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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200174	1/2	Bos taurus	1% skim milk	(d)(U)C	Sukreet, Sonal	2022	67%
Study summary Full title Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-defined Rodent Diet All authors Sonal Sukreet, Camila Pereira Braga, Thuy T An, Jiri Adamec, Juan Cui, Janos Zempleni Journal J Nutr Abstract Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumptio (show more...)Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumption of an exosome and RNA-depleted (ERD) diet elicited phenotypes compared to controls fed an exosome and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and non-ultrasonicated milk, respectively, for casein in the AIN-93G formulation. Objective: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. Methods: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation (USE); controls were not ultrasonicated (NSE). Exosome count, size and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids and proteins were analyzed by RNA-sequencing and mass spectrometry. Intestinal transport, bioavailability and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells and C57BL/6J mice (n = 3; age 6 - 8 weeks). Results: The exosome count was 76 ± 22% lower in USE compared to NSE (P < 0.05). Ultrasonication caused a degradation of up to 100% of microRNAs. USE and NSE contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected total of 525 and 484 proteins in USE and NSE. The uptake of USEs decreased by 46 ± 30% and 40 ± 27% compared to NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). Conclusions: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells and C57BL/6J mice and causes a near-complete degradation of microRNA cargos. (hide) EV-METRIC 67% (66th 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 1% skim milk Sample origin Control condition Focus vesicles exosome 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 No extra separation steps Protein markers EV: TSG101/ Alix/ CD63/ CD9/ CD81 non-EV: Beta-integrin/ lactalbumin/ Histone-H3 Proteomics yes Show all info Study aim Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type 1% skim milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Fiberlite-F37L-8x100 rotor Pelleting: speed (g) 120,000 Wash: volume per pellet (ml) 30 mL Wash: time (min) 90 Wash: Rotor Type Fiberlite-F37L-8x100 rotor Wash: speed (g) 120,000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA;Qubit Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Detected contaminants lactalbumin Not detected contaminants Beta-integrin/ Histone-H3 Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 117+/-44 EV concentration Yes Particle yield Yes, per milliliter of starting sample 5.90E+11 EM EM-type Transmission-EM/ Scanning-EM Image type Close-up, Wide-field

	EV200174	2/2	Bos taurus	1% skim milk	(d)(U)C	Sukreet, Sonal	2022	67%
Study summary Full title Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-defined Rodent Diet All authors Sonal Sukreet, Camila Pereira Braga, Thuy T An, Jiri Adamec, Juan Cui, Janos Zempleni Journal J Nutr Abstract Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumptio (show more...)Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumption of an exosome and RNA-depleted (ERD) diet elicited phenotypes compared to controls fed an exosome and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and non-ultrasonicated milk, respectively, for casein in the AIN-93G formulation. Objective: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. Methods: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation (USE); controls were not ultrasonicated (NSE). Exosome count, size and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids and proteins were analyzed by RNA-sequencing and mass spectrometry. Intestinal transport, bioavailability and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells and C57BL/6J mice (n = 3; age 6 - 8 weeks). Results: The exosome count was 76 ± 22% lower in USE compared to NSE (P < 0.05). Ultrasonication caused a degradation of up to 100% of microRNAs. USE and NSE contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected total of 525 and 484 proteins in USE and NSE. The uptake of USEs decreased by 46 ± 30% and 40 ± 27% compared to NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). Conclusions: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells and C57BL/6J mice and causes a near-complete degradation of microRNA cargos. (hide) EV-METRIC 67% (66th 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 1% skim milk Sample origin Sonicated milk Focus vesicles exosome 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 Protein markers EV: CD81/ Alix/ TSG101/ CD63/ CD9 non-EV: Beta-integrin/ lactalbumin/ Histone H3 Proteomics yes Show all info Study aim Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type 1% skim milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Fiberlite-F37L-8x100 rotor Pelleting: speed (g) 120,000 Wash: volume per pellet (ml) 30 mL Wash: time (min) 90 Wash: Rotor Type Fiberlite-F37L-8x100 rotor Wash: speed (g) 120,000 Characterization: Protein analysis Protein Concentration Method BCA;Qubit Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected EV-associated proteins CD81/ Alix Detected contaminants lactalbumin Not detected contaminants Beta-integrin/ Histone H3 Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 218+/-99 EV concentration Yes Particle yield Yes, per milliliter of starting sample 1.40E+11 EM EM-type Transmission-EM/ Scanning-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV200174
species	Bos taurus
sample type	1% skim milk
condition	Control condition	Sonicated milk
separation protocol	dUC No extra separation steps	dUC
Exp. nr.	1	2
EV-METRIC %	67	67