RESUMO
Objective: aggregation of the TAU proteins in the form of neurofibrillary tangles [NFTs] in the brain is a common risk factor in tauopathies including Alzheimer's disease [AD]. Several strategies have been implemented to target NFTs, among which chaperones, which facilitate the proper folding of proteins, appear to hold great promise in effectively inhibiting TAU polymerization. The aim of this study was to analyze the impact of the chaperone Artemin on TAU aggregation in vitro
Materials and Methods: in this experimental study, recombinant TAU- or Artemin proteins were expressed in E.coli bacteria, and purified using ion-exchange and affinity chromatography. Sodium dodecyl sulfate-poly acrylamide gel electrophoresis [SDS-PAGE] was used to run the extracted proteins and check their purity. Heparin was used as an aggregation inducer. The interaction kinetics of TAU aggregation and disassembly was performed using thioflavin T [ThT] fluorescence analysis and circular dichroism [CD] spectroscopy
Results: ion-exchange and affinity chromatography yielded highly pure TAU and Artemin proteins for subsequent analyses. In addition, we found that heparin efficiently induced TAU fibrillization 48 hours post-incubation, as evidenced by ThT assay. Importantly, Artemin was observed to effectively block the aggregation of both physiologic- and supraphysiologic TAU concentrations in a dose-dependent manner, as judged by ThT and CD spectroscopy analyses
Conclusion: our collective results show, for the first time, that the chaperone Artemin could significantly inhibit aggregation of the TAU proteins in a dose-dependent manner, and support Artemin as a potential potent blocker of TAU aggregation in people with AD
RESUMO
Objective: Embryonic stem cells [ESCs] are regulated by a gene regulatory circuitry composed of transcription factors, signaling pathways, metabolic mediators, and non-coding RNAs [ncRNAs]. MicroRNAs [miRNAs] are short ncRNAs which play crucial roles in ESCs. Here, we explored the impact of miR-302b-3p on ESC self-renewal in the absence of leukemia inhibitory factor [LIF]
Materials and Methods: In this experimental study, ESCs were cultured in the presence of 15% fetal bovine serum [FBS] and induced to differentiate by LIF removal. miR-302b-3p overexpression was performed by transient transfection of mature miRNA mimics. Cell cycle profiling was done using propidium iodide [PI] staining followed by flow cytometry. miRNA expression was quantified using a miR-302b-3p-specific TaqMan assay. Data were analyzed using t test, and a P<0.05 was considered statistically significant
Results: We observed that miR-302b-3p promoted the viability of both wild-type and LIF-withdrawn ESCs. It also increased ESC clonogenicity and alkaline phosphatase [AP] activity. The defective cell cycling of LIF-deprived ESCs was completely rescued by miR-302b-3p delivery. Moreover, miR-302b-3p inhibited the increased cell death rate induced by LIF removal
Conclusion: miR-302b-3p, as a pluripotency-associated miRNA, promotes diverse features of ESC self-renewal in the absence of extrinsic LIF signals