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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230285	1/2	Sus scrofa	Stool	(d)(U)C Filtration UF DG	Lagos L	2020	57%
Study summary Full title Isolation and Characterization of Extracellular Vesicles Secreted In Vitro by Porcine Microbiota. All authors Lagos L, Leanti La Rosa S, Ø Arntzen M, Ånestad R, Terrapon N, Gaby JC, Westereng B Journal Microorganisms Abstract The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic (show more...)The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic cells for intercellular communication, survival, and pathogenesis. Previous studies have illustrated the presence of EVs in supernatants from pure cultures of bacteria, including Gram-positive and Gram-negative glycan-degrading gut commensals. However, the isolation and characterization of EVs secreted by a complex microbial community have not been clearly reported. In a recent paper, we showed that wood-derived, complex β-mannan, which shares a structural similarity with conventional dietary fibers, can be used to modulate the porcine gut microbiota composition and activity. In this paper, we investigated the production, size, composition, and proteome of EVs secreted by pig fecal microbiota after 24 h enrichment on complex β-mannan. Using transmission electron microscopy and nanoparticle tracking analysis, we identified EVs with an average size of 165 nm. We utilized mass spectrometry-based metaproteomic profiling of EV proteins against a database of 355 metagenome-assembled genomes (MAGs) from the porcine colon and thereby identified 303 proteins. For EVs isolated from the culture grown on β-mannan, most proteins mapped to two MAGs, MAG53 and MAG272, belonging to the orders and , respectively. Furthermore, the MAG with the third-most-detected protein was MAG 343, belonging to the order . The most abundant proteins detected in the β-mannan EVs proteome were involved in translation, energy production, amino acid, and carbohydrate transport, as well as metabolism. Overall, this proof-of-concept study demonstrates the successful isolation of EVs released from a complex microbial community/ furthermore, the protein content of the EVs reflects the response of specific microbes to the available carbohydrate source. (hide) EV-METRIC 57% (88th 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 Stool Sample origin No carbohydrate source Focus vesicles extracellular vesicle 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 (Differential) (ultra)centrifugation Filtration Ultrafiltration Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Stool Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 50.1 Pelleting: speed (g) 114000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mode Reported size (nm) 105/ 350 EM EM-type Transmission-EM Image type Wide-field

	EV230285	2/2	Sus scrofa	Stool	(d)(U)C Filtration UF DG	Lagos L	2020	57%
Study summary Full title Isolation and Characterization of Extracellular Vesicles Secreted In Vitro by Porcine Microbiota. All authors Lagos L, Leanti La Rosa S, Ø Arntzen M, Ånestad R, Terrapon N, Gaby JC, Westereng B Journal Microorganisms Abstract The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic (show more...)The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic cells for intercellular communication, survival, and pathogenesis. Previous studies have illustrated the presence of EVs in supernatants from pure cultures of bacteria, including Gram-positive and Gram-negative glycan-degrading gut commensals. However, the isolation and characterization of EVs secreted by a complex microbial community have not been clearly reported. In a recent paper, we showed that wood-derived, complex β-mannan, which shares a structural similarity with conventional dietary fibers, can be used to modulate the porcine gut microbiota composition and activity. In this paper, we investigated the production, size, composition, and proteome of EVs secreted by pig fecal microbiota after 24 h enrichment on complex β-mannan. Using transmission electron microscopy and nanoparticle tracking analysis, we identified EVs with an average size of 165 nm. We utilized mass spectrometry-based metaproteomic profiling of EV proteins against a database of 355 metagenome-assembled genomes (MAGs) from the porcine colon and thereby identified 303 proteins. For EVs isolated from the culture grown on β-mannan, most proteins mapped to two MAGs, MAG53 and MAG272, belonging to the orders and , respectively. Furthermore, the MAG with the third-most-detected protein was MAG 343, belonging to the order . The most abundant proteins detected in the β-mannan EVs proteome were involved in translation, energy production, amino acid, and carbohydrate transport, as well as metabolism. Overall, this proof-of-concept study demonstrates the successful isolation of EVs released from a complex microbial community/ furthermore, the protein content of the EVs reflects the response of specific microbes to the available carbohydrate source. (hide) EV-METRIC 57% (88th 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 Stool Sample origin ?-mannan Focus vesicles extracellular vesicle 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 (Differential) (ultra)centrifugation Filtration Ultrafiltration Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Stool Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 50.1 Pelleting: speed (g) 114000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mode Reported size (nm) 165/ 950 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV230285
species	Sus scrofa
sample type	Stool

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Study summary

Study data