{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,25]],"date-time":"2025-10-25T04:42:32Z","timestamp":1761367352692,"version":"build-2065373602"},"reference-count":50,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T00:00:00Z","timestamp":1761177600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100005701","name":"\u00c5hl\u00e9n-Foundation","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100005701","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Frimurarna Foundation"},{"name":"Ulla-Carin Linquist Foundation"},{"DOI":"10.13039\/100000957","name":"Alzheimer\u2019s Association","doi-asserted-by":"crossref","award":["AARG-24-1310046"],"award-info":[{"award-number":["AARG-24-1310046"]}],"id":[{"id":"10.13039\/100000957","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Dementia Foundation"},{"DOI":"10.13039\/100010815","name":"Foundation for Old Servants","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100010815","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Karolinska Institutet\u2019s Research Foundations"},{"name":"Olle Engkvist Byggm\u00e4stare Foundation"},{"name":"Magnus Bergvalls Foundation"},{"DOI":"10.13039\/100009673","name":"Gun and Bertil Stohnes Foundation","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100009673","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Swedish government","award":["ALF Med N 20200330"],"award-info":[{"award-number":["ALF Med N 20200330"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The cholinergic system is one of the most ancient and widespread signaling systems in the body, implicated in a range of pathological conditions\u2014from neurodegenerative disorders to cancer. Given its broad relevance, there is growing interest in characterizing this system across diverse cellular models to enable drug screening, mechanistic studies, and exploration of new therapeutic avenues. In this study, we investigated four cancer cell lines: one of neuroblastoma origin previously used in cholinergic signaling studies (SH-SY5Y), one non-small cell lung adenocarcinoma line (A549), and two small cell lung carcinoma lines (H69 and H82). We assessed the expression and localization of key components of the cholinergic system, along with the cellular capacity for acetylcholine (ACh) synthesis and release. Whole-cell flow cytometry following membrane permeabilization revealed that all cell lines expressed the ACh-synthesizing enzyme choline acetyltransferase (ChAT). HPLC-MS analysis confirmed that ChAT was functionally active, as all cell lines synthesized and released ACh into the conditioned media, suggesting the presence of autocrine and\/or paracrine ACh signaling circuits, consistent with previous reports. The cell lines also demonstrated choline uptake, indicative of functional choline and\/or organic cation transporters. Additionally, all lines expressed the ACh-degrading enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), as well as the alfa seven (\u03b17) nicotinic and M1 muscarinic ACh receptor subtypes. Notably, flow cytometry of intact SH-SY5Y cells revealed two novel findings: (1) ChAT was localized to the extracellular membrane, a feature not observed in the lung cancer cell lines, and (2) BChE, rather than AChE, was the predominant membrane-bound ACh-degrading enzyme. These results were corroborated by both whole-cell and surface-confocal microscopy. In conclusion, our findings suggest that a functional cholinergic phenotype is a shared feature of several carcinoma cell lines, potentially serving as a survival checkpoint that could be therapeutically explored. The discovery of extracellular membrane-bound ChAT uniquely in neuroblastoma SH-SY5Y cells points to a novel form of in situ ACh signaling that warrants further investigation.<\/jats:p>","DOI":"10.3390\/ijms262110311","type":"journal-article","created":{"date-parts":[[2025,10,24]],"date-time":"2025-10-24T00:47:36Z","timestamp":1761266856000},"page":"10311","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells"],"prefix":"10.3390","volume":"26","author":[{"given":"Banita","family":"Thakur","sequence":"first","affiliation":[{"name":"Division of Clinical Geriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, SE-141 86 Stockholm, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2374-5887","authenticated-orcid":false,"given":"Samar","family":"Tarazi","sequence":"additional","affiliation":[{"name":"Division of Clinical Geriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, SE-141 86 Stockholm, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lada","family":"Dole\u017ealov\u00e1","sequence":"additional","affiliation":[{"name":"Division of Clinical Geriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, SE-141 86 Stockholm, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Homira","family":"Behbahani","sequence":"additional","affiliation":[{"name":"Division of Clinical Geriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, SE-141 86 Stockholm, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Taher","family":"Darreh-Shori","sequence":"additional","affiliation":[{"name":"Division of Clinical Geriatrics, Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, SE-141 86 Stockholm, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Vijayaraghavan, S., Karami, A., Aeinehband, S., Behbahani, H., Grandien, A., Nilsson, B., Ekdahl, K.N., Lindblom, R.P., Piehl, F., and Darreh-Shori, T. (2013). Regulated Extracellular Choline Acetyltransferase Activity- The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0065936"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1093\/biolre\/ioad127","article-title":"Presence of key cholinergic enzymes in human spermatozoa and seminal fluiddagger","volume":"110","author":"Thakur","year":"2024","journal-title":"Biol. Reprod."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4124","DOI":"10.1002\/cne.23415","article-title":"Cholinergic circuitry of the human nucleus basalis and its fate in Alzheimer\u2019s disease","volume":"521","author":"Mesulam","year":"2013","journal-title":"J. Comp. Neurol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1038\/nrn1035","article-title":"Neurobiology of butyrylcholinesterase","volume":"4","author":"Darvesh","year":"2003","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1073\/pnas.1703601115","article-title":"Generation of a whole-brain atlas for the cholinergic system and mesoscopic projectome analysis of basal forebrain cholinergic neurons","volume":"115","author":"Li","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1016\/0022-510X(82)90045-4","article-title":"Alzheimer\u2019s disease. Correlation of cortical choline acetyltransferase activity with the severity of dementia and histological abnormalities","volume":"57","author":"Wilcock","year":"1982","journal-title":"J. Neurol. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1136\/jnnp.53.2.161","article-title":"A quantitative study of the neurofibrillary tangles and the choline acetyltransferase activity in the cerebral cortex and the amygdala in Alzheimer\u2019s disease","volume":"53","author":"Esiri","year":"1990","journal-title":"J. Neurol. Neurosurg. Psychiatry"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1745","DOI":"10.1001\/archneur.60.12.1745","article-title":"Cortical cholinergic function is more severely affected in parkinsonian dementia than in Alzheimer disease: An in vivo positron emission tomographic study","volume":"60","author":"Bohnen","year":"2003","journal-title":"Arch Neurol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1097\/NEN.0b013e31827c5710","article-title":"Synaptic proteins and choline acetyltransferase loss in visual cortex in dementia with Lewy bodies","volume":"72","author":"Andras","year":"2013","journal-title":"J. Neuropathol. Exp. Neurol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Yates, C.M., Simpson, J., Maloney, A.F., Gordon, A., and Reid, A.H. (1980). Alzheimer-like cholinergic deficiency in Down syndrome. Lancet, 2.","DOI":"10.1016\/S0140-6736(80)92137-6"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1469","DOI":"10.1007\/s00702-022-02547-9","article-title":"Differential cholinergic systems\u2019 changes in progressive supranuclear palsy versus Parkinson\u2019s disease: An exploratory analysis","volume":"129","author":"Kanel","year":"2022","journal-title":"J. Neural. Transm."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1093\/brain\/awh391","article-title":"Cholinergic systems in progressive supranuclear palsy","volume":"128","author":"Warren","year":"2005","journal-title":"Brain"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1002\/1531-8249(199907)46:1<62::AID-ANA10>3.0.CO;2-P","article-title":"Positron emission tomographic measurement of acetylcholinesterase activity reveals differential loss of ascending cholinergic systems in Parkinson\u2019s disease and progressive supranuclear palsy","volume":"46","author":"Shinotoh","year":"1999","journal-title":"Ann. Neurol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1002\/ana.410180503","article-title":"Dopaminergic and cholinergic lesions in progressive supranuclear palsy","volume":"18","author":"Ruberg","year":"1985","journal-title":"Ann. Neurol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1111\/j.1440-1827.1995.tb03418.x","article-title":"Immunohistochemical study on choline acetyltransferase in the spinal cord of patients with amyotrophic lateral sclerosis","volume":"45","author":"Oda","year":"1995","journal-title":"Pathol. Int."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1016\/0006-8993(82)90439-5","article-title":"Choline acetyltransferase and substance P-like immuno-reactivity in the human spinal cord: Changes in amyotrophic lateral sclerosis","volume":"250","author":"Gillberg","year":"1982","journal-title":"Brain Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/j.proghi.2009.10.001","article-title":"Cholinergic neurotransmission and muscarinic receptors in the enteric nervous system","volume":"44","author":"Harrington","year":"2010","journal-title":"Prog. Histochem. Cytochem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1038\/nrgastro.2016.107","article-title":"The bowel and beyond: The enteric nervous system in neurological disorders","volume":"13","author":"Rao","year":"2016","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2055","DOI":"10.1016\/S0024-3205(03)00083-3","article-title":"The non-neuronal cholinergic system in humans: Expression, function and pathophysiology","volume":"72","author":"Wessler","year":"2003","journal-title":"Life Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2159","DOI":"10.1016\/S0024-3205(03)00078-X","article-title":"Synthesis of acetylcholine by lung cancer","volume":"72","author":"Song","year":"2003","journal-title":"Life Sci."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Chernyavsky, A.I., Shchepotin, I.B., Galitovkiy, V., and Grando, S.A. (2015). Mechanisms of tumor-promoting activities of nicotine in lung cancer: Synergistic effects of cell membrane and mitochondrial nicotinic acetylcholine receptors. BMC Cancer, 15.","DOI":"10.1186\/s12885-015-1158-4"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"G591","DOI":"10.1152\/ajpgi.00055.2008","article-title":"Acetylcholine release by human colon cancer cells mediates autocrine stimulation of cell proliferation","volume":"295","author":"Cheng","year":"2008","journal-title":"Am. J. physiology. Gastrointest. Liver Physiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1016\/j.pharmthera.2018.10.002","article-title":"Acetylcholine signaling system in progression of lung cancers","volume":"194","author":"Friedman","year":"2019","journal-title":"Pharmacol. Ther."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2910","DOI":"10.1111\/bph.13570","article-title":"Protection from interferon-beta-induced neuronal apoptosis through stimulation of muscarinic acetylcholine receptors coupled to ERK1\/2 activation","volume":"173","author":"Olianas","year":"2016","journal-title":"Br. J. Pharmacol."},{"key":"ref_25","first-page":"214","article-title":"Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma","volume":"63","author":"Song","year":"2003","journal-title":"Cancer Res."},{"key":"ref_26","first-page":"1375","article-title":"alpha7 nicotinic acetylcholine receptors in lung cancer","volume":"16","author":"Wang","year":"2018","journal-title":"Oncol. Lett."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1016\/0006-8993(80)91074-4","article-title":"A comparison of solubilized and membrane bound forms of choline-O-acetyltransferase (EC 2.3.1.6) in mouse brain nerve endings","volume":"185","author":"Smith","year":"1980","journal-title":"Brain Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1016\/0006-8993(94)91529-6","article-title":"Membrane-bound choline-O-acetyltransferase in rat hippocampal tissue is associated with synaptic vesicles","volume":"633","author":"Carroll","year":"1994","journal-title":"Brain Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1644","DOI":"10.1046\/j.1460-9568.1998.00177.x","article-title":"Study of subcellular localization of membrane-bound choline acetyltransferase in Drosophila central nervous system and its association with membranes","volume":"10","author":"Pahud","year":"1998","journal-title":"Eur. J. Neurosci."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Dante, D., Jangra, J., Baidya, A.T.K., Kumar, R., and Darreh-Shori, T. (2024). Micellar Choline-Acetyltransferase Complexes Exhibit Ultra-Boosted Catalytic Rate for Acetylcholine Synthesis-Mechanistic Insights for Development of Acetylcholine-Enhancing Micellar Nanotherapeutics. Int. J. Mol. Sci., 25.","DOI":"10.20944\/preprints202411.1836.v1"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"19417","DOI":"10.1074\/jbc.274.27.19417","article-title":"Nuclear localization of the 82-kDa form of human choline acetyltransferase","volume":"274","author":"Resendes","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"20217","DOI":"10.1074\/jbc.M213153200","article-title":"Identification of a novel nuclear localization signal common to 69- and 82-kDa human choline acetyltransferase","volume":"278","author":"Gill","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1016\/j.trecan.2024.07.008","article-title":"Small cell lung cancer profiling: An updated synthesis of subtypes, vulnerabilities, and plasticity","volume":"10","author":"Redin","year":"2024","journal-title":"Trends Cancer"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Kumar, A., Lana, E., Kumar, R., Lithner, C.U., and Darreh-Shori, T. (2018). Soluble Abeta42 Acts as Allosteric Activator of the Core Cholinergic Enzyme Choline Acetyltransferase. Front. Mol. Neurosci., 11.","DOI":"10.3389\/fnmol.2018.00327"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1031","DOI":"10.1002\/alz.12113","article-title":"Proton pump inhibitors act with unprecedented potencies as inhibitors of the acetylcholine biosynthesizing enzyme-A plausible missing link for their association with incidence of dementia","volume":"16","author":"Kumar","year":"2020","journal-title":"Alzheimers Dement"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/j.phrs.2013.07.011","article-title":"Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: A target molecule for lung cancer therapy","volume":"76","author":"Inazu","year":"2013","journal-title":"Pharmacol. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3285","DOI":"10.3892\/mmr.2015.3200","article-title":"Use of (1)(1)C-Choline positron emission tomography\/computed tomography to investigate the mechanism of choline metabolism in lung cancer","volume":"11","author":"Huang","year":"2015","journal-title":"Mol. Med. Rep."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.pbiomolbio.2020.12.001","article-title":"The multiple biological roles of the cholinesterases","volume":"162","author":"Silman","year":"2021","journal-title":"Prog. Biophys Mol. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"672","DOI":"10.1046\/j.1432-1327.1999.00693.x","article-title":"Structural roles of acetylcholinesterase variants in biology and pathology","volume":"264","author":"Grisaru","year":"1999","journal-title":"Eur. J. Biochem."},{"key":"ref_40","first-page":"106","article-title":"A flow-cytometric approach to quantitative estimation of platelet surface immunoglobulin G","volume":"64","author":"Christopoulos","year":"1993","journal-title":"Vox Sang"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3936","DOI":"10.1158\/0008-5472.CAN-06-2484","article-title":"M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion","volume":"67","author":"Song","year":"2007","journal-title":"Cancer Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"7355","DOI":"10.1007\/s12035-019-1605-3","article-title":"Cholinergic Differentiation of Human Neuroblastoma SH-SY5Y Cell Line and Its Potential Use as an In vitro Model for Alzheimer\u2019s Disease Studies","volume":"56","author":"Rico","year":"2019","journal-title":"Mol. Neurobiol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Chen, V.P., Luk, W.K., Chan, W.K., Leung, K.W., Guo, A.J., Chan, G.K., Xu, S.L., Choi, R.C., and Tsim, K.W. (2011). Molecular Assembly and Biosynthesis of Acetylcholinesterase in Brain and Muscle: The Roles of t-peptide, FHB Domain, and N-linked Glycosylation. Front. Mol. Neurosci., 4.","DOI":"10.3389\/fnmol.2011.00036"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/S0306-4522(01)00355-4","article-title":"Identification of hybrid cholinesterase forms consisting of acetyl- and butyrylcholinesterase subunits in human glioma","volume":"107","author":"Vidal","year":"2001","journal-title":"Neuroscience"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1002\/1097-0142(19880215)61:4<727::AID-CNCR2820610416>3.0.CO;2-L","article-title":"Modified properties of serum cholinesterases in primary carcinomas","volume":"61","author":"Zakut","year":"1988","journal-title":"Cancer"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"4676","DOI":"10.1158\/1078-0432.CCR-14-3163","article-title":"Autocrine Activation of CHRM3 Promotes Prostate Cancer Growth and Castration Resistance via CaM\/CaMKK-Mediated Phosphorylation of Akt","volume":"21","author":"Wang","year":"2015","journal-title":"Clin. Cancer Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2105","DOI":"10.1007\/s13277-015-4011-0","article-title":"Muscarinic receptor M3 mediates cell proliferation induced by acetylcholine and contributes to apoptosis in gastric cancer","volume":"37","author":"Wang","year":"2016","journal-title":"Tumor Biol. J. Int. Soc. Oncodev. Biol. Med."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"4601","DOI":"10.1007\/s12035-018-1383-3","article-title":"Increased Active OMI\/HTRA2 Serine Protease Displays a Positive Correlation with Cholinergic Alterations in the Alzheimer\u2019s Disease Brain","volume":"56","author":"Rezaeianyazdi","year":"2019","journal-title":"Mol. Neurobiol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1080\/0284186X.2019.1711165","article-title":"Treatment patterns and survival outcomes for small-cell lung cancer patients\u2013A Swedish single center cohort study","volume":"59","author":"Tendler","year":"2020","journal-title":"Acta Oncol."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Vernay, A., and Cosson, P. (2013). Immunofluorescence labeling of cell surface antigens in Dictyostelium. BMC Res. Notes, 6.","DOI":"10.1186\/1756-0500-6-317"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/21\/10311\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,25]],"date-time":"2025-10-25T04:39:32Z","timestamp":1761367172000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/21\/10311"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,23]]},"references-count":50,"journal-issue":{"issue":"21","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["ijms262110311"],"URL":"https:\/\/doi.org\/10.3390\/ijms262110311","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2025,10,23]]}}}