{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T19:36:31Z","timestamp":1772825791005,"version":"3.50.1"},"reference-count":56,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,4,30]],"date-time":"2021-04-30T00:00:00Z","timestamp":1619740800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Science Center, Poland","award":["2016\/21\/B\/NZ1\/00288"],"award-info":[{"award-number":["2016\/21\/B\/NZ1\/00288"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.<\/jats:p>","DOI":"10.3390\/ijms22094758","type":"journal-article","created":{"date-parts":[[2021,4,30]],"date-time":"2021-04-30T05:10:55Z","timestamp":1619759455000},"page":"4758","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9832-2344","authenticated-orcid":false,"given":"Piotr","family":"Maj","sequence":"first","affiliation":[{"name":"Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., PL-02-093 Warszawa, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6485-8231","authenticated-orcid":false,"given":"Adam","family":"Jarmu\u0142a","sequence":"additional","affiliation":[{"name":"Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., PL-02-093 Warszawa, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7217-4163","authenticated-orcid":false,"given":"Piotr","family":"Wilk","sequence":"additional","affiliation":[{"name":"Helmholtz-Zentrum Berlin, Macromolecular Crystallography (NP-GMX) Elektronenspeicherring BESSY II, 15 Albert-Einstein-St., D-12489 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0160-9884","authenticated-orcid":false,"given":"Ma\u0142gorzata","family":"Prokopowicz","sequence":"additional","affiliation":[{"name":"College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences and Faculty of Physics, University of Warsaw, PL-02-097 Warszawa, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6097-5518","authenticated-orcid":false,"given":"Wojciech","family":"Rypniewski","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry, Polish Academy of Sciences, PL- 61-704 Pozna\u0144, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zbigniew","family":"Zieli\u0144ski","sequence":"additional","affiliation":[{"name":"Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., PL-02-093 Warszawa, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna","family":"Dowiercia\u0142","sequence":"additional","affiliation":[{"name":"Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., PL-02-093 Warszawa, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Agnieszka","family":"Bzowska","sequence":"additional","affiliation":[{"name":"Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, PL-02-093 Warszawa, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4809-4571","authenticated-orcid":false,"given":"Wojciech","family":"Rode","sequence":"additional","affiliation":[{"name":"Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., PL-02-093 Warszawa, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1146\/annurev.bi.64.070195.003445","article-title":"The catalytic mechanism and structure of thymidylate synthase","volume":"64","author":"Carreras","year":"1995","journal-title":"Annu. Rev. Biochem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"133","DOI":"10.18388\/abp.1996_4524","article-title":"Molecular mechanism of thymidylate synthase-catalyzed reaction and interaction of the enzyme with 2- and\/or 4-substituted analogues of dUMP and 5-fluoro-dUMP","volume":"43","author":"Rode","year":"1996","journal-title":"Acta Biochim. Pol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2241","DOI":"10.2174\/0929867054864868","article-title":"Thymidylate synthase structure, function and implication in drug discovery","volume":"12","author":"Costi","year":"2005","journal-title":"Curr. Med. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1211","DOI":"10.2174\/13895575110091211","article-title":"Antifolate inhibitors of thymidylate synthase as anticancer drugs","volume":"10","year":"2010","journal-title":"Mini Rev. Med. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.drup.2015.10.003","article-title":"Inside the biochemical pathways of thymidylate synthase perturbed by anticancer drugs: Novel strategies to overcome cancer chemoresistance","volume":"23","author":"Taddia","year":"2015","journal-title":"Drug Resist. Updat."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/0968-0004(82)90157-8","article-title":"The slow-binding and slow, tight-binding inhibition of enzyme-catalysed reactions","volume":"7","author":"Morrison","year":"1982","journal-title":"Trends Biochem. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3332","DOI":"10.1016\/S0021-9258(18)95914-4","article-title":"The purification and properties of thymidylate synthetase from chick embryo extracts","volume":"242","author":"Lorenson","year":"1967","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1259","DOI":"10.1021\/jm00376a004","article-title":"Interaction of N4-hydroxy-2\u2032-deoxycytidilic acid with thymidylate synthetase","volume":"27","author":"Goldstein","year":"1984","journal-title":"J. Med. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"10835","DOI":"10.1021\/bi00500a017","article-title":"Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N4-hydroxy-dCMP, N4-hydroxy-5-fluoro-dCMP, and related analogues","volume":"29","author":"Rode","year":"1990","journal-title":"Biochemistry"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"593","DOI":"10.1017\/S0031182099005880","article-title":"Trichinella spiralis and Trichinella pseudospiralis: Developmental patterns of enzymes involved in thymidylate biosynthesis and pyrimidine salvage","volume":"120","author":"Rode","year":"2000","journal-title":"Parasitology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"4647","DOI":"10.1021\/jm000975u","article-title":"5-Substituted N4-hydroxy-2\u2032-deoxycytidines and their 5\u2032-monophosphates: Synthesis, conformation, interaction with tumor thymidylate synthase, and in vitro antitumor activity","volume":"43","author":"Felczak","year":"2000","journal-title":"J. Med. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1021\/bi020598i","article-title":"Conformational Dynamics along an Enzymatic Reaction Pathway: Thymidylate Synthase, \u201cthe Movie\u201d","volume":"42","author":"Stroud","year":"2003","journal-title":"Biochemistry"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1007\/s11224-016-0840-8","article-title":"Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme","volume":"28","author":"Wilk","year":"2017","journal-title":"Struct. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1515\/pterid-2013-0010","article-title":"Crystal structures of complexes of mouse thymidylate synthase crystallized with N4-OH-dCMP alone or in the presence of N5,10-methylenetetrahydrofolate","volume":"24","author":"Wilk","year":"2013","journal-title":"Pteridines"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1333","DOI":"10.1039\/C6MB00026F","article-title":"Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N4-hydroxy-dCMP","volume":"12","author":"Ludwiczak","year":"2016","journal-title":"Mol. BioSyst."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1002\/prot.20921","article-title":"MUSTANG: A multiple structural alignment algorithm","volume":"64","author":"Konagurthu","year":"2006","journal-title":"Proteins"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1021\/bi981881d","article-title":"Crystal structures of rat thymidylate synthase inhibited by Tomudex, a potent anticancer drug","volume":"38","author":"Dzik","year":"1999","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.jmgm.2017.08.008","article-title":"Crystal structures of nematode (parasitic T. spiralis and free living C. elegans), compared to mammalian, thymidylate synthases (TS). Molecular docking and molecular dynamics simulations in search for nematode-specific inhibitors of TS","volume":"77","author":"Wilk","year":"2017","journal-title":"J. Mol. Graph. Model."},{"key":"ref_19","first-page":"945803","article-title":"Crystal Structure of Mouse Thymidylate Synthase in Tertiary Complex with dUMP and Raltitrexed Reveals N-Terminus Architecture and Two Different Active Site Conformations","volume":"2014","author":"Wilk","year":"2014","journal-title":"BioMed Res. Int."},{"key":"ref_20","first-page":"163","article-title":"Crystal structures of substrate- and sulfate-bound mouse thymidylate synthase","volume":"20","author":"Rypniewski","year":"2009","journal-title":"Pteridines"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1515\/pterid-2013-0011","article-title":"Crystal structures of thymidylate synthase from nematodes, Trichinella spiralis and Caenorhabditis elegans, as a potential template for species-specific drug design","volume":"24","author":"Wilk","year":"2013","journal-title":"Pteridines"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.bioorg.2013.11.003","article-title":"Crystal structure of phosphoramide-phosphorylated thymidylate synthase reveals pSer127, reflecting probably pHis to pSer phosphotransfer","volume":"52","author":"Wilk","year":"2014","journal-title":"Bioorg. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"108106","DOI":"10.1016\/j.abb.2019.108106","article-title":"Thymidylate synthase-catalyzed, tetrahydrofolate-dependent self-inactivation by 5-FdUMP","volume":"674","author":"Sobich","year":"2019","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"17356","DOI":"10.1038\/srep17356","article-title":"Conservation and role of electrostatics in thymidylate synthase","volume":"5","author":"Garg","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"7485","DOI":"10.1038\/ncomms8485","article-title":"Caenorhabditis elegans is a useful model for anthelmintic discovery","volume":"6","author":"Burns","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1041","DOI":"10.1002\/bmc.3214","article-title":"5,10-Methylene-tetrahydrofolate dissociates into tetrahydrofolate and formaldehyde at physiological pH and acidic pH, typical conditions used during sample extraction and LC-MS\/MS analysis of biological samples","volume":"28","author":"Jastrebova","year":"2014","journal-title":"Biomed. Chromatogr."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"14188","DOI":"10.1021\/bi701363s","article-title":"Role of Y94 in proton and hydride transfers catalyzed by thymidylate synthase","volume":"46","author":"Hong","year":"2007","journal-title":"Biochemistry"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1016\/0959-440X(95)80059-X","article-title":"Freeze trapping of reaction intermediates","volume":"5","author":"Moffat","year":"1995","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1042\/BJ20041804","article-title":"Observation of a tetrahedral reaction intermediate in the HIV-1 protease-substrate complex","volume":"389","author":"Kumar","year":"2005","journal-title":"Biochem. J."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1080\/08893110802604868","article-title":"Kinetic protein crystallography: A tool to watch proteins in action","volume":"15","author":"Bourgeois","year":"2009","journal-title":"Crystallogr. Rev."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1038\/nature11181","article-title":"Watching DNA polymerase \u03b7 make a phosphodiester bond","volume":"487","author":"Nakamura","year":"2012","journal-title":"Nature"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1209","DOI":"10.1021\/acschembio.7b01104","article-title":"Crystallographic trapping of reaction intermediates in quinolinic acid synthesis by NadA","volume":"13","author":"Volbeda","year":"2018","journal-title":"ACS Chem. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1016\/bs.mie.2019.02.034","article-title":"Quick-soaking of crystals reveals unprecedented insights into the catalytic mechanism of glycosyltransferases","volume":"621","author":"Cifuente","year":"2019","journal-title":"Methods Enzymol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1006\/jmbi.1997.1495","article-title":"Contributions of orientation and hydrogen bonding to catalysis in Asn229 mutants of thymidylate synthase","volume":"276","author":"Liu","year":"1998","journal-title":"J. Mol. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"651","DOI":"10.18388\/abp.2002_3774","article-title":"The effect of Arg 209 to Lys mutation in mouse thymidylate synthase","volume":"49","author":"Jagielska","year":"2002","journal-title":"Acta Biochim. Pol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1017\/S0031182005007274","article-title":"Developmental arrest in Caenorhabditis elegans dauer larvae causes high expression of enzymes involved in thymidylate biosynthesis, similar to that found in Trichinella muscle larvae","volume":"131","author":"Rode","year":"2005","journal-title":"Parasitology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.bioorg.2010.02.001","article-title":"Phosphorylation of thymidylate synthase from various sources by human protein kinase CK2 and its catalytic subunits","volume":"38","author":"Hellman","year":"2010","journal-title":"Bioorg. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1922","DOI":"10.1016\/j.bbapap.2015.08.007","article-title":"Properties of phosphorylated thymidylate synthase","volume":"1854","author":"Ruman","year":"2015","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3794","DOI":"10.1016\/S0021-9258(19)84524-6","article-title":"The enzymatic synthesis of thymidylate. I. Early steps in the purification of thymidylate synthetase of Escherichia coli","volume":"237","author":"Wahba","year":"1962","journal-title":"J. Biol. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/0006-2952(87)90690-3","article-title":"Studies on the interaction with thymidylate synthase of analogues of 2\u2032-deoxyuridine-5\u2032-phosphate and 5-fluoro-2\u2032-deoxyuridine-5\u2032-phosphate with modified phosphate groups","volume":"36","author":"Rode","year":"1987","journal-title":"Biochem. Pharmacol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4389","DOI":"10.1002\/pmic.200402049","article-title":"Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide\/hydroxide affinity chromatography (MOAC)","volume":"5","author":"Wolschin","year":"2005","journal-title":"Proteomics"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/0076-6879(90)82008-P","article-title":"Quantitation of protein","volume":"182","author":"Stoscheck","year":"1990","journal-title":"Methods Enzymol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"PC11","DOI":"10.1016\/S0021-9258(18)64341-8","article-title":"Direct spectrophotometric evidence for the oxidation of tetrahydrofolate during the enzymatic synthesis of thymidylate","volume":"236","author":"Wahba","year":"1961","journal-title":"J. Biol. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Rossmann, M.G., and Arnold, E. (2006). DENZO and SCALEPACK. International Tables for Crystallography Volume F: Crystallography of Biological Macromolecules, Springer.","DOI":"10.1107\/97809553602060000106"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1107\/S0907444905036693","article-title":"Scaling and assessment of data quality","volume":"62","author":"Evans","year":"2006","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1107\/S0907444910045749","article-title":"Overview of the CCP4 suite and current developments","volume":"67","author":"Winn","year":"2011","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1107\/S0907444910007493","article-title":"Features and development of Coot","volume":"66","author":"Emsley","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1107\/S0907444996012255","article-title":"Refinement of macromolecular structures by the maximum-likelihood method","volume":"53","author":"Murshudov","year":"1997","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1107\/S0907444912001308","article-title":"Towards automated crystallographic structure refinement with phenix.refine","volume":"68","author":"Afonine","year":"2012","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1107\/S0907444909052925","article-title":"PHENIX: A comprehensive Python-based system for macromolecular structure solution","volume":"66","author":"Adams","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1107\/S0021889892009944","article-title":"PROCHECK\u2014A program to check the stereochemical quality of protein structures","volume":"26","author":"Laskowski","year":"1993","journal-title":"J. Appl. Cryst."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1107\/S0907444909042073","article-title":"MolProbity: All-atom structure validation for macromolecular crystallography","volume":"66","author":"Chen","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1107\/S0907444908044296","article-title":"Crystallographic model quality at a glance","volume":"65","author":"Urzhumtseva","year":"2009","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1107\/S0907444998006684","article-title":"SFCHECK: A unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model","volume":"55","author":"Vaguine","year":"1999","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"646","DOI":"10.1107\/S1399004714028132","article-title":"FEM: Feature-enhanced map","volume":"71","author":"Afonine","year":"2015","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"1626","DOI":"10.1021\/acs.chemrev.8b00290","article-title":"Utility of B-factors in protein science: Interpreting rigidity, flexibility, and internal motion and engineering thermostability","volume":"119","author":"Sun","year":"2019","journal-title":"Chem. Rev."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/9\/4758\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:55:52Z","timestamp":1760162152000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/9\/4758"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,30]]},"references-count":56,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["ijms22094758"],"URL":"https:\/\/doi.org\/10.3390\/ijms22094758","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,4,30]]}}}