{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T04:54:55Z","timestamp":1768452895293,"version":"3.49.0"},"reference-count":44,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2024,10,16]],"date-time":"2024-10-16T00:00:00Z","timestamp":1729036800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002241","name":"Japan Science and Technology Agency","doi-asserted-by":"publisher","award":["JPMJPF2213"],"award-info":[{"award-number":["JPMJPF2213"]}],"id":[{"id":"10.13039\/501100002241","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002241","name":"Japan Science and Technology Agency","doi-asserted-by":"publisher","award":["JP24K08627"],"award-info":[{"award-number":["JP24K08627"]}],"id":[{"id":"10.13039\/501100002241","id-type":"DOI","asserted-by":"publisher"}]},{"name":"JSPS KAKENHI","award":["JPMJPF2213"],"award-info":[{"award-number":["JPMJPF2213"]}]},{"name":"JSPS KAKENHI","award":["JP24K08627"],"award-info":[{"award-number":["JP24K08627"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Serum amyloid A (SAA) is one of the most important precursor amyloid proteins discovered during the study of amyloidosis, but its underlying aggregation mechanism has not yet been well elucidated. Since SAA aggregation is a key step in the pathogenesis of AA amyloidosis, amyloid inhibitors can be used as a tool to study its pathogenesis. Previously, we reported a novel microliter-scale high-throughput screening (MSHTS) system for screening amyloid \u03b2 (A\u03b2) aggregation inhibitors based on quantum dot (QD) fluorescence imaging technology. In this study, we report the aggregation of human SAA (hSAA) in phosphate-buffered saline, in which we successfully visualized hSAA aggregation by QD using fluorescence microscopy and confocal microscopy. Two-dimensional and three-dimensional image analyses showed that most aggregations were observed at 40 \u03bcM hSAA, which was the optimal aggregation concentration in vitro. The accuracy of this finding was verified by a Thioflavin T assay. The transmission electron microscopy results showed that QD uniformly bound to hSAA aggregation. hSAA aggregation inhibitory activity was also evaluated by rosmarinic acid (RA). The results showed that RA, which is a compound with high inhibitory activity against A\u03b2 aggregation, also exhibited high inhibitory activity against 40 \u03bcM hSAA. These results indicate that the MSHTS system is an effective tool for visualizing hSAA aggregation and for screening highly active inhibitors.<\/jats:p>","DOI":"10.3390\/ijms252011128","type":"journal-article","created":{"date-parts":[[2024,10,16]],"date-time":"2024-10-16T11:28:51Z","timestamp":1729078131000},"page":"11128","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Real-Time 3D Imaging and Inhibition Analysis of Human Serum Amyloid A Aggregations Using Quantum Dots"],"prefix":"10.3390","volume":"25","author":[{"given":"Liangquan","family":"Shi","sequence":"first","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gegentuya","family":"Huanood","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuto","family":"Miura","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4958-1286","authenticated-orcid":false,"given":"Masahiro","family":"Kuragano","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7747-4808","authenticated-orcid":false,"given":"Kiyotaka","family":"Tokuraku","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,10,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1259","DOI":"10.1007\/s00709-020-01513-0","article-title":"Renal amyloidosis: An update on diagnosis and pathogenesis","volume":"257","author":"Gupta","year":"2020","journal-title":"Protoplasma"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"57","DOI":"10.2174\/1568010053622786","article-title":"Amyloidosis and auto-inflammatory syndromes","volume":"4","author":"Grateau","year":"2005","journal-title":"Curr. Drug Targets Inflamm. Allergy"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1146\/annurev-pathol-052016-100200","article-title":"Amyloidosis: Insights from proteomics","volume":"12","author":"Dogan","year":"2017","journal-title":"Annu. Rev. Pathol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1080\/15216540701283882","article-title":"Soluble protein oligomers as emerging toxins in Alzheimer\u2019s and other amyloid diseases","volume":"59","author":"Ferreira","year":"2007","journal-title":"IUBMB Life"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Lin, X., Galaqin, N., Tainaka, R., Shimamori, K., Kuragano, M., Noguchi, T.Q.P., and Tokuraku, K. (2020). Real-time 3D imaging and inhibition analysis of various amyloid aggregations using quantum dots. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21061978"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"221","DOI":"10.3109\/13506129.2014.964858","article-title":"Nomenclature 2014: Amyloid fibril proteins and clinical classification of the amyloidosis","volume":"21","author":"Sipe","year":"2014","journal-title":"Amyloid"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2641","DOI":"10.1016\/S0140-6736(15)01274-X","article-title":"Systemic amyloidosis","volume":"387","author":"Wechalekar","year":"2016","journal-title":"Lancet"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Fedotov, S.A., Khrabrova, M.S., Anpilova, A.O., Dobronravov, V.A., and Rubel, A.A. (2022). Noninvasive diagnostics of renal amyloidosis: Current state and perspectives. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms232012662"},{"key":"ref_9","first-page":"w13580","article-title":"AA amyloidosis: Basic knowledge, unmet needs and future treatments","volume":"142","author":"Obici","year":"2012","journal-title":"Swiss. Med. Wkly."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1016\/j.rdc.2018.06.004","article-title":"Secondary AA amyloidosis","volume":"44","author":"Papa","year":"2018","journal-title":"Rheum. Dis. Clin. N. Am."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1013","DOI":"10.1038\/s41467-021-21129-z","article-title":"AA amyloid fibrils from diseased tissue are structurally different from in vitro formed SAA fibrils","volume":"12","author":"Bansal","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.gene.2016.02.044","article-title":"Serum amyloid A1: Structure, function and gene polymorphism","volume":"583","author":"Sun","year":"2016","journal-title":"Gene"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1007\/978-3-030-41769-7_17","article-title":"Serum amyloid A (SAA) proteins","volume":"94","author":"Sack","year":"2020","journal-title":"Subcell Biochem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1007\/978-1-4419-6612-4_15","article-title":"Pathogenic role of IL-6 combined with TNF-\u03b1 or IL-1 in the induction of acute phase proteins SAA and CRP in chronic inflammatory diseases","volume":"691","author":"Yoshizaki","year":"2011","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1080\/14397595.2018.1515145","article-title":"AA amyloidosis\u2014Benefits and prospects of IL-6 inhibitors","volume":"29","author":"Okuda","year":"2019","journal-title":"Mod. Rheumatol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Wakai, M., Hayashi, R., Ueno, Y., Onishi, K., Takasago, T., Uchida, T., Takigawa, H., Yuge, R., Urabe, Y., and Oka, S. (2022). Promoting mechanism of serum amyloid a family expression in mouse intestinal epithelial cells. PLoS ONE, 17.","DOI":"10.1371\/journal.pone.0264836"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"764","DOI":"10.1292\/jvms.19-0473","article-title":"Differential expression of serum amyloid A1 and A3 in bovine epithelia","volume":"82","author":"Murata","year":"2020","journal-title":"J. Vet. Med. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Den Hartigh, L.J., May, K.S., Zhang, X.S., Chait, A., and Blaser, M.J. (2023). Serum amyloid A and metabolic disease: Evidence for a critical role in chronic inflammatory conditions. Front. Cardiovasc. Med., 10.","DOI":"10.3389\/fcvm.2023.1197432"},{"key":"ref_19","first-page":"369","article-title":"Systemic AA amyloidosis: Epidemiology, diagnosis, and management","volume":"6","author":"Costa","year":"2014","journal-title":"Clin. Epidemiol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Tanaka, M., Takarada, T., Nadanaka, S., Kojima, R., Hosoi, K., Machiba, Y., Kitagawa, H., and Yamada, T. (2023). Influences of amino-terminal modifications on amyloid fibril formation of human serum amyloid A. Arch. Biochem. Biophys., 742.","DOI":"10.1016\/j.abb.2023.109615"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1186\/s13023-017-0642-0","article-title":"Tocilizumab in the treatment of twelve cases with aa amyloidosis secondary to familial Mediterranean fever","volume":"12","author":"Ugurlu","year":"2017","journal-title":"Orphanet. J. Rare Dis."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Ishigaki, Y., Tanaka, H., Akama, H., Ogara, T., Uwai, K., and Tokuraku, K. (2013). A microliter-scale high-throughput screening system with quantum-dot nanoprobes for amyloid-\u03b2 aggregation inhibitors. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0072992"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Tokuraku, K., Marquardt, M., and Ikezu, T. (2009). Real-time imaging and quantification of amyloid-beta peptide aggregates by novel quantum-dot nanoprobes. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0008492"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Sasaki, R., Tainaka, R., Ando, Y., Hashi, Y., Deepak, H.V., Suga, Y., Murai, Y., Anetai, M., Monde, K., and Ohta, K. (2019). An automated microliter-scale high-throughput screening system (MSHTS) for real-time monitoring of protein aggregation using quantum-dot nanoprobes. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-38958-0"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Kuragano, M., Yoshinari, W., Lin, X., Shimamori, K., Uwai, K., and Tokuraku, K. (2020). Evaluation of amyloid \u03b242 aggregation inhibitory activity of commercial dressings by a microliter-scale high-throughput screening system using quantum-dot nanoprobes. Foods, 9.","DOI":"10.3390\/foods9060825"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"106421","DOI":"10.1016\/j.phrs.2022.106421","article-title":"Anxiolytic and antidepressive potentials of rosmarinic acid: A review with a focus on antioxidant and anti-inflammatory effects","volume":"184","author":"Dahchour","year":"2022","journal-title":"Pharmacol. Res."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Lin, X., Watanabe, K., Kuragano, M., Kurotaki, Y., Nakanishi, U., and Tokuraku, K. (2020). Dietary intake of rosmarinic acid increases serum inhibitory activity in amyloid A aggregation and suppresses deposition in the organs of mice. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21176031"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"16115","DOI":"10.1073\/pnas.1306621110","article-title":"Pathogenetic mechanisms of amyloid A amyloidosis","volume":"110","author":"Simons","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1016\/bs.pmbts.2024.03.008","article-title":"Therapeutic approaches in proteinopathies","volume":"206","author":"Afjadi","year":"2024","journal-title":"Prog. Mol. Biol. Transl. Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1111\/joim.12506","article-title":"Current and future treatment of amyloid diseases","volume":"280","author":"Ankarcrona","year":"2016","journal-title":"J. Intern. Med."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"32301","DOI":"10.1074\/jbc.274.45.32301","article-title":"Uptake, degradation, and release of fibrillar and soluble forms of Alzheimer\u2019s amyloid beta-peptide by microglial cells","volume":"274","author":"Chung","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3877","DOI":"10.4049\/jimmunol.181.6.3877","article-title":"Cytokine-mediated inhibition of fibrillar amyloid-beta peptide degradation by human mononuclear phagocytes","volume":"181","author":"Yamamoto","year":"2008","journal-title":"J. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"800","DOI":"10.1021\/nl0603350","article-title":"Cellular effect of high doses of silica-coated quantum dot profiled with high throughput gene expression analysis and high content cellomics measurements","volume":"6","author":"Zhang","year":"2006","journal-title":"Nano Lett."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"5765","DOI":"10.1039\/D1TB00729G","article-title":"Quantum dots in cell imaging and their safety issues","volume":"9","author":"Xu","year":"2021","journal-title":"J. Mater. Chem. B"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1177\/15353702211057513","article-title":"Carbon \u201cquantum\u201d dots for bioapplications","volume":"247","author":"Yuan","year":"2022","journal-title":"Exp. Biol. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1066","DOI":"10.1016\/j.ejmech.2017.07.026","article-title":"Structure\u2013activity relations of rosmarinic acid derivatives for the amyloid \u03b2 aggregation inhibition and antioxidant properties","volume":"138","author":"Taguchi","year":"2017","journal-title":"Eur. J. Med. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"9948","DOI":"10.1039\/D1CP00193K","article-title":"Effect of counter-anions on the aggregation of Thioflavin-T","volume":"23","author":"Desai","year":"2021","journal-title":"Phys. Chem. Chem. Phys."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Malisauskas, R., Botyriute, A., Cannon, J.G., and Smirnovas, V. (2015). Flavone derivatives as inhibitors of insulin amyloid-like fibril formation. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0121231"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1111\/bcpt.14055","article-title":"Drugs targeting APOE4 that regulate beta-amyloid aggregation in the brain: Therapeutic potential for Alzheimer\u2019s disease","volume":"135","author":"Poblano","year":"2024","journal-title":"Basic Clin. Pharmacol. Toxicol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1007\/s00726-015-2167-y","article-title":"Designing peptidic inhibitors of serum amyloid A aggregation process","volume":"48","author":"Sosnowska","year":"2016","journal-title":"Amino Acids"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1613","DOI":"10.1021\/acsmedchemlett.1c00456","article-title":"Small peptides for inhibiting serum amyloid A aggregation","volume":"12","author":"Jana","year":"2021","journal-title":"ACS Med. Chem. Lett."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"742","DOI":"10.1002\/jnr.20025","article-title":"Curcumin has potent anti-amyloidogenic effects for Alzheimer\u2019s beta-amyloid fibrils in vitro","volume":"75","author":"Ono","year":"2004","journal-title":"J. Neurosci. Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"23212","DOI":"10.1074\/jbc.M113.464222","article-title":"Site-specific inhibitory mechanism for amyloid \u03b242 aggregation by catechol-type flavonoids targeting the Lys residues","volume":"288","author":"Sato","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1346","DOI":"10.1126\/science.1063522","article-title":"Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct","volume":"294","author":"Conway","year":"2001","journal-title":"Science"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/20\/11128\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:14:42Z","timestamp":1760112882000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/20\/11128"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,10,16]]},"references-count":44,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2024,10]]}},"alternative-id":["ijms252011128"],"URL":"https:\/\/doi.org\/10.3390\/ijms252011128","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,10,16]]}}}