{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T22:34:27Z","timestamp":1775082867436,"version":"3.50.1"},"reference-count":53,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2023,6,2]],"date-time":"2023-06-02T00:00:00Z","timestamp":1685664000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Edmond J Safra Philanthropic Foundation"},{"name":"Krembil Foundation"},{"name":"Maybank Foundation"},{"name":"Rossy Foundation"},{"name":"Centre for Research in Neurodegenerative Disease (CRND) Graduate Student Endowment, Unilever\/Lipton Graduate Fellowship Award in Neurosciences"},{"name":"Jesse Keshin Graduate Student Award"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Recent studies have been able to detect \u03b1-synuclein (\u03b1Syn) seeding in formaldehyde-fixed paraffin-embedded (FFPE) tissues from patients with synucleinopathies using seed amplification assays (SAAs), but with relatively low sensitivity due to limited protein extraction efficiency. With the aim of introducing an alternative option to frozen tissues, we developed a streamlined protein extraction protocol for evaluating disease-specific seeding in FFPE human brain. We evaluated the protein extraction efficiency of different tissue preparations, deparaffinizations, and protein extraction buffers using formaldehyde-fixed and FFPE tissue of a single Lewy body disease (LBD) subject. Alternatively, we incorporated heat-induced antigen retrieval and dissociation using a commercially available kit. Our novel protein extraction protocol has been optimized to work with 10 sections of 4.5-\u00b5m-thickness or 2-mm-diameter micro-punch of FFPE tissue that can be used to seed SAAs. We demonstrated that extracted proteins from FFPE still preserve seeding potential and further show disease-specific seeding in LBD and multiple system atrophy. To the best of our knowledge, our study is the first to recapitulate disease-specific \u03b1Syn seeding behaviour in FFPE human brain. Our findings open new perspectives in re-evaluating archived human brain tissue, extending the disease-specific seeding assays to larger cohorts to facilitate molecular subtyping of synucleinopathies.<\/jats:p>","DOI":"10.3390\/biom13060936","type":"journal-article","created":{"date-parts":[[2023,6,2]],"date-time":"2023-06-02T10:08:41Z","timestamp":1685700521000},"page":"936","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Disease-Specific \u03b1-Synuclein Seeding in Lewy Body Disease and Multiple System Atrophy Are Preserved in Formaldehyde-Fixed Paraffin-Embedded Human Brain"],"prefix":"10.3390","volume":"13","author":[{"given":"Ain","family":"Kim","sequence":"first","affiliation":[{"name":"Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON M5T 0S8, Canada"},{"name":"Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6463-6321","authenticated-orcid":false,"given":"Ivan","family":"Martinez-Valbuena","sequence":"additional","affiliation":[{"name":"Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON M5T 0S8, Canada"},{"name":"Krembil Brain Institute, University Health Network, Toronto, ON M5T 0S8, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jun","family":"Li","sequence":"additional","affiliation":[{"name":"Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON M5T 0S8, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anthony E.","family":"Lang","sequence":"additional","affiliation":[{"name":"Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON M5T 0S8, Canada"},{"name":"Krembil Brain Institute, University Health Network, Toronto, ON M5T 0S8, Canada"},{"name":"Edmond J. Safra Program in Parkinson\u2019s Disease and the Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, Toronto, ON M5T 2S6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gabor G.","family":"Kovacs","sequence":"additional","affiliation":[{"name":"Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON M5T 0S8, Canada"},{"name":"Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada"},{"name":"Krembil Brain Institute, University Health Network, Toronto, ON M5T 0S8, Canada"},{"name":"Edmond J. Safra Program in Parkinson\u2019s Disease and the Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, Toronto, ON M5T 2S6, Canada"},{"name":"Laboratory Medicine Program, University Health Network, Toronto, ON M5G 2C4, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,6,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2804","DOI":"10.1523\/JNEUROSCI.08-08-02804.1988","article-title":"Synuclein: A Neuron-Specific Protein Localized to the Nucleus and Presynaptic Nerve Terminal","volume":"8","author":"Maroteaux","year":"1988","journal-title":"J. Neurosci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1038\/ncb748","article-title":"\u03b1-Synuclein Is Phosphorylated in Synucleinopathy Lesions","volume":"4","author":"Fujiwara","year":"2002","journal-title":"Nat. Cell Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"8637","DOI":"10.1073\/pnas.0712179105","article-title":"The Fold of \u03b1-Synuclein Fibrils","volume":"105","author":"Vilar","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1136\/jclinpath-2019-205952","article-title":"Molecular Pathology of Neurodegenerative Diseases: Principles and Practice","volume":"72","author":"Kovacs","year":"2019","journal-title":"J. Clin. Pathol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1038\/42166","article-title":"\u03b1-Synuclein in Lewy Bodies","volume":"388","author":"Spillantini","year":"1997","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/0022-510X(89)90219-0","article-title":"Glial Cytoplasmic Inclusions in the CNS of Patients with Multiple System Atrophy (Striatonigral Degeneration, Olivopontocerebellar Atrophy and Shy-Drager Syndrome)","volume":"94","author":"Papp","year":"1989","journal-title":"J. Neurol. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"670","DOI":"10.1212\/01.wnl.0000324625.00404.15","article-title":"Second Consensus Statement on the Diagnosis of Multiple System Atrophy","volume":"71","author":"Gilman","year":"2008","journal-title":"Neurology"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2575","DOI":"10.1038\/ncomms3575","article-title":"Structural and Functional Characterization of Two Alpha-Synuclein Strains","volume":"4","author":"Bousset","year":"2013","journal-title":"Nat. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"977","DOI":"10.1007\/s00401-020-02157-3","article-title":"The Structural Differences between Patient-Derived \u03b1-Synuclein Strains Dictate Characteristics of Parkinson\u2019s Disease, Multiple System Atrophy and Dementia with Lewy Bodies","volume":"139","author":"Gelders","year":"2020","journal-title":"Acta Neuropathol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1038\/s41586-020-2317-6","article-title":"Structures of \u03b1-Synuclein Filaments from Multiple System Atrophy","volume":"585","author":"Schweighauser","year":"2020","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1038\/s41586-022-05319-3","article-title":"Structures of \u03b1-Synuclein Filaments from Human Brains with Lewy Pathology","volume":"610","author":"Yang","year":"2022","journal-title":"Nature"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1038\/s41593-019-0541-x","article-title":"\u03b1-Synuclein Strains Target Distinct Brain Regions and Cell Types","volume":"23","author":"Lau","year":"2020","journal-title":"Nat. Neurosci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2079","DOI":"10.1126\/science.274.5295.2079","article-title":"Evidence for the Conformation of the Pathologic Isoform of the Prion Protein Enciphering and Propagating Prion Diversity","volume":"274","author":"Telling","year":"1996","journal-title":"Science"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1186\/s40478-018-0508-2","article-title":"Rapid and Ultra-Sensitive Quantitation of Disease-Associated \u03b1-Synuclein Seeds in Brain and Cerebrospinal Fluid by ASyn RT-QuIC","volume":"6","author":"Groveman","year":"2018","journal-title":"Acta Neuropathol. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1001\/jamaneurol.2020.3311","article-title":"Skin \u03b1-Synuclein Aggregation Seeding Activity as a Novel Biomarker for Parkinson Disease","volume":"78","author":"Wang","year":"2021","journal-title":"JAMA Neurol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1016\/j.bbapap.2010.04.001","article-title":"Molecular Mechanism of Thioflavin-T Binding to Amyloid Fibrils","volume":"1804","author":"Biancalana","year":"2010","journal-title":"Biochim. Biophys. Acta-Proteins Proteom."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"7671","DOI":"10.1073\/pnas.1315346111","article-title":"Solution Conditions Determine the Relative Importance of Nucleation and Growth Processes in \u03b1-Synuclein Aggregation","volume":"111","author":"Buell","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"5200","DOI":"10.1021\/acs.biomac.0c01292","article-title":"Unraveling the NaCl Concentration Effect on the First Stages of \u03b1-Synuclein Aggregation","volume":"21","author":"Ramis","year":"2020","journal-title":"Biomacromolecules"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"23029","DOI":"10.1073\/pnas.1909322116","article-title":"Million-Fold Sensitivity Enhancement in Proteopathic Seed Amplification Assays for Biospecimens by Hofmeister Ion Comparisons","volume":"116","author":"Metrick","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1186\/s40035-022-00283-4","article-title":"Alpha-Synuclein Seeding Shows a Wide Heterogeneity in Multiple System Atrophy","volume":"11","author":"Visanji","year":"2022","journal-title":"Transl. Neurodegener."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1007\/s00401-022-02453-0","article-title":"\u03b1-Synuclein Molecular Behavior and Nigral Proteomic Profiling Distinguish Subtypes of Lewy Body Disorders","volume":"144","author":"Swinkin","year":"2022","journal-title":"Acta Neuropathol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1002\/jms.1415","article-title":"Utility of Formaldehyde Cross-Linking and Mass Spectrometry in the Study of Protein\u2013Protein Interactions","volume":"43","author":"Sutherland","year":"2008","journal-title":"J. Mass Spectrom."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"684","DOI":"10.1177\/104063871002200502","article-title":"Detection of PrP Sc in Formalin-Fixed, Paraffin-Embedded Tissue by Western Blot Differentiates Classical Scrapie, Nor98 Scrapie, and Bovine Spongiform Encephalopathy","volume":"22","author":"Loiacono","year":"2010","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"9501","DOI":"10.1128\/JVI.00692-13","article-title":"Lack of Prion Infectivity in Fixed Heart Tissue from Patients with Creutzfeldt-Jakob Disease or Amyloid Heart Disease","volume":"87","author":"Priola","year":"2013","journal-title":"J. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"25098","DOI":"10.1038\/srep25098","article-title":"Detection and Quantification of CWD Prions in Fixed Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion","volume":"6","author":"Hoover","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"105504","DOI":"10.1016\/j.nbd.2021.105504","article-title":"Formalin RT-QuIC Assay Detects Prion-Seeding Activity in Formalin-Fixed Brain Samples from Sporadic Creutzfeldt\u2013Jakob Disease Patients","volume":"159","author":"Dong","year":"2021","journal-title":"Neurobiol. Dis."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1007\/s00401-019-02058-0","article-title":"Calling \u03b1-Synuclein a Prion Is Scientifically Justifiable","volume":"138","author":"Watts","year":"2019","journal-title":"Acta Neuropathol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2230","DOI":"10.1002\/mds.28242","article-title":"Blinded RT-QuIC Analysis of \u03b1-Synuclein Biomarker in Skin Tissue from Parkinson\u2019s Disease Patients","volume":"35","author":"Manne","year":"2020","journal-title":"Mov. Disord."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1002\/mds.27907","article-title":"A-Synuclein Real-Time Quaking-Induced Conversion in the Submandibular Glands of Parkinson\u2019s Disease Patients","volume":"35","author":"Manne","year":"2020","journal-title":"Mov. Disord."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.parkreldis.2022.08.012","article-title":"In Vivo and Autopsy Validation of Alpha-Synuclein Seeding Activity Using RT-QuIC Assay in the Gastrointestinal Tract of Patients with Parkinson\u2019s Disease","volume":"103","author":"Shin","year":"2022","journal-title":"Park. Relat. Disord."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1586\/14789450.2013.820531","article-title":"Toward Improving the Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissue","volume":"10","author":"Fowler","year":"2013","journal-title":"Expert Rev. Proteom."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"26404","DOI":"10.1074\/jbc.R115.651679","article-title":"Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes","volume":"290","author":"Hoffman","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1038\/s42004-019-0224-2","article-title":"How Formaldehyde Reacts with Amino Acids","volume":"2","author":"Kamps","year":"2019","journal-title":"Commun. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1586\/epr.13.3","article-title":"Proteomic Studies of Formalin-Fixed Paraffin-Embedded Tissues","volume":"10","author":"Giusti","year":"2013","journal-title":"Expert Rev. Proteom."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/1559-0275-11-4","article-title":"Efficient Extraction of Proteins from Formalin-Fixed Paraffin-Embedded Tissues Requires Higher Concentration of Tris(Hydroxymethyl)Aminomethane","volume":"11","author":"Kawashima","year":"2014","journal-title":"Clin. Proteom."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Shen, K., Sun, J., Cao, X., Zhou, D., and Li, J. (2015). Comparison of Different Buffers for Protein Extraction from Formalin-Fixed and Paraffin-Embedded Tissue Specimens. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0142650"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"229","DOI":"10.2144\/000114414","article-title":"Analysis of Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue via Proteomic Techniques and Misconceptions of Antigen Retrieval","volume":"60","author":"Padula","year":"2016","journal-title":"Biotechniques"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"108995","DOI":"10.1016\/j.jneumeth.2020.108995","article-title":"Unlocking the Brain: A New Method for Western Blot Protein Detection from Fixed Brain Tissue","volume":"348","author":"Thacker","year":"2021","journal-title":"J. Neurosci. Methods"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3815","DOI":"10.1002\/pmic.200800971","article-title":"Generation of High-Quality Protein Extracts from Formalin-Fixed, Paraffin-Embedded Tissues","volume":"9","author":"Addis","year":"2009","journal-title":"Proteomics"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2555","DOI":"10.1002\/pmic.201000809","article-title":"A Novel Procedure for Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissues","volume":"11","author":"Blanco","year":"2011","journal-title":"Proteomics"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.talanta.2013.10.019","article-title":"A Comprehensive Factorial Design Study of Variables Affecting Protein Extraction from Formalin-Fixed Kidney Tissue Samples","volume":"119","author":"Oliveira","year":"2014","journal-title":"Talanta"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1186\/s12014-022-09346-0","article-title":"Systematic Evaluation and Optimization of Protein Extraction Parameters in Diagnostic FFPE Specimens","volume":"19","author":"Dressler","year":"2022","journal-title":"Clin. Proteom."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s00401-011-0910-3","article-title":"National Institute on Aging\u2013Alzheimer\u2019s Association Guidelines for the Neuropathologic Assessment of Alzheimer\u2019s Disease: A Practical Approach","volume":"123","author":"Montine","year":"2012","journal-title":"Acta Neuropathol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1007\/s00401-012-0964-x","article-title":"An Antibody with High Reactivity for Disease-Associated \u03b1-Synuclein Reveals Extensive Brain Pathology","volume":"124","author":"Kovacs","year":"2012","journal-title":"Acta Neuropathol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1016\/j.bbrc.2008.01.140","article-title":"Sequence Determinants Regulating Fibrillation of Human \u03b1-Synuclein","volume":"368","author":"Koo","year":"2008","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_46","first-page":"307","article-title":"An Optimized Xylene-Free Protein Extraction Method Adapted to Formalin-Fixed Paraffin Embedded Tissue Sections for Western Blot Analysis","volume":"32","author":"Mansour","year":"2017","journal-title":"Histol. Histopathol."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Wolff, C., Schott, C., Porschewski, P., Reischauer, B., and Becker, K.-F. (2011). Successful Protein Extraction from Over-Fixed and Long-Term Stored Formalin-Fixed Tissues. PLoS ONE, 6.","DOI":"10.1371\/annotation\/a42e114f-a708-4423-8a3e-a1d8919b9b60"},{"key":"ref_48","unstructured":"Geoui, T., Urlaub, H., Plessmann, U., and Porschewski, P. (2010). Current Protocols in Molecular Biology, John Wiley & Sons, Inc."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.mex.2014.07.006","article-title":"A Novel Xylene-Free Deparaffinization Method for the Extraction of Proteins from Human Derived Formalin-Fixed Paraffin Embedded (FFPE) Archival Tissue Blocks","volume":"1","author":"Mansour","year":"2014","journal-title":"MethodsX"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"657313","DOI":"10.3389\/fmed.2021.657313","article-title":"Protein Extraction from FFPE Kidney Tissue Samples: A Review of the Literature and Characterization of Techniques","volume":"8","author":"Agra","year":"2021","journal-title":"Front. Med."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"8728","DOI":"10.1007\/s12035-018-1007-y","article-title":"Detecting Alpha Synuclein Seeding Activity in Formaldehyde-Fixed MSA Patient Tissue by PMCA","volume":"55","author":"Becker","year":"2018","journal-title":"Mol. Neurobiol."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Fenyi, A., Duyckaerts, C., Bousset, L., Braak, H., Del Tredici, K., and Melki, R. (2021). Seeding Propensity and Characteristics of Pathogenic ASyn Assemblies in Formalin-Fixed Human Tissue from the Enteric Nervous System, Olfactory Bulb, and Brainstem in Cases Staged for Parkinson\u2019s Disease. Cells, 10.","DOI":"10.3390\/cells10010139"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1087982","DOI":"10.3389\/fmolb.2023.1087982","article-title":"New Protocol for Kinetic Assay Seeding Ability Recovery \u201cKASAR\u201d from Formalin-Fixed Paraffin-Embedded Tissues","volume":"10","author":"Hepker","year":"2023","journal-title":"Front. Mol. Biosci."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/13\/6\/936\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:48:05Z","timestamp":1760125685000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/13\/6\/936"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,6,2]]},"references-count":53,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2023,6]]}},"alternative-id":["biom13060936"],"URL":"https:\/\/doi.org\/10.3390\/biom13060936","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,6,2]]}}}