Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular ...compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy.
Graphite oxides (GOs) synthesized by Brodie’s and Hummers’ methods are significantly different with respect to hydration, solvation and exfoliation properties. Hummers GO is more easily intercalated ...by liquid water and alcohols, exhibiting osmotic type of swelling. In contrast, Brodie GO shows crystalline swelling in alcohol solvents with step-like insertion of methanol or ethanol monolayers. However, the stronger hydration and easier dispersion in water observed for Hummers GO do not correlate with better dispersion of graphene powder obtained by thermal exfoliation. Higher surface area graphene powder was obtained by exfoliation of Brodie GO, while the temperature of its exfoliation is about 75°C higher than that for the studied sample of Hummers GO. It is suggested that higher exfoliation temperature and better crystallinity of GO are important factors for preparation of graphene powder using thermal exfoliation.
Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune ...disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures.
In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals.
EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+) and CD8(+) T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction).
Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.
In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and ...quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.
Charging and aggregation processes were studied in aqueous dispersions of halloysite nanotubes (HNTs) in the presence of monovalent inorganic electrolytes and ionic liquid (IL) constituents. The same ...type of co-ion (same sign of charge as HNT) was used in all systems, while the type of counterions (opposite sign of charge as HNT) was systematically varied. The affinity of the inorganic cations to the HNT surface influenced their destabilizing power leading to an increase in the critical coagulation concentration (CCC) of HNT dispersions in the Cs+ < K+ < Na+ order. This trend agrees with the classical Hofmeister series for negatively charged hydrophobic surfaces. For the IL cations, the CCCs increased in the order BMPY+ < BMPIP+ < BMPYR+ < BMIM+. An unexpectedly strong adsorption of BMPY+ cations on the HNT surface was observed giving rise to charge neutralization and reversal of the oppositely charged outer surface of HNT. The direct Hofmeister series was extended with these IL cations. The main aggregation mechanism was rationalized within the classical theory developed by Derjaguin, Landau, Verwey, and Overbeek, while ion specific effects resulted in remarkable variation in the CCC values. The results unambiguously proved that the hydration level of the surface and the counterions plays a crucial role in the formation of the ionic composition at the solid–liquid interface and consequently, in the colloidal stability of the HNT particles in both inorganic salt and IL solutions.
Homoaggregation of polystyrene microplastics (MPs) and heteroaggregation of MPs with anionic clay minerals, namely, layered double hydroxide (LDH), in different salt (NaCl, CaCl2, and Na2SO4) ...solutions were systematically investigated using light scattering techniques. The salt type and ionic strength had significant effects on the stability of both MPs and LDH particles individually and the results could be explained by DLVO theory and the Schulze–Hardy rule. However, once stable colloidal dispersions of the individual particles were mixed, heteroaggregation occurred between the oppositely charged MPs and LDH, which was also confirmed by transmission electron microscopy and X-ray scattering. Adsorption of the LDH particles resulted in neutralization and reversal of MPs surface charge at appropriate LDH doses. Once LDH adsorption neutralized the negative charges of the MP spheres, rapid aggregation was observed in the dispersions, whereas stable samples formed at high and low LDH concentrations. The governing interparticle interactions included repulsive electrical double-layer forces, as well as van der Waals and patch-charge attractions, the strength of which depended on the mass ratio of the interacting particles and the composition of the aqueous solvent. Our results shed light on the colloidal behavior of MPs in a complex aquatic environment and, in the long term, are also useful for developing LDH-based approaches for water remediation to remove contamination with MP particles.
Blood serum fractions are hotly debated adjuvants in bone replacement therapies. In the present experiment, we coated demineralized bone matrices (DBM) with serum albumin and investigated stem cell ...attachment in vitro and bone formation in a rat calvaria defect model. In the in vitro experiments, we observed that significantly more cells adhere to the serum albumin coated DBMs at every time point. In vivo bone formation with albumin coated and uncoated DBM was monitored biweekly by computed tomography until 11 weeks postoperatively while empty defects served as controls. By the seventh week, the bone defect in the albumin group was almost completely closed (remaining defect 3.0 ± 2.3%), while uncoated DBM and unfilled control groups still had significant defects (uncoated: 40.2 ± 9.1%, control: 52.4 ± 8.9%). Higher density values were also observed in the albumin coated DBM group. In addition, the serum albumin enhanced group showed significantly higher volume of newly formed bone in the microCT analysis and produced significantly higher breaking force and stiffness compared to the uncoated grafts (peak breaking force: uncoated: 15.7 ± 4 N, albumin 46.1 ± 11 N). In conclusion, this investigation shows that implanting serum albumin coated DBM significantly reduces healing period in nonhealing defects and results in mechanically stronger bone. These results also support the idea that serum albumin coating provides a convenient milieu for stem cell function, and a much improved bone grafting success can be achieved without the use of exogenous stem cells.
This study contributes to the sustained effort to unravel the chemical structure of graphite oxide (GO) by proposing a model based on elemental analysis, transmission electron microscopy, X-ray ...diffraction, 13C magic-angle spinning NMR, diffuse reflectance infrared Fourier transform spectroscopy, X-ray photoelectron spectroscopy, and electron spin resonance investigations. The model exhibits a carbon network consisting of two kinds of regions (of trans linked cyclohexane chairs and ribbons of flat hexagons with CC double bonds) and functional groups such as tertiary OH, 1,3-ether, ketone, quinone, and phenol (aromatic diol). The latter species give clear explanation for the observed planar acidity of GO, which could not be interpreted by the previous models. The above methods also confirmed the evolution of the surface functional groups upon successive oxidation steps.
•UF shell microcapsules were prepared with core material: linseed oil, Co-octoate and octadecylamine.•Combination of linseed oil, Co-octoate and octadecylamine was optimized for fast ...drying.•Self-healing was monitored on mild steel plates with complex (primer and topcoat) paint.•Electrochemical impedance spectroscopy measurements were carried out in NaCl solution.•Self-healing efficiency was improved by encapsulated corrosion inhibitor and drier.
Core–shell microcapsules of urea-resorcinol-formaldehyde shell and linseed oil (LO) core material as paint additives for self-healing coatings were prepared. The capsules contained LO either with or without Co-octoate as drier material and/or octadecylamine (ODA) as corrosion inhibitor. The microcapsules embedded in a commercial paint were applied on sandblasted mild steel sheets. After scratching the coated surface, the inhibition efficiency of core–shell microcapsule-containing coat, dipped into corrosive media, was followed visually and evaluated numerically by electrochemical impedance spectroscopy (EIS). In separate experiments, to optimize for the self-healing process, the composition of the core material, the effect of the drier and/or the inhibitor ODA on drying process of LO films were monitored by infrared spectroscopy. Pure LO needed 6–7 days to dry completely. The drying period could be shortened (to 5h) via application of a dryer, but the addition of the corrosion inhibitor alone increased significantly the time needed for solifidication. To minimize the drying period we have found the proper combination of the ODA and the dryer of the LO.
The EIS measurements, in accordance with the drying tests, resulted in the next order of self-healing ability: LO<LO(+ODA)<LO(+Co-octoate)<LO(+ODA+Co-octoate).
Electrodeposited conducting, photoluminescent poly-3,4-ethylenedioxy-thiophene (PEDOT) layers were functionally reconstituted on non-conducting surfaces through spin-coating process. The different ...electrochemical methods, including cyclic voltammetry and potentiostatic deposition on three different oxidation potentials, resulted in chemically invariant PEDOT layers. PEDOT films were removed from conducting electrode surfaces and ultrasonically homogenized before layer reformation on non-conducting support. PEDOT particles have shown photoluminescent activities both in dispersion and redeposited form. Conductance measurements of the transferred layers reflected to an ohmic behavior of the reformed films, which exhibited reversible, one magnitude conductance change upon the change of ambient atmosphere.
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•Chemical invariance of electrochemically deposited PEDOT film.•PEDOT can be functionally reconstructed non-conducting surfaces.•Conductance of the transferred layer showed ohmic behavior.•The transferred layer showed reversible sensitivity to the change of atmosphere.
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