{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T14:48:21Z","timestamp":1790434101931,"version":"4.1.0"},"reference-count":54,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2020,4,28]],"date-time":"2020-04-28T00:00:00Z","timestamp":1588032000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of University and Research","award":["PRIN to R. Pierantoni 2017"],"award-info":[{"award-number":["PRIN to R. Pierantoni 2017"]}]},{"name":"Universit\u00e0 degli Studi della Campania","award":["Dip. Medicina Sperimentale-2017 to G. Cobellis"],"award-info":[{"award-number":["Dip. Medicina Sperimentale-2017 to G. Cobellis"]}]},{"name":"Universit\u00e0 degli Studi della Campania","award":["VALERE, Vanvitelli per la Ricerca  2019 to G. Cobellis"],"award-info":[{"award-number":["VALERE, Vanvitelli per la Ricerca  2019 to G. Cobellis"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The cannabinoid receptor CB1 regulates differentiation of spermatids. We recently characterized spermatozoa from caput epididymis of CB1-knock-out mice and identified a considerable number of sperm cells with chromatin abnormality such as elevated histone content and poorly condensed chromatin. In this paper, we extended our findings and studied the role of CB1 in the epididymal phase of chromatin condensation of spermatozoa by analysis of spermatozoa from caput and cauda epididymis of wild-type and CB1-knock-out mouse in both a homozygous or heterozygous condition. Furthermore, we studied the impact of CB1-gene deletion on histone displacement mechanism by taking into account the hyperacetylation of histone H4 and players of displacement such as Chromodomain Y Like protein (CDYL) and Bromodomain testis-specific protein (BRDT). Our results show that CB1, via local and\/or endocrine cell-to-cell signaling, modulates chromatin remodeling mechanisms that orchestrate a nuclear condensation extent of mature spermatozoa. We show that CB1-gene deletion affects the epididymal phase of chromatin condensation by interfering with inter-\/intra-protamine disulphide bridges formation, and deranges the efficiency of histone removal by reducing the hyper-acetylation of histone H4. This effect is independent by gene expression of Cdyl and Brdt mRNA. Our results reveal a novel and important role for CB1 in sperm chromatin condensation mechanisms.<\/jats:p>","DOI":"10.3390\/ijms21093117","type":"journal-article","created":{"date-parts":[[2020,4,28]],"date-time":"2020-04-28T10:30:58Z","timestamp":1588069858000},"page":"3117","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["The Cannabinoid Receptor CB1 Stabilizes Sperm Chromatin Condensation Status During Epididymal Transit by Promoting Disulphide Bond Formation"],"prefix":"10.3390","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1720-1496","authenticated-orcid":false,"given":"Teresa","family":"Chioccarelli","sequence":"first","affiliation":[{"name":"Department of Experimental Medicine, Sez. Bottazzi, Universit\u00e0 degli Studi della Campania \u201cL. Vanvitelli\u201d, Via Costantinopoli 16, 80138 Napoli, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesco","family":"Manfrevola","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine, Sez. Bottazzi, Universit\u00e0 degli Studi della Campania \u201cL. Vanvitelli\u201d, Via Costantinopoli 16, 80138 Napoli, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veronica","family":"Porreca","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine, Sez. Bottazzi, Universit\u00e0 degli Studi della Campania \u201cL. Vanvitelli\u201d, Via Costantinopoli 16, 80138 Napoli, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia","family":"Fasano","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine, Sez. Bottazzi, Universit\u00e0 degli Studi della Campania \u201cL. Vanvitelli\u201d, Via Costantinopoli 16, 80138 Napoli, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7312-5387","authenticated-orcid":false,"given":"Lucia","family":"Altucci","sequence":"additional","affiliation":[{"name":"Department of Precision Medicine, Universit\u00e0 degli Studi della Campania \u201cL. Vanvitelli\u201d, Via L. De Crecchio 7, 80138 Napoli, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8246-2177","authenticated-orcid":false,"given":"Riccardo","family":"Pierantoni","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine, Sez. Bottazzi, Universit\u00e0 degli Studi della Campania \u201cL. Vanvitelli\u201d, Via Costantinopoli 16, 80138 Napoli, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5884-9208","authenticated-orcid":false,"given":"Gilda","family":"Cobellis","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine, Sez. Bottazzi, Universit\u00e0 degli Studi della Campania \u201cL. Vanvitelli\u201d, Via Costantinopoli 16, 80138 Napoli, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,4,28]]},"reference":[{"key":"ref_1","unstructured":"Knobil, E., and Neill, J.D. (1999). Spermatogenesis: An overview. Encyclopedia of Reproduction, Academic Press."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"501350","DOI":"10.1155\/2013\/501350","article-title":"Estrogens and Spermiogenesis: New Insights from Type 1 Cannabinoid Receptor Knockout Mice","volume":"2013","author":"Cacciola","year":"2013","journal-title":"Int. J. Endocrinol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/S0079-6603(08)60839-9","article-title":"Vertebrate protamine genes and the histone-to-protamine replacement reaction","volume":"40","author":"Oliva","year":"1991","journal-title":"Prog. Nucleic Acid Res. Mol. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"910","DOI":"10.1095\/biolreprod.103.022541","article-title":"Transient DNA strand breaks during mouse and human spermiogenesis: New insights in stage specificity and link to chromatin remodeling","volume":"70","author":"Marcon","year":"2004","journal-title":"Biol. Reprod."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/j.gene.2004.12.004","article-title":"Establishment of male-specific epigenetic information","volume":"345","author":"Rousseaux","year":"2005","journal-title":"Gene"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1038\/aja.2010.65","article-title":"Proteomics and the genetics of sperm chromatin condensation","volume":"13","author":"Oliva","year":"2011","journal-title":"Asian J. Androl."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.mce.2012.01.014","article-title":"The role of endocannabinoids in gonadal function and fertility along the evolutionary axis","volume":"355","author":"Battista","year":"2012","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Chioccarelli, T., Pierantoni, R., Manfrevola, F., Porreca, V., Fasano, S., Chianese, R., and Cobellis, G. (2020). Histone post-translational modifications and circRNAs in mouse and human spermatozoa: Potential epigenetic marks to assess human sperm quality. J. Clin. Med., 9.","DOI":"10.3390\/jcm9030640"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/j.molcel.2016.03.014","article-title":"Dynamic competing histone H4 K5K8 acetylation and butyrylation are hallmarks of highly active gene promoters","volume":"62","author":"Goudarzi","year":"2016","journal-title":"Mol. Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"853","DOI":"10.1016\/j.molcel.2017.07.011","article-title":"Chromodomain protein CDYL acts as a crotonyl-CoA hydratase to regulate histone crotonylation and spermatogenesis","volume":"67","author":"Liu","year":"2017","journal-title":"Mol. Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1801","DOI":"10.1016\/j.str.2015.08.004","article-title":"A Subset of Human Bromodomains Recognizes Butyryllysine and Crotonyllysine Histone Peptide Modifications","volume":"23","author":"Flynn","year":"2015","journal-title":"Structure"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1016\/j.molcel.2016.03.028","article-title":"Molecular Coupling of Histone Crotonylation and Active Transcription by AF9 YEATS Domain","volume":"62","author":"Li","year":"2016","journal-title":"Mol. Cell"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3537","DOI":"10.1242\/jcs.112.20.3537","article-title":"A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA","volume":"112","author":"Pittoggi","year":"1999","journal-title":"J. Cell Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1159\/000078190","article-title":"Male germline-specific histones in mouse and man","volume":"105","author":"Churikov","year":"2004","journal-title":"Cytogenet. Genome Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1093\/humupd\/dml009","article-title":"Protamines and male infertility","volume":"12","author":"Oliva","year":"2006","journal-title":"Hum. Reprod. Update"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1093\/humupd\/dml057","article-title":"Altered protamine expression and diminished spermatogenesis: What is the link?","volume":"13","author":"Carrell","year":"2007","journal-title":"Hum. Reprod. Update"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1111\/j.1365-2605.1994.tb01253.x","article-title":"Development of sperm motility patterns in the murine epididymis","volume":"17","author":"Soler","year":"1994","journal-title":"Int. J. Androl."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1016\/0003-9861(92)90588-N","article-title":"Formation of intraprotamine disulfides in vitro","volume":"296","author":"Balhorn","year":"1992","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/j.bbagrm.2013.08.004","article-title":"Chromatin dynamics during spermiogenesis","volume":"1839","author":"Rathke","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"236","DOI":"10.4172\/2161-0940.1000236","article-title":"Key features of genomic imprinting during mammalian spermatogenesis: Perspectives for human assisted reproductive therapy: A review","volume":"6","author":"Stiavnicka","year":"2016","journal-title":"Anat. Physiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1083","DOI":"10.1095\/biolreprod49.5.1083","article-title":"Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa","volume":"49","author":"Bianchi","year":"1993","journal-title":"Biol. Reprod."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"54","DOI":"10.3389\/fendo.2014.00054","article-title":"Endocannabinoids are Involved in Male Vertebrate Reproduction: Regulatory Mechanisms at Central and Gonadal Level","volume":"5","author":"Bovolin","year":"2014","journal-title":"Front. Endocrinol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"269","DOI":"10.3389\/fendo.2018.00269","article-title":"Analysis of Endocannabinoid System in Rat Testis During the First Spermatogenetic Wave","volume":"9","author":"Migliaccio","year":"2018","journal-title":"Front. Endocrinol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1095\/biolreprod.109.079210","article-title":"A gradient of 2-arachidonoylglycerol regulates mouse epididymal sperm cell start-up","volume":"82","author":"Cobellis","year":"2010","journal-title":"Biol. Reprod."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1006\/bbrc.2001.4977","article-title":"The central cannabinoid receptor inactivation suppresses endocrine reproductive functions","volume":"284","author":"Wenger","year":"2001","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1210\/en.2002-220544","article-title":"Anandamide activity and degradation are regulated by early postnatal aging and follicle-stimulating hormone in mouse Sertoli cells","volume":"144","author":"Maccarrone","year":"2003","journal-title":"Endocrinology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4393","DOI":"10.1242\/jcs.02536","article-title":"Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction","volume":"118","author":"Maccarrone","year":"2005","journal-title":"J. Cell Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"984","DOI":"10.1210\/jc.2004-1287","article-title":"Human sperm express cannabinoid receptor Cb1, the activation of which inhibits motility, acrosome reaction, and mitochondrial function","volume":"90","author":"Rossato","year":"2005","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1095\/biolreprod.106.051730","article-title":"Endocannabinoid system in frog and rodent testis: Type-1 cannabinoid receptor and fatty acid amide hydrolase activity in male germ cells","volume":"75","author":"Cobellis","year":"2006","journal-title":"Biol. Reprod."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"758","DOI":"10.1095\/biolreprod.108.070128","article-title":"Expression of type-1 cannabinoid receptor during rat postnatal testicular development: Possible involvement in adult leydig cell differentiation","volume":"79","author":"Cacciola","year":"2008","journal-title":"Biol. Reprod."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"11131","DOI":"10.1073\/pnas.0812789106","article-title":"The endocannabinoid system and pivotal role of the CB2 receptor in mouse spermatogenesis","volume":"106","author":"Grimaldi","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_32","first-page":"E475","article-title":"Anandamide regulates the expression of GnRH1, GnRH2, and GnRH-Rs in frog testis","volume":"303","author":"Chianese","year":"2012","journal-title":"Am. J. Physiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1095\/biolreprod.112.105726","article-title":"Low 17beta-estradiol levels in Cnr1 knock-out mice affect spermatid chromatin remodeling by interfering with chromatin reorganization","volume":"88","author":"Cacciola","year":"2013","journal-title":"Biol. Reprod."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1016\/j.ygcen.2013.07.018","article-title":"Nuclear size as estrogen-responsive chromatin quality parameter of mouse spermatozoa","volume":"193","author":"Cacciola","year":"2013","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"47","DOI":"10.3389\/fendo.2016.00047","article-title":"Effects of Neuroendocrine CB1 Activity on Adult Leydig Cells","volume":"7","author":"Cobellis","year":"2016","journal-title":"Front. Endocrinol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1016\/S0083-6729(09)81014-5","article-title":"CB1 activity in male reproduction: Mammalian and nonmammalian animal models","volume":"81","author":"Pierantoni","year":"2009","journal-title":"Vitam. Horm."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"69","DOI":"10.3389\/fendo.2014.00069","article-title":"Intra-testicular signals regulate germ cell progression and production of qualitatively mature spermatozoa in vertebrates","volume":"5","author":"Meccariello","year":"2014","journal-title":"Front. Endocrinol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"5017","DOI":"10.1210\/en.2010-0133","article-title":"Cannabinoid receptor 1 influences chromatin remodeling in mouse spermatids by affecting content of transition protein 2 mRNA and histone displacement","volume":"151","author":"Chioccarelli","year":"2010","journal-title":"Endocrinology"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1016\/j.ygcen.2007.02.003","article-title":"Endocannabinoid control of sperm motility: The role of epididymus","volume":"153","author":"Ricci","year":"2007","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4692","DOI":"10.1210\/en.2009-0057","article-title":"Characterization of the endocannabinoid system in human spermatozoa and involvement of transient receptor potential vanilloid 1 receptor in their fertilizing ability","volume":"150","author":"Francavilla","year":"2009","journal-title":"Endocrinology"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1126\/science.283.5400.401","article-title":"Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice","volume":"283","author":"Ledent","year":"1999","journal-title":"Science"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2399","DOI":"10.1210\/endo.143.6.8873","article-title":"ER function in the adult male rat: Short\u2014And long-term effects of the antiestrogen ICI 182,780 on the testis and efferent ductules, without changes in testosterone","volume":"143","author":"Oliveira","year":"2002","journal-title":"Endocrinology"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1093\/humrep\/14.4.1039","article-title":"Utility of sperm chromatin structure assay as a diagnostic tool in the human fertility clinic","volume":"14","author":"Evenson","year":"1999","journal-title":"Hum. Reprod."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1002\/j.1939-4640.2002.tb02599.x","article-title":"Sperm chromatin structure assay: Its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques","volume":"23","author":"Evenson","year":"2002","journal-title":"J. Androl."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1017\/S0967199499000635","article-title":"DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes","volume":"7","author":"Said","year":"1999","journal-title":"Zygote"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1016\/j.phrs.2016.09.005","article-title":"Bisphenol A induces hypothalamic down-regulation of the the cannabinoid receptor 1 and anorexigenic effects in male mice","volume":"113","author":"Suglia","year":"2016","journal-title":"Pharmacol. Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/j.placenta.2008.11.018","article-title":"Low type I cannabinoid receptor levels characterize placental villous in labouring delivery","volume":"30","author":"Acone","year":"2009","journal-title":"Placenta"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1016\/j.placenta.2009.03.015","article-title":"Endocannabinoid system in first trimester placenta: Low FAAH and high CB1 expression characterize spontaneous miscarriage","volume":"30","author":"Trabucco","year":"2009","journal-title":"Placenta"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1677\/joe.0.1020387","article-title":"Seasonal testosterone profile and testicular responsiveness to pituitary factors and gonadotrophin releasing hormone during two different phases of the sexual cycle of the frog (Rana esculenta)","volume":"102","author":"Pierantoni","year":"1984","journal-title":"J. Endocrinol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.anireprosci.2016.01.017","article-title":"The Sperm Chromatin Structure Assay (SCSA (\u00ae)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related tofertility","volume":"169","author":"Evenson","year":"2016","journal-title":"Anim. Reprod. Sci."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1095\/biolreprod44.4.569","article-title":"DNA packaging and organization in mammalian spermatozoa: Comparison with somatic cells","volume":"44","author":"Ward","year":"1991","journal-title":"Biol. Reprod."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"629","DOI":"10.2164\/jandrol.05040","article-title":"Nonprotein thiols and disulfides in rat epididymal spermatozoa and epididymal fluid: Role of g-Glutamyl-Transpeptidase in sperm maturation","volume":"26","author":"Seligman","year":"2005","journal-title":"J. Androl."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"12866","DOI":"10.1038\/s41598-019-49177-y","article-title":"Characterisation and localisation of the endocannabinoid system components in the adult human testis","volume":"9","author":"Nielsen","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Errico, S., Chioccarelli, T., Moggio, M., Diano, N., and Cobellis, G. (2019). A New LC-MS\/MS method for simultaneous and quantitative detection of bisphenol-A and steroids in target tissues: A power tool to characterize the interference of bisphenol-A exposure on steroid levels. Molecules, 25.","DOI":"10.3390\/molecules25010048"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/9\/3117\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:51:33Z","timestamp":1760363493000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/9\/3117"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,4,28]]},"references-count":54,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2020,5]]}},"alternative-id":["ijms21093117"],"URL":"https:\/\/doi.org\/10.3390\/ijms21093117","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,4,28]]}}}