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Article: Integration of miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism

TitleIntegration of miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism
Authors
Issue Date2022
PublisherNature Research: Fully open access journals. The Journal's web site is located at http://www.nature.com/srep/index.html
Citation
Scientific Reports, 2022, v. 12, article no. 1083 How to Cite?
AbstractDefective biosynthesis or function of proteoglycans causes pathological conditions in a variety of tissue systems. Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by progressive cartilage destruction caused by imbalanced proteoglycan synthesis and degradation. Identifying agents that regulate proteoglycan metabolism may benefit the development of OA-modifying therapeutics. High-throughput screening (HTS) of chemical libraries has paved the way for achieving this goal. However, the implementation and adaptation of HTS assays based on proteoglycan measurement remain underexploited. Using primary porcine chondrocytes as a model, we report a miniaturized dimethyl-methylene blue (DMMB) assay, which is commonly used to quantitatively evaluate sulfated glycosaminoglycan (GAG) content, with an optimized detection range and reproducibility and its integration with HTS. Treatment with TGF-β1 and IL1-α, known as positive and negative proteoglycan regulators, respectively, supported the assay specificity. A pre-test of chemical screening of 960 compounds identified both stimulators (4.48%) and inhibitors (6.04%) of GAG production. Fluorophore-assisted carbohydrate electrophoresis validated the activity of selected hits on chondroitin sulfate expression in an alginate culture system. Our findings support the implementation of this simple colorimetric assay in HTS to discover modifiers of OA or other diseases related to dysregulated proteoglycan metabolism.
Persistent Identifierhttp://hdl.handle.net/10722/310894
ISSN
2021 Impact Factor: 4.996
2020 SCImago Journal Rankings: 1.240
PubMed Central ID

 

DC FieldValueLanguage
dc.contributor.authorSun, Y-
dc.contributor.authorTsui, YK-
dc.contributor.authorYU, M-
dc.contributor.authorLyu, M-
dc.contributor.authorCheung, KCM-
dc.contributor.authorKao, RYT-
dc.contributor.authorLeung, VYL-
dc.date.accessioned2022-02-25T04:56:36Z-
dc.date.available2022-02-25T04:56:36Z-
dc.date.issued2022-
dc.identifier.citationScientific Reports, 2022, v. 12, article no. 1083-
dc.identifier.issn2045-2322-
dc.identifier.urihttp://hdl.handle.net/10722/310894-
dc.description.abstractDefective biosynthesis or function of proteoglycans causes pathological conditions in a variety of tissue systems. Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by progressive cartilage destruction caused by imbalanced proteoglycan synthesis and degradation. Identifying agents that regulate proteoglycan metabolism may benefit the development of OA-modifying therapeutics. High-throughput screening (HTS) of chemical libraries has paved the way for achieving this goal. However, the implementation and adaptation of HTS assays based on proteoglycan measurement remain underexploited. Using primary porcine chondrocytes as a model, we report a miniaturized dimethyl-methylene blue (DMMB) assay, which is commonly used to quantitatively evaluate sulfated glycosaminoglycan (GAG) content, with an optimized detection range and reproducibility and its integration with HTS. Treatment with TGF-β1 and IL1-α, known as positive and negative proteoglycan regulators, respectively, supported the assay specificity. A pre-test of chemical screening of 960 compounds identified both stimulators (4.48%) and inhibitors (6.04%) of GAG production. Fluorophore-assisted carbohydrate electrophoresis validated the activity of selected hits on chondroitin sulfate expression in an alginate culture system. Our findings support the implementation of this simple colorimetric assay in HTS to discover modifiers of OA or other diseases related to dysregulated proteoglycan metabolism.-
dc.languageeng-
dc.publisherNature Research: Fully open access journals. The Journal's web site is located at http://www.nature.com/srep/index.html-
dc.relation.ispartofScientific Reports-
dc.titleIntegration of miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism-
dc.typeArticle-
dc.identifier.emailSun, Y: hkusunyi@hku.hk-
dc.identifier.emailCheung, KCM: cheungmc@hku.hk-
dc.identifier.emailKao, RYT: rytkao@hkucc.hku.hk-
dc.identifier.emailLeung, VYL: vicleung@hku.hk-
dc.identifier.authorityCheung, KCM=rp00387-
dc.identifier.authorityKao, RYT=rp00481-
dc.identifier.authorityLeung, VYL=rp01764-
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1038/s41598-022-04805-y-
dc.identifier.pmid35058478-
dc.identifier.pmcidPMC8776954-
dc.identifier.hkuros332013-
dc.identifier.volume12-
dc.identifier.spagearticle no. 1083-
dc.identifier.epagearticle no. 1083-
dc.publisher.placeUnited Kingdom-

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