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1.
J Biosci ; 472022.
Article in English | MEDLINE | ID: mdl-36210730

ABSTRACT

Tuberculosis (TB) is a common disease caused by Mycobacterium tuberculosis (M.tb) infection. Our study was to explore the function and mechanism of circular RNA WD repeat domain 27 (circ-WDR27) in TB progression. Cell viability and apoptosis were detected by 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide assay and flow cytometry. Protein quantification was performed by Western blot. Inflammatory cytokines were examined using enzyme-linked immunosorbent assay. RNA levels were assayed via quantitative reverse-transcription polymerase chain reaction. M.tb survival was assessed using colony-forming unit assay. Target binding was analyzed via dual-luciferase reporter assay and RNA immunoprecipitation assay. Cell damages were induced by M.tb infection, and inflammatory cytokines were secreted in human macrophages. Circ-WDR27 was downregulated in TB patients and M.tb-infected macrophages. Circ-WDR27 overexpression reduced M.tb survival and released inflammatory cytokines in macrophages. Circ-WDR27 acted as a sponge for miR-370-3p. Circ-WDR27-mediated inhibition of TB progression was partly achieved by sponging miR-370-3p. miR-370-3p directly targeted Follistatin-like protein 1 (FSTL1). FSTL1 suppressed M.tb-induced cell damages, and reversed the protective role of miR-370-3p inhibition in TB progression. Circ- WDR27 regulated FSTL1 expression by targeting miR-370-3p. These results showed that circ-WDR27 repressed M.tb vitality and stimulated pro-inflammatory cytokines in M.tb-infected macrophages by affecting the miR-370-3p/FSTL1 axis.


Subject(s)
Follistatin-Related Proteins , MicroRNAs , Mycobacterium tuberculosis , Tuberculosis , Apoptosis/genetics , Bromides/metabolism , Cell Proliferation , Cytokines/genetics , Cytokines/metabolism , Follistatin-Related Proteins/genetics , Follistatin-Related Proteins/metabolism , Humans , Macrophages/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Mycobacterium tuberculosis/metabolism , RNA, Circular/genetics , Tuberculosis/genetics , Tuberculosis/metabolism
2.
Assay Drug Dev Technol ; 1(4): 507-19, 2003 Aug.
Article in English | MEDLINE | ID: mdl-15090247

ABSTRACT

We describe a new approach to affinity selection based on the application of centrifugal force to macromolecules in solution. The method relies on the well known macromolecular hydrodynamic principles of centrifugation. It can be automated and operated in a centralized fashion, or it can be decentralized and used by single researchers or networks of researchers with a minimal additional capital investment. In this method, a centrifugal driving force is used to establish a differential and selective concentration gradient between a therapeutic target and potential ligands in compound libraries. This concentration gradient, in turn, drives the binding of ligands. Once formed, the differential concentration gradient of target macromolecules and ligands is fractionated to capture the self-sorting binding events. Ligand binding is defined by the individual ligand binding constants, so tight binding ligands will essentially distribute identically with the protein target, and weaker binding ligands will not. The level of affinity needed to operationally define tight binding can be adjusted by selecting the initial concentration conditions or centrifugal force. A variety of rapid, commonly available, detection methods can be used to assess binding in the fractionated samples. The method can be broadly applied in drug discovery efforts to examine most types of cell-cell, protein-protein, and protein-small molecule interactions. We describe the application of this method to systems of small molecule interactions with several macromolecules of therapeutic interest.


Subject(s)
Centrifugation/methods , Chemical Fractionation/methods , Automation/economics , Automation/methods , Carrier Proteins/chemistry , Carrier Proteins/isolation & purification , Centrifugation, Density Gradient/methods , Chromatography, High Pressure Liquid , Ligands , Macromolecular Substances , Mass Spectrometry , Protein Binding , Proteins/chemistry , Proteins/isolation & purification , Solubility
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