{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:05:47Z","timestamp":1768467947158,"version":"3.49.0"},"reference-count":63,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2022,3,16]],"date-time":"2022-03-16T00:00:00Z","timestamp":1647388800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31572068"],"award-info":[{"award-number":["31572068"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Key Technology Research and Development Program of the Ministry of Science and Technology of China","award":["2015BAD08B03"],"award-info":[{"award-number":["2015BAD08B03"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Agasicles hygrophila is a classical biological agent used to control alligator weed (Alternanthera philoxeroides). Previous research has indicated that the heat shock factor (HSF) is involved in regulating the transcriptional expression of Hsp70 in response to heat resistance in A. hygrophila. However, the regulatory mechanism by which AhHsf regulates the expression of AhHsp70 remains largely unknown. Here, we identified and cloned a 944 bp AhHsp70 promoter (AhHsp70p) region from A. hygrophila. Subsequent bioinformatics analysis revealed that the AhHsp70p sequence contains multiple functional elements and has a common TATA box approximately 30 bp upstream of the transcription start site, with transcription commencing at a purine base approximately 137 bp upstream of ATG. Promoter deletion analyses revealed that the sequence from \u2212944 to \u2212744 bp was the core regulatory region. A dual-luciferase reporter assay indicated that overexpressed AhHsf significantly enhanced the activity of AhHsp70p. Furthermore, qPCR showed that AhHsp70 expression increased with time in Spodoptera frugiperda (Sf9) cells, and AhHsf overexpression significantly upregulated AhHsp70 expression in vitro. Characterization of the upstream regulatory mechanisms demonstrated that AhHsf binds to upstream cis-acting elements in the promoter region of AhHsp70 from \u2212944 to \u2212744 bp to activate the AhHSF\u2013AhHSP pathway at the transcriptional level to protect A. hygrophila from high temperature damage. Furthermore, we proposed a molecular model of AhHsf modulation of AhHsp70 transcription following heat shock in A. hygrophila. The findings of this study suggest that enhancing the heat tolerance of A. hygrophila by modulating the upstream pathways of the Hsp family can improve the biocontrol of A. philoxeroides.<\/jats:p>","DOI":"10.3390\/ijms23063210","type":"journal-article","created":{"date-parts":[[2022,3,16]],"date-time":"2022-03-16T22:09:58Z","timestamp":1647468598000},"page":"3210","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Regulatory Mechanism of Transcription Factor AhHsf Modulates AhHsp70 Transcriptional Expression Enhancing Heat Tolerance in Agasicles hygrophila (Coleoptera: Chrysomelidae)"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3265-4071","authenticated-orcid":false,"given":"Jisu","family":"Jin","sequence":"first","affiliation":[{"name":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yiran","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaocui","family":"Liang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yiming","family":"Pei","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fanghao","family":"Wan","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China"},{"name":"Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianying","family":"Guo","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,3,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1046\/j.1365-2486.2002.00451.x","article-title":"Herbivory in global climate change research: Direct effects of rising temperature on insect herbivores","volume":"8","author":"Bale","year":"2002","journal-title":"Glob. Chang. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1016\/S2095-3119(12)60013-6","article-title":"cDNA Cloning of Heat Shock Protein Genes and Their Expression in an Indigenous Cryptic Species of the Whitefly Bemisia tabaci Complex from China","volume":"11","author":"Yu","year":"2012","journal-title":"J. Integr. Agric."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1111\/j.1469-185X.2008.00046.x","article-title":"Insect thermal tolerance: What is the role of ontogeny, ageing and senescence?","volume":"83","author":"Bowler","year":"2008","journal-title":"Biol. Rev. Camb. Philos. Soc."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Chevin, L.M., Lande, R., and Mace, G.M. (2010). Adaptation, plasticity, and extinction in a changing environment: Towards a predictive theory. PLoS Biol., 8.","DOI":"10.1371\/journal.pbio.1000357"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1038\/nature09670","article-title":"Climate change and evolutionary adaptation","volume":"470","author":"Hoffmann","year":"2011","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1146\/annurev.cb.11.110195.002301","article-title":"Heat shock transcription factors: Structure and regulation","volume":"11","author":"Wu","year":"1995","journal-title":"Annu. Rev. Cell Dev. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1038\/36237","article-title":"Chaperoning extended life","volume":"390","author":"Tatar","year":"1997","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1146\/annurev.physiol.61.1.243","article-title":"Heat-shock proteins, molecular chaperones, and the stress response: Evolutionary and ecological physiology","volume":"61","author":"Feder","year":"1999","journal-title":"Annu. Rev. Physiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1007\/BF02715811","article-title":"Mild heat stress at a young age in Drosophila melanogaster leads to increased Hsp70 synthesis after stress exposure later in Life","volume":"82","author":"Kristensen","year":"2003","journal-title":"J. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1025","DOI":"10.1046\/j.1461-0248.2003.00528.x","article-title":"The evolutionary and ecological role of heat shock proteins","volume":"6","author":"Kristensen","year":"2003","journal-title":"Ecol. Lett."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"850","DOI":"10.1126\/science.2188360","article-title":"Interaction of Hsp70 with newly synthesized proteins: Implications for protein folding and assembly","volume":"248","author":"Beckmann","year":"1990","journal-title":"Science"},{"key":"ref_12","first-page":"105","article-title":"Stress-induced activation of the heat-shock response: Cell and molecular biology of heat-shock factors","volume":"64","author":"Cotto","year":"1999","journal-title":"Biochem. Soc. Symp."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3380","DOI":"10.1093\/nar\/27.16.3380","article-title":"Silk worm Bm1 SINE RNA increases following cellular insults","volume":"27","author":"Kimura","year":"1999","journal-title":"Nucleic Acids Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1007\/s00427-002-0221-8","article-title":"Heat-inducible transgenic expression in the silkmoth Bombyx mori","volume":"212","author":"Uhlirova","year":"2002","journal-title":"Dev. Genes Evol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1146\/annurev.ge.22.120188.003215","article-title":"The heat shock proteins","volume":"22","author":"Lindquist","year":"1988","journal-title":"Annu. Rev. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"510319","DOI":"10.1155\/2015\/510319","article-title":"Roles of Hsp70s in stress responses of microorganisms, plants, and animals","volume":"2015","author":"Yu","year":"2015","journal-title":"Biomed Res. Int."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1007\/s00405-003-0621-z","article-title":"Inducible promoters for gene therapy of head and neck cancer: An in vitro study","volume":"261","author":"Schmidt","year":"2004","journal-title":"Eur. Arch. Otorhinolaryngol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"108691","DOI":"10.1016\/j.vetmic.2020.108691","article-title":"Cellular microRNA, miR-1343-5p, modulates IFN-I responses to facilitate feline panleukopenia virus replication by directly targeting IRAK1 gene","volume":"245","author":"Zhang","year":"2020","journal-title":"Vet. Microbiol."},{"key":"ref_19","first-page":"235","article-title":"The homologous region 3 from Bombyx mori nucleopolyhedrovirus enhancing the transcriptional activity of Drosophila hsp70 promoter","volume":"9","author":"Tang","year":"2004","journal-title":"Int. J. Ind. Entomol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1146\/annurev.immunol.021908.132641","article-title":"Regulation and function of NF-kappaB transcription factors in the immune system","volume":"27","author":"Vallabhapurapu","year":"2009","journal-title":"Annu. Rev. Immunol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.coi.2011.12.008","article-title":"Transcriptional regulation in the innate immune system","volume":"24","author":"Smale","year":"2012","journal-title":"Curr. Opin. Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/j.fsi.2012.09.022","article-title":"Functional analysis of the promoter of the heat shock cognate 70 gene of the Pacific white shrimp, Litopenaeus vannamei","volume":"34","author":"Zhao","year":"2013","journal-title":"Fish Shellfish. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1126\/science.1253168","article-title":"HSF-1-mediated cytoskeletal integrity determines thermotolerance and life span","volume":"346","author":"Baird","year":"2014","journal-title":"Science"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"546","DOI":"10.1126\/science.2200124","article-title":"Antibody-mediated activation of Drosophila heat shock factor in vitro","volume":"249","author":"Zimarino","year":"1990","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1360\/062005-119","article-title":"AtHsfA2 modulates expression of stress responsive genes and enhances tolerance to heat and oxidative stress in Arabidopsis","volume":"48","author":"Li","year":"2005","journal-title":"Sci. China C. Life Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1007\/s12192-008-0020-x","article-title":"New candidate genes for heat resistance in Drosophila melanogaster are regulated by HSF","volume":"13","author":"Jensen","year":"2008","journal-title":"Cell Stress Chaperones"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1063","DOI":"10.1126\/science.1165946","article-title":"Stress-inducible regulation of heat shock factor 1 by the deacetylase SIRT1","volume":"323","author":"Westerheide","year":"2009","journal-title":"Science"},{"key":"ref_28","first-page":"323","article-title":"Stress proteins in eukaryotic cells","volume":"42","author":"Margulis","year":"2000","journal-title":"Tsitologiia"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10689","DOI":"10.1074\/jbc.REV120.011666","article-title":"Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code","volume":"295","author":"Nitika","year":"2020","journal-title":"J. Biol. Chem."},{"key":"ref_30","first-page":"31","article-title":"Biological control of alligator weed with Agasicles n. sp. in Florida","volume":"6","author":"Zeiger","year":"1967","journal-title":"Hyacinth Control J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/0304-3770(76)90019-X","article-title":"The biological control of alligator weed, Alternanthera philoxeroides, in the United States of America","volume":"2","author":"Spencer","year":"1976","journal-title":"Aquat. Bot."},{"key":"ref_32","unstructured":"Coulson, J.R. (1977). Biological control of alligator weed, 1959\u20131972. A Review and Evaluation, Technical Bulletins, United States Department of Agriculture."},{"key":"ref_33","unstructured":"Zhao, X. (2009). Thermal Adaptation of Agasicles Hygrophila (Coleoptera: Chrysomelidae) in Responses to Temperature Stress, Southwest University."},{"key":"ref_34","first-page":"275","article-title":"Effects of release density on the population dynamics of the biocontrol agent, Agasicles hygrophila (Coleoptera:Chrysomelidae)","volume":"20","author":"Li","year":"2011","journal-title":"J. Biosaf."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1586","DOI":"10.1093\/jee\/tow093","article-title":"Effects of Periodically Repeated Heat Events on Reproduction and Ovary Development of Agasicles hygrophila (Coleoptera: Chrysomelidae)","volume":"109","author":"Zhao","year":"2016","journal-title":"J. Econ. Entomol."},{"key":"ref_36","unstructured":"Zhao, M.T. (2017). Ecological Performance and Transcriptome Analyses of the Two Geographic Population of Agasicles Hygrophila in Response to Heat Stresses, Chinese Academy of Agricultural Sciences."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1127\/entomologia\/2020\/0968","article-title":"Impact of summer temperatures on Agasicles hygrophila, a key biocontrol agent of the invasive weed Alternanthera philoxeroides in Hunan province, China","volume":"41","author":"Jin","year":"2021","journal-title":"Entomol. Gen."},{"key":"ref_38","first-page":"232","article-title":"Biological control of alligator weed, Alternanthera philoxeroides, the world\u2019s first aquatic weed success story","volume":"61","author":"Buckingham","year":"1996","journal-title":"Castanea"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.aquabot.2005.01.004","article-title":"Genetic diversity of Alternanthera philoxeroides in China","volume":"81","author":"Wang","year":"2005","journal-title":"Aquat. Bot."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1046\/j.1365-3180.2003.00346.x","article-title":"Genetic uniformity of Alternanthera philoxeroides in South China","volume":"43","author":"Ye","year":"2003","journal-title":"Weed Res."},{"key":"ref_41","unstructured":"State Environmental Protection Administration of China, and Chinese Academy of Sciences (2003). The Announcement about the First List if Invasive Species in China, Chinese Academy of Sciences."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1593","DOI":"10.3389\/fphys.2019.01593","article-title":"Induced thermotolerance and expression of three key Hsp genes (Hsp70, Hsp21, and sHsp21) and their roles in the high temperature tolerance of Agasicles hygrophila","volume":"10","author":"Jin","year":"2019","journal-title":"Front. Physiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"562204","DOI":"10.3389\/fphys.2020.562204","article-title":"Heat shock factor is involved in regulating the transcriptional expression of twopotential Hsps (AhHsp70 and AhsHsp21) and its role in heat shock response of Agasicles hygrophila","volume":"11","author":"Jin","year":"2020","journal-title":"Front. Physiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"822","DOI":"10.1038\/353822a0","article-title":"Stress-induced oligomerization and chromosomal relocalization of heat-shock factor","volume":"353","author":"Westwood","year":"1991","journal-title":"Nature"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"12272","DOI":"10.1021\/bi00163a042","article-title":"Trimerization of the heat shock transcription factor by a triple-stranded alpha-helical coiled-coil","volume":"31","author":"Peteranderl","year":"1992","journal-title":"Biochemistry"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1126\/science.8421783","article-title":"Regulation of heat shock factor trimer formation: Role of a conserved leucine zipper","volume":"259","author":"Rabindran","year":"1993","journal-title":"Science"},{"key":"ref_47","first-page":"1392","article-title":"Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress","volume":"13","author":"Sarge","year":"1993","journal-title":"Mol. Cell Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1627","DOI":"10.1128\/MCB.19.3.1627","article-title":"Cooperative binding of heat shock factor to the yeast HSP82 promoter in vivo and in vitro","volume":"19","author":"Erkine","year":"1999","journal-title":"Mol. Cell Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/978-1-62703-284-1_7","article-title":"Promoter deletion analysis using a dual-luciferase reporter system","volume":"977","author":"Xu","year":"2013","journal-title":"Methods Mol. Biol."},{"key":"ref_50","unstructured":"Liu, H.J. (2014). Clone of Bmall Gene Promoter in Mice and its Construction of Luciferase Reporter Gene Vector, Hebei Medical University."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1082","DOI":"10.1038\/nature08924","article-title":"CpG islands influence chromatin structure via the CpG-binding protein Cfp1","volume":"464","author":"Thomson","year":"2010","journal-title":"Nature"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1010","DOI":"10.1101\/gad.2037511","article-title":"CpG islands and the regulation of transcription","volume":"25","author":"Deaton","year":"2011","journal-title":"Genes Dev."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"147","DOI":"10.4161\/epi.6.2.13640","article-title":"CpG island chromatin: A platform for gene regulation","volume":"6","author":"Blackledge","year":"2011","journal-title":"Epigenetics"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1134\/S1022795418060042","article-title":"Activities analysis and polymorphisms identification of GPIHBP1 promoter region in porcine","volume":"54","author":"Chen","year":"2018","journal-title":"Russ. J. Genet."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1972","DOI":"10.1093\/jee\/toaa105","article-title":"Induction of heat shock protein genes is the hallmark of egg heat tolerance in Agasicles hygrophila (Coleoptera: Chrysomelidae)","volume":"113","author":"Jia","year":"2020","journal-title":"J. Econ. Entomol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"321","DOI":"10.2108\/zsj.25.321","article-title":"Characteristic expression of three heat shock-responsive genes during larval diapause in the bamboo borer Omphisa fuscidentalis","volume":"25","author":"Tungjitwitayakul","year":"2008","journal-title":"Zool. Sci."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1007\/s00223-020-00685-4","article-title":"Identification of a 22 bp DNA cis element that plays a critical role in colony stimulating factor 1-dependent transcriptional activation of the SPHK1 gene","volume":"107","author":"Yao","year":"2020","journal-title":"Calcif. Tissue Int."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.gene.2019.01.023","article-title":"Bioinformatic and functional analysis of promoter region of human SLC25A13 gene","volume":"693","author":"Chen","year":"2019","journal-title":"Gene"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1071\/FP18237","article-title":"Promoter deletion analysis reveals root-specific expression of the alkenal reductase gene (OsAER1) in Oryza sativa","volume":"46","author":"Apriana","year":"2019","journal-title":"Funct. Plant Biol."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1007\/s10725-019-00504-3","article-title":"Functional characterization of OsHAK1 promoter in response to osmotic\/drought stress by deletion analysis in transgenic rice","volume":"88","author":"Chen","year":"2019","journal-title":"Plant Growth Regul."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"1123","DOI":"10.1016\/j.exger.2007.09.001","article-title":"Sex specific effects of heat induced hormesis in Hsf-deficient Drosophila melanogaster","volume":"42","author":"Sorensen","year":"2007","journal-title":"Exp. Gerontol."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"1597","DOI":"10.1007\/s10530-010-9932-3","article-title":"Performance of Agasicles hygrophila (Coleoptera: Chrysomelidae), a biological control agent of invasive alligator weed, at low non-freezing temperatures","volume":"14","author":"Guo","year":"2012","journal-title":"Biol. Invasions"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/S0022-2836(83)80320-9","article-title":"Chromosomal walking and jumping to isolate DNA from the Ace and rosy loci and the bithorax complex in Drosophila melanogaster","volume":"168","author":"Bender","year":"1983","journal-title":"J. Mol. Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/6\/3210\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:37:51Z","timestamp":1760135871000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/6\/3210"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,3,16]]},"references-count":63,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,3]]}},"alternative-id":["ijms23063210"],"URL":"https:\/\/doi.org\/10.3390\/ijms23063210","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,3,16]]}}}