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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120105	1/1	Cryptococcus neoformans Bacillus anthracis	Bacteria	(d)(U)C DG Filtration UF	Wolf JM	2012	14%
Study summary Full title Serum albumin disrupts Cryptococcus neoformans and Bacillus anthracis extracellular vesicles All authors Wolf JM, Rivera J, Casadevall A Journal Cell Microbiol Abstract For both pathogenic fungi and bacteria, extracellular vesicles have been shown to contain many micro (show more...)For both pathogenic fungi and bacteria, extracellular vesicles have been shown to contain many microbial components associated with virulence, suggesting a role in pathogenesis. However, there are many unresolved issues regarding vesicle synthesis and stability, including the fact that vesicular packaging for extracellular factors involved in virulence must also have a mechanism for vesicle unloading. Consequently, we studied the kinetics of vesicle production and stability using [1-(14) C] palmitic acid metabolic labelling and dynamic light scattering techniques. Cryptococcus neoformans vesicles were produced throughout all stages of fungal culture growth and they were stable once isolated. Density gradient analysis revealed that only a portion of the vesicle population carried cryptococcal polysaccharide, implying heterogeneity in vesicular cargo. Vesicle incubation with macrophages resulted in rapid vesicle instability, a phenomenon that was ultimately associated with serum albumin. Additionally, albumin, along with mouse serum and murine immunoglobulin destabilized Bacillus anthracis vesicles, but the effect was not observed with ovalbumin or keyhole limpet haemocyanin, demonstrating that this phenomenon is neither host-, microbe- nor protein-specific. Our findings strongly suggest that cryptococcal vesicles are short-lived in vivo and vesicle destabilization is mediated by albumin. The ability of albumin to promote vesicular offload through destabilization indicates a new activity for this abundant serum protein. (hide) EV-METRIC 14% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bacteria Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration UF Protein markers EV: GXM non-EV: Proteomics no Show all info Study aim Other/Bacterial vesicle stability Sample Species Cryptococcus neoformans / Bacillus anthracis Sample Type Bacteria Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 10 Highest density fraction 35 Orientation Bottom-up Filtration steps > 0.45 µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins GXM ELISA Antibody details provided? No Detected EV-associated proteins GXM Characterization: Particle analysis DLS

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EV-TRACK ID	EV120105
species	Cryptococcus neoformans Bacillus anthracis
sample type	Bacteria
condition	NAY
separation protocol	(d)(U)C DG Filtration UF
Exp. nr.	1
EV-METRIC %	14