{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T20:00:48Z","timestamp":1790712048636,"version":"4.1.0"},"reference-count":61,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2021,6,16]],"date-time":"2021-06-16T00:00:00Z","timestamp":1623801600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010665","name":"H2020 Marie Sk\u0142odowska-Curie Actions","doi-asserted-by":"publisher","award":["MSCA-ITN-2017-765441"],"award-info":[{"award-number":["MSCA-ITN-2017-765441"]}],"id":[{"id":"10.13039\/100010665","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002426","name":"Fondazione Telethon","doi-asserted-by":"publisher","award":["GGP19113"],"award-info":[{"award-number":["GGP19113"]}],"id":[{"id":"10.13039\/501100002426","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening.<\/jats:p>","DOI":"10.3390\/ijms22126440","type":"journal-article","created":{"date-parts":[[2021,6,16]],"date-time":"2021-06-16T10:27:07Z","timestamp":1623839227000},"page":"6440","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["New In Vitro Cellular Model for Molecular Studies of Retinitis Pigmentosa"],"prefix":"10.3390","volume":"22","author":[{"given":"Li","family":"Huang","sequence":"first","affiliation":[{"name":"Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meltem","family":"Kutluer","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elisa","family":"Adani","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonella","family":"Comitato","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4428-2084","authenticated-orcid":false,"given":"Valeria","family":"Marigo","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy"},{"name":"Center for Neuroscience and Neurotechnology, Via Campi 287, 41125 Modena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,6,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"4039","DOI":"10.1242\/jcs.175687","article-title":"Photoreceptors at a glance","volume":"128","author":"Molday","year":"2015","journal-title":"J. Cell Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1111\/cge.12203","article-title":"Genes and mutations causing retinitis pigmentosa","volume":"84","author":"Daiger","year":"2013","journal-title":"Clin. Genet."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"100772","DOI":"10.1016\/j.preteyeres.2019.07.005","article-title":"Cellular mechanisms of hereditary photoreceptor degeneration\u2014Focus on cGMP","volume":"74","author":"Power","year":"2020","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Paquet-Durand, F., Ekstr\u00f6m, P., and Marigo, V. (2018). CHAPTER 3. Modulation of Calcium Overload and Calpain Activity. Drug Discovery, Royal Society of Chemistry (RSC).","DOI":"10.1039\/9781788013666-00048"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Marigo, V., Ekstr\u00f6m, P., Schwede, F., Rentsch, A., and Paquet-Durand, F. (2019). Chapter 6 Modulation of cGMP-signalling to Prevent Retinal Degeneration. Therapies for Retinal Degeneration: Targeting Common Processes, The Royal Society of Chemistry.","DOI":"10.1039\/9781788013666-00088"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1007\/978-3-030-27378-1_40","article-title":"RD Genes Associated with High Photoreceptor cGMP-Levels (Mini-Review)","volume":"1185","author":"Marigo","year":"2019","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1637","DOI":"10.1016\/j.drudis.2019.03.004","article-title":"Drug delivery to retinal photoreceptors","volume":"24","author":"Himawan","year":"2019","journal-title":"Drug Discov. Today"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0169-328X(00)00224-2","article-title":"Characterization of a transformed rat retinal ganglion cell line","volume":"86","author":"Krishnamoorthy","year":"2001","journal-title":"Brain Res. Mol. Brain Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1242\/jcs.104.1.37","article-title":"Immortalization of polarized rat retinal pigment epithelium","volume":"104","author":"Nabi","year":"1993","journal-title":"J. Cell Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1006\/exer.1996.0230","article-title":"Human Papillomavirus-16 E6\/E7 Transfected Retinal Cell Line Expresses the M\u00fcller Cell Phenotype","volume":"64","author":"Roque","year":"1997","journal-title":"Exp. Eye Res."},{"key":"ref_11","first-page":"2665","article-title":"Immortalization of rabbit corneal epithelial cells by a recombinant SV40-adenovirus vector","volume":"34","author":"Araki","year":"1993","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1126\/science.2451289","article-title":"Cone cell-specific genes expressed in retinoblastoma","volume":"240","author":"Bogenmann","year":"1988","journal-title":"Science"},{"key":"ref_13","first-page":"3931","article-title":"Expression of retina-specific genes by mouse retinoblastoma cells","volume":"35","author":"Bernstein","year":"1994","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_14","first-page":"1003","article-title":"Characterization of a new continuous cell line derived from a human retinoblastoma","volume":"37","author":"McFall","year":"1977","journal-title":"Cancer Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4016","DOI":"10.1073\/pnas.92.9.4016","article-title":"Rod photoreceptor-specific gene expression in human retinoblastoma cells","volume":"92","author":"Farber","year":"1995","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1242\/bio.2012027","article-title":"The adult retinal stem cell is a rare cell in the ciliary epithelium whose progeny can differentiate into photoreceptors","volume":"1","author":"Ballios","year":"2012","journal-title":"Biol. Open"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Demontis, G.C., Aruta, C., Comitato, A., De Marzo, A., and Marigo, V. (2012). Functional and molecular characterization of rod-like cells from retinal stem cells derived from the adult ciliary epithelium. PLoS ONE, 7.","DOI":"10.1371\/annotation\/ca21f359-8e8e-4c3d-8308-e0f20fc446bb"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"E2997","DOI":"10.1073\/pnas.1718792115","article-title":"Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration","volume":"115","author":"Vighi","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1681","DOI":"10.1083\/jcb.119.6.1681","article-title":"Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter","volume":"119","author":"Alubaidi","year":"1992","journal-title":"J. Cell Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"764","DOI":"10.1167\/iovs.03-1114","article-title":"Expression of Cone-Photoreceptor\u2013Specific Antigens in a Cell Line Derived from Retinal Tumors in Transgenic Mice","volume":"45","author":"Tan","year":"2004","journal-title":"Investig. Opthalmology Vis. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"127","DOI":"10.3389\/fgene.2019.00127","article-title":"Opportunities and Challenges for Molecular Understanding of Ciliopathies-The 100,000 Genomes Project","volume":"10","author":"Wheway","year":"2019","journal-title":"Front. Genet."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1699","DOI":"10.1093\/hmg\/ddm118","article-title":"Mutations in splicing factor PRPF3, causing retinal degeneration, form detrimental aggregates in photoreceptor cells","volume":"16","author":"Comitato","year":"2007","journal-title":"Hum. Mol. Genet."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41419-018-0613-y","article-title":"Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina","volume":"9","author":"Comitato","year":"2018","journal-title":"Cell Death Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/S0959-4388(99)80005-1","article-title":"The roles of intrinsic and extrinsic cues and bHLH genes in the determination of retinal cell fates","volume":"9","author":"Cepko","year":"1999","journal-title":"Curr. Opin. Neurobiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1073\/pnas.89.1.266","article-title":"A conserved retina-specific gene encodes a basic motif\/leucine zipper domain","volume":"89","author":"Swaroop","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1073\/pnas.93.1.191","article-title":"The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression","volume":"93","author":"Rehemtulla","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"29794","DOI":"10.1074\/jbc.M003658200","article-title":"The leucine zipper of NRL interacts with the CRX homeodomain. A possible mechanism of transcriptional synergy in rhodopsin regulation","volume":"275","author":"Mitton","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1563","DOI":"10.1093\/hmg\/ddh173","article-title":"Photoreceptor-specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors","volume":"13","author":"Cheng","year":"2004","journal-title":"Hum. Mol. Genet."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2460","DOI":"10.1016\/j.celrep.2016.10.074","article-title":"NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors","volume":"17","author":"Kim","year":"2016","journal-title":"Cell Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1038\/ng774","article-title":"Nrl is required for rod photoreceptor development","volume":"29","author":"Mears","year":"2001","journal-title":"Nat. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1679","DOI":"10.1073\/pnas.0605934104","article-title":"Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL","volume":"104","author":"Oh","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2156","DOI":"10.1167\/iovs.04-1427","article-title":"Cone-like Morphological, Molecular, and Electrophysiological Features of the Photoreceptors of the Nrl Knockout Mouse","volume":"46","author":"Daniele","year":"2005","journal-title":"Investig. Opthalmology Vis. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1038\/ng0693-130","article-title":"Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa","volume":"4","author":"McLaughlin","year":"1993","journal-title":"Nat. Genet."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3060","DOI":"10.1167\/iovs.05-0257","article-title":"Efficacy and Selectivity of Phosphodiesterase-Targeted Drugs in Inhibiting Photoreceptor Phosphodiesterase (PDE6) in Retinal Photoreceptors","volume":"46","author":"Zhang","year":"2005","journal-title":"Investig. Opthalmology Vis. Sci."},{"key":"ref_35","first-page":"803","article-title":"Vasodilator-stimulated phosphoprotein (VASP) phosphorylation provides a biomarker for the action of exisulind and related agents that activate protein kinase G","volume":"1","author":"Deguchi","year":"2002","journal-title":"Mol. Cancer Ther."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"796","DOI":"10.1111\/j.1471-4159.2008.05822.x","article-title":"PKG activity causes photoreceptor cell death in two retinitis pigmentosa models","volume":"108","author":"Hauck","year":"2009","journal-title":"J. Neurochem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"5301","DOI":"10.18632\/oncotarget.23685","article-title":"New cGMP analogues restrain proliferation and migration of melanoma cells","volume":"9","author":"Vighi","year":"2018","journal-title":"Oncotarget"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1007\/s12035-019-01723-5","article-title":"Calpain Activation Is the Major Cause of Cell Death in Photoreceptors Expressing a Rhodopsin Misfolding Mutation","volume":"57","author":"Comitato","year":"2019","journal-title":"Mol. Neurobiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1007\/978-3-030-27378-1_51","article-title":"Differential Contribution of Calcium-Activated Proteases and ER-Stress in Three Mouse Models of Retinitis Pigmentosa Expressing P23H Mutant RHO","volume":"1185","author":"Comitato","year":"2019","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"930","DOI":"10.1111\/j.1471-4159.2010.06983.x","article-title":"Photoreceptor rescue and toxicity induced by different calpain inhibitors","volume":"115","author":"Sanges","year":"2010","journal-title":"J. Neurochem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1784","DOI":"10.4103\/1673-5374.280303","article-title":"Targeting molecular pathways for the treatment of inherited retinal degeneration","volume":"15","author":"Marigo","year":"2020","journal-title":"Neural Regen. Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"25241","DOI":"10.1074\/jbc.M115.645846","article-title":"Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor","volume":"290","author":"Kenealey","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"13897","DOI":"10.1073\/pnas.0404197101","article-title":"Leber congenital amaurosis linked to AIPL1: A mouse model reveals destabilization of cGMP phosphodiesterase","volume":"101","author":"Ramamurthy","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"664","DOI":"10.1126\/science.193183","article-title":"Cyclic GMP accumulation causes degeneration of photoreceptor cells: Simulation of an inherited disease","volume":"196","author":"Lolley","year":"1977","journal-title":"Science"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1111\/j.1749-6632.1999.tb07837.x","article-title":"Calcium Overload Triggers Rod Photoreceptor Apoptotic Cell Death in Chemical-Induced and Inherited Retinal Degenerations","volume":"893","author":"Fox","year":"1999","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"100760","DOI":"10.1016\/j.preteyeres.2019.05.001","article-title":"Voltage- and calcium-gated ion channels of neurons in the vertebrate retina","volume":"72","author":"Nawy","year":"2019","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1183","DOI":"10.1038\/13508","article-title":"Retinitis pigmentosa: Rod photoreceptor rescue by a calcium-channel blocker in the rd mouse","volume":"5","author":"Frasson","year":"1999","journal-title":"Nat. Med."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"17366","DOI":"10.1073\/pnas.0606276103","article-title":"Apoptosis in retinal degeneration involves cross-talk between apoptosis-inducing factor (AIF) and caspase-12 and is blocked by calpain inhibitors","volume":"103","author":"Sanges","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1007\/978-1-4419-1399-9_56","article-title":"Do Calcium Channel Blockers Rescue Dying Photoreceptors in the Pde6b rd1 Mouse?","volume":"664","author":"Barabas","year":"2010","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1016\/S0014-4835(03)00027-7","article-title":"Inhibitory action of diltiazem on voltage-gated calcium channels in cone photoreceptors","volume":"76","author":"Hart","year":"2003","journal-title":"Exp. Eye Res."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"d2023","DOI":"10.2741\/krizaj","article-title":"Calcium regulation in photoreceptors","volume":"7","author":"Krizaj","year":"2002","journal-title":"Front. Biosci."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1152\/physrev.00015.2005","article-title":"Function of cGMP-dependent protein kinases as revealed by gene deletion","volume":"86","author":"Hofmann","year":"2006","journal-title":"Physiol. Rev."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1006\/geno.1997.4743","article-title":"Characterization of the Human Gene Encoding the Type I\u03b1 and Type I\u03b2 cGMP-Dependent Protein Kinase (PRKG1)","volume":"42","author":"Natarajan","year":"1997","journal-title":"Genomics"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"2299","DOI":"10.1093\/hmg\/ddx121","article-title":"Two pathways of rod photoreceptor cell death induced by elevated cGMP","volume":"26","author":"Wang","year":"2017","journal-title":"Hum. Mol. Genet."},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Roy, A., Groten, J., Marigo, V., Tomar, T., and Hilhorst, R. (2021). Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22031180"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"308","DOI":"10.3389\/fgene.2019.00308","article-title":"661W Photoreceptor Cell Line as a Cell Model for Studying Retinal Ciliopathies","volume":"10","author":"Wheway","year":"2019","journal-title":"Front. Genet."},{"key":"ref_57","first-page":"2766","article-title":"Localization of cGMP-Dependent Protein Kinase Isoforms in Mouse Eye","volume":"41","author":"Gamm","year":"2000","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/j.ejmech.2017.09.053","article-title":"New dimeric cGMP analogues reduce proliferation in three colon cancer cell lines","volume":"141","author":"Hoffmann","year":"2017","journal-title":"Eur. J. Med. Chem."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1042\/BSR20190405","article-title":"Active PKG II inhibited the growth and migration of ovarian cancer cells through blocking Raf\/MEK and PI3K\/Akt signaling pathways","volume":"39","author":"Wu","year":"2019","journal-title":"Biosci. Rep."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/j.canlet.2015.10.024","article-title":"The cyclic GMP\/protein kinase G pathway as a therapeutic target in head and neck squamous cell carcinoma","volume":"370","author":"Tuttle","year":"2016","journal-title":"Cancer Lett."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"1758834017751635","DOI":"10.1177\/1758834017751635","article-title":"The constitutively active PKG II mutant effectively inhibits gastric cancer development via a blockade of EGF\/EGFR-associated signalling cascades","volume":"10","author":"Wu","year":"2018","journal-title":"Ther. Adv. Med. Oncol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/12\/6440\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:16:56Z","timestamp":1760163416000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/12\/6440"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,16]]},"references-count":61,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2021,6]]}},"alternative-id":["ijms22126440"],"URL":"https:\/\/doi.org\/10.3390\/ijms22126440","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,16]]}}}