{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,17]],"date-time":"2026-01-17T20:08:08Z","timestamp":1768680488611,"version":"3.49.0"},"reference-count":51,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2021,8,31]],"date-time":"2021-08-31T00:00:00Z","timestamp":1630368000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"European Structural and Investment Funds","award":["POCI-01-0145-FEDER-016385"],"award-info":[{"award-number":["POCI-01-0145-FEDER-016385"]}]},{"name":"HEALTH-UNORTE","award":["NORTE-01-0145-FEDER-000039"],"award-info":[{"award-number":["NORTE-01-0145-FEDER-000039"]}]},{"name":"FCT- Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia\/MEC \u2013 Minist\u00e9rio da Educa\u00e7\u00e3o e Ci\u00eancia","award":["SFRH\/BD\/123674\/2016"],"award-info":[{"award-number":["SFRH\/BD\/123674\/2016"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.<\/jats:p>","DOI":"10.3390\/ijms22179488","type":"journal-article","created":{"date-parts":[[2021,8,31]],"date-time":"2021-08-31T21:59:45Z","timestamp":1630447185000},"page":"9488","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["In Vitro and In Vivo Effects of SerpinA1 on the Modulation of Transthyretin Proteolysis"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1675-2317","authenticated-orcid":false,"given":"Filipa","family":"Bezerra","sequence":"first","affiliation":[{"name":"Molecular Neurobiology Group, i3S-Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Departamento de Biologia Molecular, ICBAS-Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal"}]},{"given":"Christoph","family":"Niemietz","sequence":"additional","affiliation":[{"name":"Medizinische Klinik B, Universit\u00e4tsklinikum M\u00fcnster, 48149 M\u00fcnster, Germany"}]},{"given":"Hartmut H. J.","family":"Schmidt","sequence":"additional","affiliation":[{"name":"Medizinische Klinik B, Universit\u00e4tsklinikum M\u00fcnster, 48149 M\u00fcnster, Germany"}]},{"given":"Andree","family":"Zibert","sequence":"additional","affiliation":[{"name":"Medizinische Klinik B, Universit\u00e4tsklinikum M\u00fcnster, 48149 M\u00fcnster, Germany"}]},{"given":"Shuling","family":"Guo","sequence":"additional","affiliation":[{"name":"Ionis Pharmaceuticals, Carlsbad, CA 92010, USA"}]},{"given":"Brett P.","family":"Monia","sequence":"additional","affiliation":[{"name":"Ionis Pharmaceuticals, Carlsbad, CA 92010, USA"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8110-479X","authenticated-orcid":false,"given":"Paula","family":"Gon\u00e7alves","sequence":"additional","affiliation":[{"name":"Molecular Neurobiology Group, i3S-Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"}]},{"given":"Maria Jo\u00e3o","family":"Saraiva","sequence":"additional","affiliation":[{"name":"Molecular Neurobiology Group, i3S-Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Departamento de Biologia Molecular, ICBAS-Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9289-3835","authenticated-orcid":false,"given":"Maria Ros\u00e1rio","family":"Almeida","sequence":"additional","affiliation":[{"name":"Molecular Neurobiology Group, i3S-Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Departamento de Biologia Molecular, ICBAS-Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1007\/s40120-020-00210-7","article-title":"Transthyretin Amyloidosis: Update on the Clinical Spectrum, Pathogenesis, and Disease-Modifying Therapies","volume":"9","author":"Koike","year":"2020","journal-title":"Neurol. Ther."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Nativi-Nicolau, J.N., Karam, C., Khella, S., and Maurer, M.S. (2021). Screening for ATTR amyloidosis in the clinic: Overlapping disorders, misdiagnosis, and multiorgan awareness. Heart Fail. Rev., 1\u20139.","DOI":"10.1007\/s10741-021-10080-2"},{"key":"ref_3","first-page":"215","article-title":"Amyloid nomenclature 2018: Recommendations by the International Society of Amyloidosis (ISA) nomenclature committee","volume":"25","author":"Benson","year":"2018","journal-title":"Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1002\/humu.1380050302","article-title":"Transthyretin mutations in health and disease","volume":"5","author":"Saraiva","year":"1995","journal-title":"Hum. Mutat."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6969","DOI":"10.1021\/bi800636q","article-title":"Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its disease-associated variants: The relationship between stability and amyloidosis","volume":"47","author":"Powers","year":"2008","journal-title":"Biochemistry"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.cell.2005.01.018","article-title":"The biological and chemical basis for tissue-selective amyloid disease","volume":"121","author":"Sekijima","year":"2005","journal-title":"Cell"},{"key":"ref_7","first-page":"48","article-title":"Transthyretin-derived senile systemic amyloidosis: Clinicopathologic and structural considerations","volume":"10","author":"Westermark","year":"2003","journal-title":"Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis"},{"key":"ref_8","first-page":"209","article-title":"Amyloid fibril proteins and amyloidosis: Chemical identification and clinical classification International Society of Amyloidosis 2016 Nomenclature Guidelines","volume":"23","author":"Sipe","year":"2016","journal-title":"Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"12119","DOI":"10.1021\/bi00096a024","article-title":"Transthyretin mutation Leu-55-Pro significantly alters tetramer stability and increases amyloidogenicity","volume":"32","author":"McCutchen","year":"1993","journal-title":"Biochemistry"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"27207","DOI":"10.1074\/jbc.M101024200","article-title":"Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants","volume":"276","author":"Quintas","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Si, J.B., Kim, B., and Kim, J.H. (2021). Transthyretin Misfolding, A Fatal Structural Pathogenesis Mechanism. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22094429"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1006\/jmbi.2002.5441","article-title":"Transthyretin fibrillogenesis entails the assembly of monomers: A molecular model for in vitro assembled transthyretin amyloid-like fibrils","volume":"317","author":"Cardoso","year":"2002","journal-title":"J. Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1002\/path.1759","article-title":"Amyloid deposits in transthyretin-derived amyloidosis: Cleaved transthyretin is associated with distinct amyloid morphology","volume":"206","author":"Bergstrom","year":"2005","journal-title":"J. Pathol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1002\/path.2411","article-title":"Amyloid fibril composition is related to the phenotype of hereditary transthyretin V30M amyloidosis","volume":"216","author":"Ihse","year":"2008","journal-title":"J. Pathol."},{"key":"ref_15","first-page":"142","article-title":"Amyloid fibrils containing fragmented ATTR may be the standard fibril composition in ATTR amyloidosis","volume":"20","author":"Ihse","year":"2013","journal-title":"Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1007\/s00109-010-0695-1","article-title":"Variation in amount of wild-type transthyretin in different fibril and tissue types in ATTR amyloidosis","volume":"89","author":"Ihse","year":"2011","journal-title":"J. Mol. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1017","DOI":"10.1097\/TP.0b013e31824b3749","article-title":"Amyloid fibril composition as a predictor of development of cardiomyopathy after liver transplantation for hereditary transthyretin amyloidosis","volume":"93","author":"Gustafsson","year":"2012","journal-title":"Transplantation"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"28324","DOI":"10.1074\/jbc.M114.563981","article-title":"The importance of a gatekeeper residue on the aggregation of transthyretin: Implications for transthyretin-related amyloidoses","volume":"289","author":"Braga","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1539","DOI":"10.1073\/pnas.1317488111","article-title":"Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis","volume":"111","author":"Mangione","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1337","DOI":"10.15252\/emmm.201505357","article-title":"A novel mechano-enzymatic cleavage mechanism underlies transthyretin amyloidogenesis","volume":"7","author":"Marcoux","year":"2015","journal-title":"EMBO Mol. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"E6428","DOI":"10.1073\/pnas.1802977115","article-title":"Unusual duplication mutation in a surface loop of human transthyretin leads to an aggressive drug-resistant amyloid disease","volume":"115","author":"Klimtchuk","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"14192","DOI":"10.1074\/jbc.RA118.003990","article-title":"Plasminogen activation triggers transthyretin amyloidogenesis in vitro","volume":"293","author":"Mangione","year":"2018","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2172","DOI":"10.1046\/j.1471-4159.2000.0752172.x","article-title":"Tissue plasminogen activator requires plasminogen to modulate amyloid-beta neurotoxicity and deposition","volume":"75","author":"Tucker","year":"2000","journal-title":"J. Neurochem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1046\/j.1365-2141.2000.02183.x","article-title":"Bleeding symptoms and coagulation abnormalities in 337 patients with AL-amyloidosis","volume":"110","author":"Mumford","year":"2000","journal-title":"Br. J. Haematol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1139","DOI":"10.1111\/j.1538-7836.2007.02457.x","article-title":"Increased plasmin-alpha2-antiplasmin levels indicate activation of the fibrinolytic system in systemic amyloidoses","volume":"5","author":"Bouma","year":"2007","journal-title":"J. Thromb. Haemost. JTH"},{"key":"ref_26","first-page":"89","article-title":"Excessive fibrinolysis in AL-amyloidosis is induced by urokinae-type plasminogen activator from bone marrow plasma cells","volume":"16","author":"Uchiba","year":"2009","journal-title":"Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1097\/00001756-199202000-00020","article-title":"Alpha 1-antitrypsin and alpha 1-antichymotrypsin are in the lesions of Alzheimer\u2019s disease","volume":"3","author":"Gollin","year":"1992","journal-title":"Neuroreport"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"15990","DOI":"10.1074\/jbc.274.23.15990","article-title":"Endogenous proteins controlling amyloid beta-peptide polymerization. Possible implications for beta-amyloid formation in the central nervous system and in peripheral tissues","volume":"274","author":"Bohrmann","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1677\/jme.1.01530","article-title":"Multiple plasma proteins control atrial natriuretic peptide (ANP) aggregation","volume":"33","author":"Torricelli","year":"2004","journal-title":"J. Mol. Endocrinol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1186\/1479-7364-7-22","article-title":"Update of the human and mouse SERPIN gene superfamily","volume":"7","author":"Heit","year":"2013","journal-title":"Hum. Genom."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"da Costa, G., Ribeiro-Silva, C., Ribeiro, R., Gilberto, S., Gomes, R.A., Ferreira, A., Mateus, E., Barroso, E., Coelho, A.V., and Freire, A.P. (2015). Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0125392"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4104","DOI":"10.1021\/acs.jproteome.7b00479","article-title":"Serum Proteomic Variability Associated with Clinical Phenotype in Familial Transthyretin Amyloidosis (ATTRm)","volume":"16","author":"Chan","year":"2017","journal-title":"J. Proteome Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"jcs219824","DOI":"10.1242\/jcs.219824","article-title":"Hepatocyte-like cells reveal novel role of SERPINA1 in transthyretin amyloidosis","volume":"131","author":"Niemietz","year":"2018","journal-title":"J. Cell Sci."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"112217","DOI":"10.1016\/j.yexcr.2020.112217","article-title":"SERPINA1 modulates expression of amyloidogenic transthyretin","volume":"395","author":"Niemietz","year":"2020","journal-title":"Exp. Cell Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1111\/jcmm.12024","article-title":"Presence of N-glycosylated transthyretin in plasma of V30M carriers in familial amyloidotic polyneuropathy: An escape from ERAD","volume":"17","author":"Teixeira","year":"2013","journal-title":"J. Cell. Mol. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1111\/joim.12585","article-title":"One mutation, two distinct disease variants: Unravelling the impact of transthyretin amyloid fibril composition","volume":"281","author":"Suhr","year":"2017","journal-title":"J. Intern. Med."},{"key":"ref_37","first-page":"175","article-title":"Tertiary structures of amyloidogenic and non-amyloidogenic transthyretin variants: New model for amyloid fibril formation","volume":"5","author":"Schormann","year":"1998","journal-title":"Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1295","DOI":"10.1002\/pro.3420","article-title":"Crystal structures of amyloidogenic segments of human transthyretin","volume":"27","author":"Saelices","year":"2018","journal-title":"Protein Sci. Publ. Protein Soc."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1038\/s41598-018-37230-1","article-title":"Transthyretin Aggregation Pathway toward the Formation of Distinct Cytotoxic Oligomers","volume":"9","author":"Dasari","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"9951","DOI":"10.1074\/jbc.R115.639799","article-title":"Systemic amyloidosis: Lessons from beta2-microglobulin","volume":"290","author":"Stoppini","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1159\/000265422","article-title":"Relation of vitreous amyloidosis to prealbumin","volume":"18","author":"Sandgren","year":"1986","journal-title":"Ophthalmic Res."},{"key":"ref_42","first-page":"170","article-title":"Biochemical characterization of vitreous and cardiac amyloid in Ile84Ser transthyretin amyloidosis","volume":"13","author":"Liepnieks","year":"2006","journal-title":"Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"740","DOI":"10.1136\/jnnp-2013-305973","article-title":"Changes in pathological and biochemical findings of systemic tissue sites in familial amyloid polyneuropathy more than 10 years after liver transplantation","volume":"85","author":"Oshima","year":"2014","journal-title":"J. Neurol. Neurosurg. Psychiatry"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Suhr, O.B., Wixner, J., Anan, I., Lundgren, H.E., Wijayatunga, P., Westermark, P., and Ihse, E. (2019). Amyloid fibril composition within hereditary Val30Met (p. Val50Met) transthyretin amyloidosis families. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0211983"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"14425","DOI":"10.1074\/jbc.M117.786657","article-title":"Amorphous protein aggregates stimulate plasminogen activation, leading to release of cytotoxic fragments that are clients for extracellular chaperones","volume":"292","author":"Constantinescu","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"11259","DOI":"10.1074\/jbc.RA119.007851","article-title":"A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH","volume":"294","author":"Ueda","year":"2019","journal-title":"J. Biol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"5008","DOI":"10.1038\/s41467-019-13038-z","article-title":"Cryo-EM structure of a transthyretin-derived amyloid fibril from a patient with hereditary ATTR amyloidosis","volume":"10","author":"Schmidt","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e28511","DOI":"10.4161\/rdis.28511","article-title":"Using antisense technology to develop a novel therapy for alpha-1 antitrypsin deficient (AATD) liver disease and to model AATD lung disease","volume":"2","author":"Guo","year":"2014","journal-title":"Rare Dis."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2415","DOI":"10.1021\/bi00223a017","article-title":"Production of recombinant human transthyretin with biological activities toward the understanding of the molecular basis of familial amyloidotic polyneuropathy (FAP)","volume":"30","author":"Furuya","year":"1991","journal-title":"Biochemistry"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Osorio, H., Silva, C., Ferreira, M., Gullo, I., Maximo, V., Barros, R., Mendonca, F., Oliveira, C., and Carneiro, F. (2021). Proteomics Analysis of Gastric Cancer Patients with Diabetes Mellitus. J. Clin. Med., 10.","DOI":"10.3390\/jcm10030407"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1186\/s12974-017-0891-9","article-title":"Differential expression of Cathepsin E in transthyretin amyloidosis: From neuropathology to the immune system","volume":"14","author":"Goncalves","year":"2017","journal-title":"J. Neuroinflammation"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/17\/9488\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:53:55Z","timestamp":1760165635000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/17\/9488"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,31]]},"references-count":51,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2021,9]]}},"alternative-id":["ijms22179488"],"URL":"https:\/\/doi.org\/10.3390\/ijms22179488","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,31]]}}}