scholarly journals A possible role of gas-phase electrophoretic mobility molecular analysis (nES GEMMA) in extracellular vesicle research

2021 ◽  
Author(s):  
Stephanie Steinberger ◽  
Sobha Karuthedom George ◽  
Lucia Lauková ◽  
René Weiss ◽  
Carla Tripisciano ◽  
...  
Keyword(s):  
Gas Phase ◽  
Electrophoretic Mobility ◽  
Analytical Techniques ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Label Free ◽  
Protein Size ◽  
Depth Analysis ◽  
Core Proteome

AbstractThe emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Graphical abstract

scholarly journals Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation

Biomedicines ◽  
2020 ◽  
Vol 8 (8) ◽  
pp. 246 ◽  
Author(s):  
Anders Askeland ◽  
Anne Borup ◽  
Ole Østergaard ◽  
Jesper V. Olsen ◽  
Sigrid M. Lund ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
High Speed ◽  
Proteome Analysis ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Label Free ◽  
Protein Biomarkers ◽  
Transmission Electron ◽  
Exclusion Chromatography

Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both.

scholarly journals Extracellular Vesicles: A Novel Opportunity for Precision Medicine in Respiratory Diseases

2021 ◽  
Vol 8 ◽  
Author(s):  
Jonathan M. Carnino ◽  
Zhi Hao Kwok ◽  
Yang Jin
Keyword(s):  
Precision Medicine ◽  
Extracellular Vesicles ◽  
Biological Fluids ◽  
Relative Size ◽  
Extracellular Vesicle ◽  
The Body ◽  
Size Exclusion ◽  
Physiological Status ◽  
Target Cells

Extracellular vesicles are membrane-bound nanoparticles secreted by cells which play a well-known role in cell to cell communication. The most update to date nomenclature categorizes extracellular vesicles based on their relative size, protein markers, and/or the cell type of origin. Extracellular vesicles can be isolated from biological fluids using a variety of methods, including but not limited to, ultrafiltration, size-exclusion chromatography, differential ultracentrifugation, density gradient centrifugation, precipitation-based methods, and immunoaffinity capture. These nanovesicles carry distinct “cargo,” made up of biomolecules such as nucleic acids, lipids, and protein, which is delivered to nearby target cells. The “cargo” profile carried by extracellular vesicles is critical in their role of communication and resembles the physiological status of the cell they originated from. For the purpose of this review, we will focus on the miRNA cargo. Extracellular vesicle-miRNA profiles hold the potential to be used in diagnostic panels for a variety of diseases through a novel method known as “liquid biopsy.” In addition to this, extracellular vesicles may serve as a potential method to deliver drugs to specific cells within the body. This mini-review provides background into what extracellular vesicles are, methods of isolating these nanoparticles, their potential use as a biomarker and drug delivery system for precision medicine, and a summary of the current literature covering the role of some extracellular vesicle-cargo's in various pulmonary diseases.

scholarly journals The Role of Surface Chemistry in the Efficacy of Protein and DNA Microarrays for Label-Free Detection: An Overview

Polymers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1026
Author(s):  
Elisa Chiodi ◽  
Allison M. Marn ◽  
Matthew T. Geib ◽  
M. Selim Ünlü
Keyword(s):  
Surface Chemistry ◽  
Surface Functionalization ◽  
Dna Microarrays ◽  
Label Free ◽  
Polymeric Coatings ◽  
Label Free Detection ◽  
Particle Imaging ◽  
Single Particle Imaging ◽  
The Impact

The importance of microarrays in diagnostics and medicine has drastically increased in the last few years. Nevertheless, the efficiency of a microarray-based assay intrinsically depends on the density and functionality of the biorecognition elements immobilized onto each sensor spot. Recently, researchers have put effort into developing new functionalization strategies and technologies which provide efficient immobilization and stability of any sort of molecule. Here, we present an overview of the most widely used methods of surface functionalization of microarray substrates, as well as the most recent advances in the field, and compare their performance in terms of optimal immobilization of the bioreceptor molecules. We focus on label-free microarrays and, in particular, we aim to describe the impact of surface chemistry on two types of microarray-based sensors: microarrays for single particle imaging and for label-free measurements of binding kinetics. Both protein and DNA microarrays are taken into consideration, and the effect of different polymeric coatings on the molecules’ functionalities is critically analyzed.

scholarly journals O-GlcNAcylation protein disruption by Thiamet G promotes changes on the GBM U87-MG cells secretome molecular signature

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Maria Cecilia Oliveira-Nunes ◽  
Glaucia Julião ◽  
Aline Menezes ◽  
Fernanda Mariath ◽  
John A. Hanover ◽  
...  
Keyword(s):  
Experimental Evidence ◽  
Critical Role ◽  
Molecular Signature ◽  
Label Free ◽  
Intercellular Signaling ◽  
Tumor Aggressiveness ◽  
Signaling Axis ◽  
Literature Evidence ◽  
Therapeutic Tools

AbstractGlioblastoma (GBM) is a grade IV glioma highly aggressive and refractory to the therapeutic approaches currently in use. O-GlcNAcylation plays a key role for tumor aggressiveness and progression in different types of cancer; however, experimental evidence of its involvement in GBM are still lacking. Here, we show that O-GlcNAcylation plays a critical role in maintaining the composition of the GBM secretome, whereas inhibition of OGA activity disrupts the intercellular signaling via microvesicles. Using a label-free quantitative proteomics methodology, we identified 51 proteins in the GBM secretome whose abundance was significantly altered by activity inhibition of O-GlcNAcase (iOGA). Among these proteins, we observed that proteins related to proteasome activity and to regulation of immune response in the tumor microenvironment were consistently downregulated in GBM cells upon iOGA. While the proteins IGFBP3, IL-6 and HSPA5 were downregulated in GBM iOGA cells, the protein SQSTM1/p62 was exclusively found in GBM cells under iOGA. These findings were in line with literature evidence on the role of p62/IL-6 signaling axis in suppressing tumor aggressiveness and our experimental evidence showing a decrease in radioresistance potential of these cells. Taken together, our findings provide evidence that OGA activity may regulate the p62 and IL-6 abundance in the GBM secretome. We propose that the assessment of tumor status from the main proteins present in its secretome may contribute to the advancement of diagnostic, prognostic and even therapeutic tools to approach this relevant malignancy.

scholarly journals The Role of High-Resolution Analytical Techniques in the Development of Functional Foods

2021 ◽  
Vol 22 (6) ◽  
pp. 3220
Author(s):  
Álvaro Fernández-Ochoa ◽  
Francisco Javier Leyva-Jiménez ◽  
María De la Luz Cádiz-Gurrea ◽  
Sandra Pimentel-Moral ◽  
Antonio Segura-Carretero
Keyword(s):  
High Resolution ◽  
Functional Foods ◽  
Intervention Studies ◽  
Analytical Techniques ◽  
Nutritional Intervention ◽  
Bioactive Properties ◽  
Metabolic Reactions ◽  
Bioactive Potential ◽  
Endogenous Metabolites

The approaches based on high-resolution analytical techniques, such as nuclear magnetic resonance or mass spectrometry coupled to chromatographic techniques, have a determining role in several of the stages necessary for the development of functional foods. The analyses of botanical extracts rich in bioactive compounds is one of the fundamental steps in order to identify and quantify their phytochemical composition. However, the compounds characterized in the extracts are not always responsible for the bioactive properties because they generally undergo metabolic reactions before reaching the therapeutic targets. For this reason, analytical techniques are also applied to analyze biological samples to know the bioavailability, pharmacokinetics and/or metabolism of the compounds ingested by animal or human models in nutritional intervention studies. In addition, these studies have also been applied to determine changes of endogenous metabolites caused by prolonged intake of compounds with bioactive potential. This review aims to describe the main types and modes of application of high-resolution analytical techniques in all these steps for functional food development.

scholarly journals Space Radiation-Induced Alterations in the Hippocampal Ubiquitin-Proteome System

2021 ◽  
Vol 22 (14) ◽  
pp. 7713
Author(s):  
Alyssa Tidmore ◽  
Sucharita M. Dutta ◽  
Arriyam S. Fesshaye ◽  
William K. Russell ◽  
Vania D. Duncan ◽  
...  
Keyword(s):  
Spatial Memory ◽  
Memory Performance ◽  
Space Radiation ◽  
Label Free ◽  
Proteomic Profiling ◽  
Neurocognitive Deficits ◽  
Male Wistar Rats ◽  
Radiation Induced ◽  
Induced Changes

Exposure of rodents to <20 cGy Space Radiation (SR) impairs performance in several hippocampus-dependent cognitive tasks, including spatial memory. However, there is considerable inter-individual susceptibility to develop SR-induced spatial memory impairment. In this study, a robust label-free mass spectrometry (MS)-based unbiased proteomic profiling approach was used to characterize the composition of the hippocampal proteome in adult male Wistar rats exposed to 15 cGy of 1 GeV/n 48Ti and their sham counterparts. Unique protein signatures were identified in the hippocampal proteome of: (1) sham rats, (2) Ti-exposed rats, (3) Ti-exposed rats that had sham-like spatial memory performance, and (4) Ti-exposed rats that impaired spatial memory performance. Approximately 14% (159) of the proteins detected in hippocampal proteome of sham rats were not detected in the Ti-exposed rats. We explored the possibility that the loss of the Sham-only proteins may arise as a result of SR-induced changes in protein homeostasis. SR-exposure was associated with a switch towards increased pro-ubiquitination proteins from that seen in Sham. These data suggest that the role of the ubiquitin-proteome system as a determinant of SR-induced neurocognitive deficits needs to be more thoroughly investigated.

scholarly journals Serum Extracellular Vesicle-Derived circHIPK3 and circSMARCA5 Are Two Novel Diagnostic Biomarkers for Glioblastoma Multiforme

2021 ◽  
Vol 14 (7) ◽  
pp. 618
Author(s):  
Michele Stella ◽  
Luca Falzone ◽  
Angela Caponnetto ◽  
Giuseppe Gattuso ◽  
Cristina Barbagallo ◽  
...  
Keyword(s):  
Glioblastoma Multiforme ◽  
Roc Analysis ◽  
Characteristic Curve ◽  
Area Under The Curve ◽  
Digital Pcr ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Circular Rnas ◽  
Diagnostic Biomarkers ◽  
Exclusion Chromatography

Glioblastoma multiforme (GBM) is the most frequent and deadly human brain cancer. Early diagnosis through non-invasive biomarkers may render GBM more easily treatable, improving the prognosis of this currently incurable disease. We suggest the use of serum extracellular vesicle (sEV)-derived circular RNAs (circRNAs) as highly stable minimally invasive diagnostic biomarkers for GBM diagnosis. EVs were isolated by size exclusion chromatography from sera of 23 GBM and 5 grade 3 glioma (GIII) patients, and 10 unaffected controls (UC). The expression of two candidate circRNAs (circSMARCA5 and circHIPK3) was assayed by droplet digital PCR. CircSMARCA5 and circHIPK3 were significantly less abundant in sEVs from GBM patients with respect to UC (fold-change (FC) of −2.15 and −1.92, respectively) and GIII (FC of −1.75 and −1.4, respectively). Receiver operating characteristic curve (ROC) analysis, based on the expression of sEV-derived circSMARCA5 and circHIPK3, allowed us to distinguish GBM from UC (area under the curve (AUC) 0.823 (0.667–0.979) and 0.855 (0.704 to 1.000), with a 95% confidence interval (CI), respectively). Multivariable ROC analysis, performed by combining the expression of sEV-derived circSMARCA5 and circHIPK3 with preoperative neutrophil to lymphocyte (NLR), platelet to lymphocyte (PLR) and lymphocyte to monocyte (LMR) ratios, three known diagnostic and prognostic GBM markers, allowed an improvement in the GBM diagnostic accuracy (AUC 0.901 (0.7912 to 1.000), 95% CI). Our data suggest sEV-derived circSMARCA5 and circHIPK3 as good diagnostic biomarkers for GBM, especially when associated with preoperative NLR, PLR and LMR.

scholarly journals cROStalk for Life: Uncovering ROS Signaling in Plants and Animal Systems, from Gametogenesis to Early Embryonic Development

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 525
Author(s):  
Valentina Lodde ◽  
Piero Morandini ◽  
Alex Costa ◽  
Irene Murgia ◽  
Ignacio Ezquer
Keyword(s):  
Signal Transduction ◽  
Signaling Pathways ◽  
Analytical Techniques ◽  
Antioxidant Systems ◽  
Developmental Studies ◽  
Science Field ◽  
Ros Signaling ◽  
Reproductive Processes

This review explores the role of reactive oxygen species (ROS)/Ca2+ in communication within reproductive structures in plants and animals. Many concepts have been described during the last years regarding how biosynthesis, generation products, antioxidant systems, and signal transduction involve ROS signaling, as well as its possible link with developmental processes and response to biotic and abiotic stresses. In this review, we first addressed classic key concepts in ROS and Ca2+ signaling in plants, both at the subcellular, cellular, and organ level. In the plant science field, during the last decades, new techniques have facilitated the in vivo monitoring of ROS signaling cascades. We will describe these powerful techniques in plants and compare them to those existing in animals. Development of new analytical techniques will facilitate the understanding of ROS signaling and their signal transduction pathways in plants and mammals. Many among those signaling pathways already have been studied in animals; therefore, a specific effort should be made to integrate this knowledge into plant biology. We here discuss examples of how changes in the ROS and Ca2+ signaling pathways can affect differentiation processes in plants, focusing specifically on reproductive processes where the ROS and Ca2+ signaling pathways influence the gametophyte functioning, sexual reproduction, and embryo formation in plants and animals. The study field regarding the role of ROS and Ca2+ in signal transduction is evolving continuously, which is why we reviewed the recent literature and propose here the potential targets affecting ROS in reproductive processes. We discuss the opportunities to integrate comparative developmental studies and experimental approaches into studies on the role of ROS/ Ca2+ in both plant and animal developmental biology studies, to further elucidate these crucial signaling pathways.

scholarly journals The role of radiolysis in the modelling of C2H4O2 isomers and dimethyl ether in cold dark clouds

2020 ◽  
Vol 500 (3) ◽  
pp. 3414-3424
Author(s):  
Alec Paulive ◽  
Christopher N Shingledecker ◽  
Eric Herbst
Keyword(s):  
Gas Phase ◽  
Dimethyl Ether ◽  
Recent Observation ◽  
Cosmic Ray ◽  
Thermal Method ◽  
Dark Clouds ◽  
Ice Surface ◽  
Dust Grain ◽  
Complex Organic

ABSTRACT Complex organic molecules (COMs) have been detected in a variety of interstellar sources. The abundances of these COMs in warming sources can be explained by syntheses linked to increasing temperatures and densities, allowing quasi-thermal chemical reactions to occur rapidly enough to produce observable amounts of COMs, both in the gas phase, and upon dust grain ice mantles. The COMs produced on grains then become gaseous as the temperature increases sufficiently to allow their thermal desorption. The recent observation of gaseous COMs in cold sources has not been fully explained by these gas-phase and dust grain production routes. Radiolysis chemistry is a possible non-thermal method of producing COMs in cold dark clouds. This new method greatly increases the modelled abundance of selected COMs upon the ice surface and within the ice mantle due to excitation and ionization events from cosmic ray bombardment. We examine the effect of radiolysis on three C2H4O2 isomers – methyl formate (HCOOCH3), glycolaldehyde (HCOCH2OH), and acetic acid (CH3COOH) – and a chemically similar molecule, dimethyl ether (CH3OCH3), in cold dark clouds. We then compare our modelled gaseous abundances with observed abundances in TMC-1, L1689B, and B1-b.

Sign in / Sign up

Export Citation Format

Share Document