosage for significantly improving reciliation of airway epithelia using DAPT. Given that cilia length and function are significantly compromised in COPD, these findings open up interesting avenues for further exploration.<\/jats:p>","DOI":"10.3390\/biom10081182","type":"journal-article","created":{"date-parts":[[2020,8,14]],"date-time":"2020-08-14T08:28:35Z","timestamp":1597393715000},"page":"1182","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia"],"prefix":"10.3390","volume":"10","author":[{"given":"Maliha","family":"Zahid","sequence":"first","affiliation":[{"name":"Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th St, Pittsburgh, PA 15201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy N.","family":"Feinstein","sequence":"additional","affiliation":[{"name":"Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th St, Pittsburgh, PA 15201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony","family":"Oro","sequence":"additional","affiliation":[{"name":"Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th St, Pittsburgh, PA 15201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Molly","family":"Schwartz","sequence":"additional","affiliation":[{"name":"Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th St, Pittsburgh, PA 15201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1459-4482","authenticated-orcid":false,"given":"Alex D.","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th St, Pittsburgh, PA 15201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cecilia W.","family":"Lo","sequence":"additional","affiliation":[{"name":"Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th St, Pittsburgh, PA 15201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,8,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Hessel, J., Heldrich, J., Fuller, J., Staudt, M.R., Radisch, S., Hollmann, C., Harvey, B.-G., Kaner, R.J., Salit, J., and Yee-Levin, J. (2014). Intraflagellar Transport Gene Expression Associated with Short Cilia in Smoking and COPD. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0085453"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1139","DOI":"10.1016\/j.rmed.2012.04.001","article-title":"Ciliary beating is depressed in nasal cilia from chronic obstructive pulmonary disease subjects","volume":"106","author":"Yaghi","year":"2012","journal-title":"Respir. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/j.ccm.2013.11.001","article-title":"Acute COPD exacerbations","volume":"35","author":"Wedzicha","year":"2014","journal-title":"Clin. Chest Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1080\/17476348.2018.1398086","article-title":"An overview of the current management of chronic obstructive pulmonary disease: Can we go beyond the GOLD recommendations?","volume":"12","author":"Cazzola","year":"2017","journal-title":"Expert Rev. Respir. Med."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Mathers, C.D., and Loncar, D. (2006). Projections of Global Mortality and Burden of Disease from 2002 to 2030. PLoS Med., 3.","DOI":"10.1371\/journal.pmed.0030442"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"31893","DOI":"10.1038\/srep31893","article-title":"Projecting the COPD population and costs in England and Scotland: 2011 to 2030","volume":"6","author":"McLean","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1016\/j.jval.2011.06.008","article-title":"Developing and Applying a Stochastic Dynamic Population Model for Chronic Obstructive Pulmonary Disease","volume":"14","author":"Hoogendoorn","year":"2011","journal-title":"Value Health"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2188","DOI":"10.1002\/ajmg.a.37133","article-title":"Respiratory motile cilia dysfunction in a patient with cranioectodermal dysplasia","volume":"167","author":"Li","year":"2015","journal-title":"Am. J. Med. Genet. Part A"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1002\/humu.20614","article-title":"Mutations of theCEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome","volume":"29","author":"Frank","year":"2008","journal-title":"Hum. Mutat."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1426","DOI":"10.1513\/AnnalsATS.201405-222OC","article-title":"Airway Ciliary Dysfunction and Sinopulmonary Symptoms in Patients with Congenital Heart Disease","volume":"11","author":"Garrod","year":"2014","journal-title":"Ann. Am. Thorac. Soc."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Zahid, M., Bais, A., Tian, X., Devine, W., Lee, D.M., Yau, C., Sonnenberg, D., Beerman, L., Khalifa, O., and Lo, C.W. (2018). Airway ciliary dysfunction and respiratory symptoms in patients with transposition of the great arteries. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0191605"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1542\/hpeds.2013-0074","article-title":"Hospitalists Should Have Increased Suspicion for Primary Ciliary Dyskinesia in Patients with Congenital Heart Disease","volume":"4","author":"Trost","year":"2014","journal-title":"Hosp. Pediatr."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1016\/j.jtcvs.2010.07.082","article-title":"Increased postoperative and respiratory complications in patients with congenital heart disease associated with heterotaxy","volume":"141","author":"Swisher","year":"2011","journal-title":"J. Thorac. Cardiovasc. Surg."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1016\/j.jtcvs.2017.09.050","article-title":"Airway ciliary dysfunction: Association with adverse postoperative outcomes in nonheterotaxy congenital heart disease patients","volume":"155","author":"Stewart","year":"2018","journal-title":"J. Thorac. Cardiovasc. Surg."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1007\/BF00463582","article-title":"The preservation and regeneration of cilia on human nasal epithelial cells cultured in vitro","volume":"246","author":"Jorissen","year":"1989","journal-title":"Eur. Arch. Otorhinolaryngol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"66","DOI":"10.3109\/00016489509133349","article-title":"Normal Ciliary Beat Frequency after Ciliogenesis in Nasal Epithelial Cells Cultured Sequentially as Monolayer and in Suspension","volume":"115","author":"Jorissen","year":"1995","journal-title":"Acta Otolaryngol."},{"key":"ref_17","first-page":"67","article-title":"Ciliogenesis and coordinated ciliary beating in human nasal epithelial cells cultured in vitro","volume":"43","author":"Jorissen","year":"1989","journal-title":"Acta Otolaryngol. Belg."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Method."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1076","DOI":"10.1016\/j.bone.2010.08.023","article-title":"BoneJ: Free and extensible bone image analysis in Image","volume":"47","author":"Doube","year":"2010","journal-title":"J. Bone"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1083\/jcb.130.2.393","article-title":"Identification and characterization of a fibroblast marker: FSP1","volume":"130","author":"Strutz","year":"1995","journal-title":"J. Cell Boil."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"L650","DOI":"10.1152\/ajplung.00170.2003","article-title":"Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells","volume":"286","author":"You","year":"2004","journal-title":"Am. J. Physiol. Cell. Mol. Physiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1038\/ng.726","article-title":"CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs","volume":"43","author":"Merveille","year":"2010","journal-title":"Nat. Genet."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1593\/neo.12580","article-title":"CAND1 Promotes PLK4-Mediated Centriole Overduplication and Is Frequently Disrupted in Prostate Cancer","volume":"14","author":"Korzeniewski","year":"2012","journal-title":"Neoplasia"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1751","DOI":"10.1242\/dev.029249","article-title":"Notch signaling promotes airway mucous metaplasia and inhibits alveolar development","volume":"136","author":"Guseh","year":"2009","journal-title":"Development"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2297","DOI":"10.1242\/dev.034884","article-title":"Notch signaling controls the balance of ciliated and secretory cell fates in developing airways","volume":"136","author":"Tsao","year":"2009","journal-title":"Development"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"7349","DOI":"10.1038\/s41598-018-25799-6","article-title":"A novel method for expansion and differentiation of mouse tracheal epithelial cells in culture","volume":"8","author":"Eenjes","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1165\/rcmb.2013-0237OC","article-title":"Submersion and Hypoxia Inhibit Ciliated Cell Differentiation in a Notch-Dependent Manner","volume":"51","author":"Gerovac","year":"2014","journal-title":"Am. J. Respir. Cell Mol. Boil."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1444","DOI":"10.1001\/jama.2018.0158","article-title":"The State of US Health, 1990\u20132016: Burden of Diseases, Injuries, and Risk Factors Among US States","volume":"319","author":"Collaborators","year":"2018","journal-title":"JAMA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"5212","DOI":"10.1172\/JCI69636","article-title":"Histone deacetylase 6\u2013mediated selective autophagy regulates COPD-associated cilia dysfunction","volume":"123","author":"Lam","year":"2013","journal-title":"J. Clin. Investig."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4418","DOI":"10.1038\/ncomms5418","article-title":"MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia","volume":"5","author":"Boon","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.jcf.2004.05.011","article-title":"Sequential monolayer-suspension culture of human airway epithelial cells","volume":"3","author":"Willems","year":"2004","journal-title":"J. Cyst. Fibros."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1007\/978-1-62703-125-7_9","article-title":"Culture and differentiation of mouse tracheal epithelial cells","volume":"945","author":"You","year":"2013","journal-title":"Method. Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1007\/978-1-62703-496-8_7","article-title":"Applications of Mouse Airway Epithelial Cell Culture for Asthma Research","volume":"1032","author":"Horani","year":"2013","journal-title":"Method. Mol. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1016\/B978-0-12-397944-5.00014-6","article-title":"Analysis of Ciliogenesis in Primary Culture Mouse Tracheal Epithelial Cells","volume":"525","author":"Vladar","year":"2013","journal-title":"Method. Enzymol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1165\/rcmb.2016-0220OC","article-title":"Variable Susceptibility to Cigarette Smoke\u2013Induced Emphysema in 34 Inbred Strains of Mice Implicates Abi3bp in Emphysema Susceptibility","volume":"57","author":"Radder","year":"2017","journal-title":"Am. J. Respir. Cell Mol. Boil."},{"key":"ref_36","first-page":"115","article-title":"An in vitro Model of Differentiated Human Airway Epithelia: Methods for Establishing Primary Cultures","volume":"188","author":"Karp","year":"2002","journal-title":"Method. Mol. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"You, Y., Richer, E.J., Huang, T., and Brody, S.L. (2002). Growth and differentiation of mouse tracheal epithelial cells: Selection of a proliferative population. Am. J. Physiol. Cell. Mol. Physiol., 283.","DOI":"10.1152\/ajplung.00169.2002"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Gras, D., Petit, A., Charriot, J., Knabe, L., Alagha, K., Gamez, A.S., Garulli, C., Bourdin, A., Chanez, P., and Molinari, N. (2017). Epithelial ciliated beating cells essential for ex vivo ALI culture growth. BMC Pulm. Med., 17.","DOI":"10.1186\/s12890-017-0423-5"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1038\/ng817","article-title":"Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left\u2013right asymmetry","volume":"30","author":"Olbrich","year":"2002","journal-title":"Nat. Genet."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1030","DOI":"10.1086\/319511","article-title":"Axonemal Dynein Intermediate-Chain Gene (DNAI1) Mutations Result in Situs Inversus and Primary Ciliary Dyskinesia (Kartagener Syndrome)","volume":"68","author":"Guichard","year":"2001","journal-title":"Am. J. Hum. Genet."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Francis, R.O., Chatterjee, B., Loges, N.T., Zentgraf, H., Omran, H., and Lo, C.W. (2009). Initiation and maturation of cilia-generated flow in newborn and postnatal mouse airway. Am. J. Physiol. Cell. Mol. Physiol., 296.","DOI":"10.1152\/ajplung.00001.2009"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1136\/jmedgenet-2012-100867","article-title":"Delineation of CCDC39\/CCDC40 mutation spectrum and associated phenotypes in primary ciliary dyskinesia","volume":"49","author":"Blanchon","year":"2012","journal-title":"J. Med. Genet."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1002\/humu.22261","article-title":"Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganization and absent inner dynein arms","volume":"34","author":"Antony","year":"2013","journal-title":"Hum. Mutat."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Abdelhamed, Z., Vuong, S.M., Hill, L., Shula, C., Timms, A., Beier, D., Campbell, K., Mangano, F.T., Stottmann, R.W., and Goto, J. (2018). A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice. Development, 145.","DOI":"10.1242\/dev.154500"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1038\/nature15715","article-title":"Therapeutic antibodies reveal Notch control of transdifferentiation in the adult lung","volume":"528","author":"Lafkas","year":"2015","journal-title":"Nature"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1083\/jcb.147.2.277","article-title":"Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons","volume":"147","author":"Annaert","year":"1999","journal-title":"J. Cell Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"844","DOI":"10.1016\/j.stem.2017.03.001","article-title":"Efficient Derivation of Functional Human Airway Epithelium from Pluripotent Stem Cells via Temporal Regulation of Wnt Signaling","volume":"20","author":"McCauley","year":"2017","journal-title":"Cell Stem Cell"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1186\/s13287-015-0103-4","article-title":"Wnt and Notch signaling pathway involved in wound healing by targeting c-Myc and Hes1 separately","volume":"6","author":"Shi","year":"2015","journal-title":"Stem Cell Res. Ther."},{"key":"ref_49","first-page":"1184","article-title":"Accuracy of Nasal Nitric Oxide Measurement as a Diagnostic Test for Primary Ciliary Dyskinesia. A Systematic Review and Meta-analysis","volume":"14","author":"Shapiro","year":"2017","journal-title":"Ann. Am. Thorac. Soc."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.stem.2010.01.001","article-title":"Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells","volume":"6","author":"Charles","year":"2010","journal-title":"Cell Stem Cell"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1466","DOI":"10.1242\/jcs.019018","article-title":"Downregulation by lipopolysaccharide of Notch signaling, via nitric oxide","volume":"121","author":"Kim","year":"2008","journal-title":"J. Cell Sci."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Welchering, N., Ochoa, S., Tian, X., Francis, R.O., Zahid, M., Lo, C., and Mu\u00f1oz, R. (2015). Dexmedetomidine and Fentanyl Exhibit Temperature Dependent Effects on Human Respiratory Cilia. Front. Pediatr., 3.","DOI":"10.3389\/fped.2015.00007"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"111","DOI":"10.3389\/fped.2014.00111","article-title":"The Effects of Temperature and Anesthetic Agents on Ciliary Function in Murine Respiratory Epithelia","volume":"2","author":"Christopher","year":"2014","journal-title":"Front. Pediatr."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1517\/17425247.2011.561310","article-title":"Challenges and advances in the development of inhalable drug formulations for cystic fibrosis lung disease","volume":"8","author":"Ibrahim","year":"2011","journal-title":"Expert Opin. Drug Deliv."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/10\/8\/1182\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:00:51Z","timestamp":1760176851000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/10\/8\/1182"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,8,14]]},"references-count":54,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2020,8]]}},"alternative-id":["biom10081182"],"URL":"https:\/\/doi.org\/10.3390\/biom10081182","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,8,14]]}}}