{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T17:47:13Z","timestamp":1792000033120,"version":"4.4.0"},"reference-count":53,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2021,10,6]],"date-time":"2021-10-06T00:00:00Z","timestamp":1633478400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100007052","name":"University of Verona","doi-asserted-by":"publisher","award":["JPVR184ZZ5"],"award-info":[{"award-number":["JPVR184ZZ5"]}],"id":[{"id":"10.13039\/501100007052","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca2+, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca2+ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg2+ alone and Mg2+ and Ca2+. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC50 value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca2+. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca2+ conditions, as suggested by 2 \u03bcs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca2+ levels, which would ultimately lead to toxic accumulation of cGMP and Ca2+ in the photoreceptor outer segment, thus triggering cell death.<\/jats:p>","DOI":"10.3390\/ijms221910809","type":"journal-article","created":{"date-parts":[[2021,10,7]],"date-time":"2021-10-07T16:23:58Z","timestamp":1633623838000},"page":"10809","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase"],"prefix":"10.3390","volume":"22","author":[{"given":"Amedeo","family":"Biasi","sequence":"first","affiliation":[{"name":"Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7821-3717","authenticated-orcid":false,"given":"Valerio","family":"Marino","sequence":"additional","affiliation":[{"name":"Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuditta","family":"Dal Cortivo","sequence":"additional","affiliation":[{"name":"Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1974-4937","authenticated-orcid":false,"given":"Paolo Enrico","family":"Maltese","sequence":"additional","affiliation":[{"name":"MAGI\u2019S Lab s.r.l., 38068 Rovereto, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonio Mattia","family":"Modarelli","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, ASST Santi Paolo e Carlo Hospital, University of Milan, 20142 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9552-221X","authenticated-orcid":false,"given":"Matteo","family":"Bertelli","sequence":"additional","affiliation":[{"name":"MAGI\u2019S Lab s.r.l., 38068 Rovereto, Italy"},{"name":"MAGI Euregio, 39100 Bolzano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9556-994X","authenticated-orcid":false,"given":"Leonardo","family":"Colombo","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, ASST Santi Paolo e Carlo Hospital, University of Milan, 20142 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3724-3044","authenticated-orcid":false,"given":"Daniele","family":"Dell\u2019Orco","sequence":"additional","affiliation":[{"name":"Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"67","DOI":"10.3389\/fnmol.2015.00067","article-title":"Protein and signaling networks in vertebrate photoreceptor cells","volume":"8","author":"Koch","year":"2015","journal-title":"Front. Mol. Neurosci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1021\/cn400027z","article-title":"A calcium-relay mechanism in vertebrate phototransduction","volume":"4","author":"Koch","year":"2013","journal-title":"ACS Chem. Neurosci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"442","DOI":"10.1016\/j.preteyeres.2012.05.002","article-title":"Speed, sensitivity, and stability of the light response in rod and cone photoreceptors: Facts and models","volume":"31","author":"Korenbrot","year":"2012","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"100619","DOI":"10.1016\/j.jbc.2021.100619","article-title":"Molecular properties of human guanylate cyclase\u2013activating protein 2 (GCAP2) and its retinal dystrophy\u2013associated variant G157R","volume":"296","author":"Avesani","year":"2021","journal-title":"J. Biol. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"20105","DOI":"10.1038\/s41598-019-56606-5","article-title":"Normal GCAPs partly compensate for altered cGMP signaling in retinal dystrophies associated with mutations in GUCA1A","volume":"9","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"16903","DOI":"10.1074\/jbc.C400065200","article-title":"Guanylyl cyclase-activating proteins (GCAPs) are Ca2+\/Mg2+ sensors: Implications for photoreceptor guanylyl cyclase (RetGC) regulation in mammalian photoreceptors","volume":"279","author":"Peshenko","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"23830","DOI":"10.1074\/jbc.M600257200","article-title":"Ca2+ and Mg2+ binding properties of GCAP-Evidence that Mg2+-bound form is the physiological activator of photoreceptor guanylyl cyclase","volume":"281","author":"Peshenko","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1007\/s11010-009-0330-z","article-title":"Ca2+-modulated vision-linked ROS-GC guanylate cyclase transduction machinery","volume":"334","author":"Koch","year":"2009","journal-title":"Mol. Cell. Biochem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1007\/s11010-009-0328-6","article-title":"Mg2+\/Ca2+ cation binding cycle of guanylyl cyclase activating proteins (GCAPs): Role in regulation of photoreceptor guanylyl cyclase","volume":"334","author":"Dizhoor","year":"2009","journal-title":"Mol. Cell. Biochem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5590","DOI":"10.1021\/bi200491b","article-title":"Enzymatic properties and regulation of the native isozymes of retinal membrane guanylyl cyclase (RetGC) from mouse photoreceptors","volume":"50","author":"Peshenko","year":"2011","journal-title":"Biochemistry"},{"key":"ref_11","first-page":"198","article-title":"Cone dystrophy or macular dystrophy associated with novel autosomal dominant GUCA1A mutations","volume":"23","author":"Manes","year":"2017","journal-title":"Mol. Vis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1458","DOI":"10.1021\/acschemneuro.0c00111","article-title":"The neuronal calcium sensor GCAP1 encoded by GUCA1A exhibits heterogeneous functional properties in two cases of retinitis pigmentosa","volume":"11","author":"Abbas","year":"2020","journal-title":"ACS Chem. Neurosci."},{"key":"ref_13","first-page":"4204","article-title":"A novel p. (Glu111Val) missense mutation in GUCA1A associated with cone-rod dystrophy leads to impaired calcium sensing and perturbed second messenger homeostasis in photoreceptors","volume":"27","author":"Marino","year":"2018","journal-title":"Hum. Mol. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"6653","DOI":"10.1093\/hmg\/ddv370","article-title":"Two retinal dystrophy-associated missense mutations inGUCA1Awith distinct molecular properties result in a similar aberrant regulation of the retinal guanylate cyclase","volume":"24","author":"Marino","year":"2015","journal-title":"Hum. Mol. Genet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"17311","DOI":"10.1074\/jbc.273.28.17311","article-title":"Constitutive activation of photoreceptor guanylate cyclase by Y99C mutant of GCAP-1: Possible role in causing human autosomal dominant cone degeneration","volume":"273","author":"Dizhoor","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/S1097-2765(00)80121-5","article-title":"GCAP1(Y99C) Mutant is constitutively active in autosomal dominant cone dystrophy","volume":"2","author":"Sokal","year":"1998","journal-title":"Mol. Cell"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1086\/323265","article-title":"Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy","volume":"69","author":"Wilkie","year":"2001","journal-title":"Am. J. Hum. Genet."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3863","DOI":"10.1167\/iovs.04-0590","article-title":"A novel mutation (I143NT) in guanylate cyclase-activating protein 1 (GCAP1) associated with autosomal dominant cone degeneration","volume":"45","author":"Nishiguchi","year":"2004","journal-title":"Investig. Opthalmol. Vis. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1167\/iovs.04-1431","article-title":"A novel GCAP1 missense mutation (L151F) in a large family with autosomal dominant cone-rod dystrophy (adCORD)","volume":"46","author":"Sokal","year":"2005","journal-title":"Investig. Opthalmol. Vis. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2425","DOI":"10.1016\/j.visres.2008.07.016","article-title":"A novel GCAP1(N104K) mutation in EF-hand 3 (EF3) linked to autosomal dominant cone dystrophy","volume":"48","author":"Jiang","year":"2008","journal-title":"Vis. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1007\/s00018-009-0243-8","article-title":"Calcium binding, structural stability and guanylate cyclase activation in GCAP1 variants associated with human cone dystrophy","volume":"67","author":"Behnen","year":"2010","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"517570","DOI":"10.1155\/2013\/517570","article-title":"Novel GUCA1A mutations suggesting possible mechanisms of pathogenesis in cone, cone-rod, and macular dystrophy patients","volume":"2013","author":"Kamenarova","year":"2013","journal-title":"Biomed. Res. Int."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"E782","DOI":"10.1002\/humu.21055","article-title":"Mutations in the GUCA1A gene involved in hereditary cone dystrophies impair calcium-mediated regulation of guanylate cyclase","volume":"30","author":"Kitiratschky","year":"2009","journal-title":"Hum. Mutat."},{"key":"ref_24","first-page":"133","article-title":"Dysfunction of cGMP signalling in photoreceptors by a macular dystrophy-related mutation in the calcium sensor GCAP1","volume":"26","author":"Vocke","year":"2016","journal-title":"Hum. Mol. Genet."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3476","DOI":"10.1074\/jbc.RA118.006180","article-title":"A G86R mutation in the calcium-sensor protein GCAP1 alters regulation of retinal guanylyl cyclase and causes dominant cone-rod degeneration","volume":"294","author":"Peshenko","year":"2019","journal-title":"J. Biol. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"711","DOI":"10.1136\/bjophthalmol-2018-313278","article-title":"Progressive cone and cone-rod dystrophies: Clinical features, molecular genetics and prospects for therapy","volume":"103","author":"Gill","year":"2019","journal-title":"Br. J. Ophthalmol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"118794","DOI":"10.1016\/j.bbamcr.2020.118794","article-title":"Missense mutations affecting Ca2+-coordination in GCAP1 lead to cone-rod dystrophies by altering protein structural and functional properties","volume":"1867","author":"Marino","year":"2020","journal-title":"Biochim Biophys Acta Mol Cell Res."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Marino, V., Dal Cortivo, G., Maltese, P., Placidi, G., De Siena, E., Falsini, B., Bertelli, M., and Dell\u2019Orco, D. (2021). Impaired Ca2+ sensitivity of a novel GCAP1 variant causes cone dystrophy and leads to abnormal Synaptic transmission between photoreceptors and bipolar cells. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22084030"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1978","DOI":"10.1093\/bioinformatics\/bty897","article-title":"VarSome: The human genomic variant search engine","volume":"35","author":"Kopanos","year":"2019","journal-title":"Bioinformatics"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1038\/gim.2015.30","article-title":"Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology","volume":"17","author":"Richards","year":"2015","journal-title":"Genet. Med."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1006\/abio.2002.5661","article-title":"Measurement of Ca2+-binding constants of proteins and presentation of the CaLigator software","volume":"305","author":"Linse","year":"2002","journal-title":"Anal. Biochem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/j.ab.2015.11.005","article-title":"Electrophoretic mobility shift in native gels indicates calcium-dependent structural changes of neuronal calcium sensor proteins","volume":"494","author":"Viviano","year":"2016","journal-title":"Anal. Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2982","DOI":"10.1021\/ac300213j","article-title":"Dynamics of conformational Ca2+ -switches in signaling Networks Detected by a Planar Plasmonic Device","volume":"84","author":"Sulmann","year":"2012","journal-title":"Anal. Chem."},{"key":"ref_34","first-page":"15","article-title":"Calcium binding to proteins studied via competition with chromophoric chelators","volume":"173","author":"Linse","year":"2002","journal-title":"Methods Mol. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Lim, S., Roseman, G., Peshenko, I., Manchala, G., Cudia, D., Dizhoor, A., Millhauser, G., and Ames, J.B. (2018). Retinal guanylyl cyclase activating protein 1 forms a functional dimer. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0193947"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Bon\u00ec, F., Marino, V., Bidoia, C., Mastrangelo, E., Barbiroli, A., Dell\u2019Orco, D., and Milani, M. (2020). Modulation of guanylate cyclase activating protein 1 (GCAP1) dimeric assembly by Ca2+ or Mg2+: Hints to understand protein activity. Biomolecules, 10.","DOI":"10.3390\/biom10101408"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Lim, S., Peshenko, I.V., Dizhoor, A., and Ames, J.B. (2013). Structural insights for activation of retinal guanylate cyclase by GCAP1. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0081822"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"10140","DOI":"10.1074\/jbc.M113.540716","article-title":"Identification of target binding site in photoreceptor guanylyl cyclase-activating protein 1 (GCAP1)","volume":"289","author":"Peshenko","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"6244","DOI":"10.1074\/jbc.274.10.6244","article-title":"Identification of a domain in guanylyl cyclase-activating protein 1 that interacts with a complex of guanylyl cyclase and tubulin in photoreceptors","volume":"274","author":"Schrem","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1515\/bc.2010.063","article-title":"Involvement of the calcium sensor GCAP1 in hereditary cone dystrophies","volume":"391","author":"Behnen","year":"2010","journal-title":"Biol. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s00424-021-02556-9","article-title":"The role of cGMP-signalling and calcium-signalling in photoreceptor cell death: Perspectives for therapy development","volume":"473","author":"Das","year":"2021","journal-title":"Pflugers Arch"},{"key":"ref_42","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":"2019","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_43","first-page":"6753","article-title":"PipeMAGI: An integrated and validated workflow for analysis of NGS data for clinical diagnostics","volume":"23","author":"Marceddu","year":"2019","journal-title":"Eur Rev. Med. Pharmacol. Sci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"13021","DOI":"10.1021\/bi026618y","article-title":"Calcium- and Myristoyl-Dependent properties of guanylate cyclase-activating protein-1 and protein-2","volume":"41","author":"Hwang","year":"2002","journal-title":"Biochemistry"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"5065","DOI":"10.1021\/bi400288p","article-title":"The Dimerization domain in outer segment guanylate cyclase is a Ca2+-sensitive control switch module","volume":"52","author":"Koch","year":"2013","journal-title":"Biochemistry"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"274","DOI":"10.3389\/fnmol.2018.00274","article-title":"Preferential Binding of Mg2+ Over Ca2+ to CIB2 triggers an allosteric switch impaired in usher syndrome type 1J","volume":"11","author":"Vallone","year":"2018","journal-title":"Front. Mol. Neurosci."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"11773","DOI":"10.1039\/C7NR03288A","article-title":"CaF2 nanoparticles as surface carriers of GCAP1, a calcium sensor protein involved in retinal dystrophies","volume":"9","author":"Marino","year":"2017","journal-title":"Nanoscale"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1392","DOI":"10.1016\/j.str.2007.09.013","article-title":"Stabilizing function for myristoyl group revealed by the crystal structure of a neuronal calcium sensor, guanylate cyclase-activating protein","volume":"15","author":"Stephen","year":"2007","journal-title":"Structure"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2055","DOI":"10.1016\/j.bbamcr.2014.10.026","article-title":"Structural effects of Mg2+ on the regulatory states of three neuronal calcium sensors operating in vertebrate phototransduction","volume":"1853","author":"Marino","year":"2015","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers","volume":"1","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1038\/nmeth.4067","article-title":"CHARMM36m: An improved force field for folded and intrinsically disordered proteins","volume":"14","author":"Huang","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"34277","DOI":"10.1038\/srep34277","article-title":"Allosteric communication pathways routed by Ca2+\/Mg2+ exchange in GCAP1 selectively switch target regulation modes","volume":"6","author":"Marino","year":"2016","journal-title":"Sci. Rep."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/19\/10809\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:09:59Z","timestamp":1760166599000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/19\/10809"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,6]]},"references-count":53,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2021,10]]}},"alternative-id":["ijms221910809"],"URL":"https:\/\/doi.org\/10.3390\/ijms221910809","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,6]]}}}