Cells secrete extracellular vesicles (EVs) into their microenvironment that act as mediators of intercellular communication under physiological conditions and in this context also actively participate in spreading various diseases. Large efforts are currently made to produce reliable EV samples and to develop, improve, and standardize techniques allowing their biophysical characterization. Here, we used ultrafiltration and size-exclusion chromatography for the isolation and a model-free fluorescence fluctuation analysis for the investigation of the physical and biological properties of EVs secreted by mammalian cells. Our purification strategy produced enriched samples of morphologically intact EVs free of extravesicular proteins and allowed labeling of marker molecules on the vesicle surface for single-vesicle analysis with single-molecule sensitivity. This novel approach provides information on the distribution profile of both EV size and relative expression level of a specific exosomal marker, deciphering the overall heterogeneity of EV preparations.
Ozone is an effective disinfectant against all types of waterborne pathogens. However, accurate and quantitative kinetic data regarding virus inactivation by ozone are scarce, because of the experimental challenges associated with the high reactivity of ozone toward viruses. Here, we established an experimental batch system that allows tailoring and quantifying of very low ozone exposures and simultaneously measuring virus inactivation. Second-order ozone inactivation rate constants (k) of five enteric viruses [laboratory and two environmental strains of coxsackievirus B5 (CVF, CVEnv1, and CVEnv2), human adenovirus (HAdV), and echovirus 11 (EV)] and four bacteriophages (MS2, Qβ, T4, and Φ174) were measured in buffered solutions. The k values of all tested viruses ranged from 4.5 × 10 to 3.3 × 10 M s. For MS2, k depended only weakly on temperature (2-22 °C; E = 22.2 kJ mol) and pH (6.5-8.5), with an increase in k with increasing pH. The susceptibility of the selected viruses toward ozone decreases in the following order: Qβ > CVEnv2 > EV ≈ MS2 > Φ174 ≈ T4 > HAdV > CVF ≈ CVEnv1. On the basis of the measured k and typical ozone exposures applied in water and wastewater treatment, we conclude that ozone is a highly effective disinfectant for virus control.
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