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Proteomic profiling of exosomes derived from pancreatic beta-cells cultured under hyperglycemia

Rząca Carina, Jankowska Urszula, Stępień Ewa Ł.

Bio-Algorithms and Med-Systems

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2022

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Vol. 18, no. 1

151--157

EN

Cargo carried by extracellular vesicles (EVs) is considered a promising diagnostic marker, especially proteins. EVs can be divided according to their size and way of biogenesis into exosomes (diameter < 200 nm) and ectosomes (diameter > 200 nm). Exosomes are considered to be of endocytic origin, and ectosomes are produced by budding and shedding from the plasma membrane [1]. Methods The first step of this study was a characterization of the exosome sample. Using Tunable Resistive Pulse Sensing (qNano) size distribution and concentration were measured. The mean size of exosomes was 120±9.17 nm. In the present study, a nano liquid chromatography coupled with tandem mass spectrometry (nanoLCMS/MS) was used to compare protein profiles of exosomes secreted by pancreatic beta cells (1.1B4) grown under normal glucose (NG, 5 mM D-glucose) and high glucose (HG, 25 mM D-glucose) conditions. The EV samples were lysed, and proteins were denatured, digested, and analyzed using a Q-Exactive mass spectrometer coupled with the UltiMate 3000 RSLC nano system. The nanoLC-MS/MS data were searched against the SwissProt Homo sapiens database using MaxQuant software and protein quantitation was done by the MaxLFQ algorithm. Statistical analysis was carried out with Perseus software. Further bioinformatic analysis was performed using the FunRich 3.1.4 software with the UniProt protein database and String [2]. Results As a result of the nanoLC-MS/MS analysis more than 1,000 proteins were identified and quantified in each sample. The average number of identified proteins in exosomes was 1,397. Label-free quantitative analysis showed that exosome composition differed significantly between those isolated under NG and HG conditions. Many pathways were down-regulated in HG, particularly the ubiquitin-proteasome pathway. In addition, a significant up-regulation of the Ras-proteins pathway was observed in HG. Conclusion Our description of exosomes protein content and its related functions provides the first insight into the EV interactome and its role in glucose intolerance development and diabetic complications. The results also indicate the applicability of EV proteins for further investigation regarding their potential as circulating in vivo biomarkers.

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Comparison of qNANO results from the isolation of extracellular microvesicles with the theoretical model

Durak-Kozica Martyna, Wróbel Andrzej, Platt Mark, Stępień Ewa Ł.

Bio-Algorithms and Med-Systems

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2022

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Vol. 18, no. 1

171--179

EN

Objectives: Extracellular vesicles (EVs) are heterogeneous membrane vesicles in diameter of 30-5000 nm, that transport proteins, non-coding RNAs (miRNAs), lipids and metabolites. Major populations include exosomes, ectosomes and apoptotic bodies. The purpose of this study was to compare the distribution of EVs obtained under different conditions of differential centrifugation, including ultracentrifugation, with the results developed based on a theoretical model. Methods: Immortalized endothelial cell line that expresses h-TERT (human telomerase) was used to release of EVs: microvascular TIME. EVs were isolated from the culture medium at different centrifugation parameters. The size distribution of the EVs was measured using TRPS technology on a qNano instrument. Surface markers were evaluated using flow cytometry. The isolated EV subpopulations were compared with the theoretical model developed by Livshits. Results: EVs isolated from endothelial cells show strong aggregating properties, which was confirmed by TEM, TRPS imaging and flow cytometry. Conclusions: Obtaining pure EV subpopulations is difficult because of the small differences in the diameter of ectosomes and exosomes, and the strong aggregating properties of EVs.

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Novel biomarker and drug delivery systems for theranostics - extracellular vesicles

Stępień Ewa Ł., Rząca Carina, Moskal Paweł

Bio-Algorithms and Med-Systems

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2021

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Vol. 17, no. 4

301--309

EN

Extracellular vesicles (EVs) are nano- and micro-sized double-layered membrane entities derived from most cell types and released into biological fluids. Biological properties (cell-uptake, biocompatibility), and chemical (composition, structure) or physical (size, density) characteristics make EVs a good candidate for drug delivery systems (DDS). Recent advances in the field of EVs (e.g., scaling-up production, purification) and developments of new imaging methods (total-body positron emission tomography [PET]) revealed benefits of radiolabeled EVs in diagnostic and interventional medicine as a potential DDs in theranostics.