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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200050	1/4	Homo sapiens	Blood plasma	qEV UF	Vestad, Beate	2021	75%
Study summary Full title Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk All authors Beate Vestad, Tuula A Nyman, Malene Hove-Skovsgaard, Maria Stensland, Hedda Hoel, Anne-Marie Siebke Trøseid, Trude Aspelin, Hans Christian D Aass, Maija Puhka, Johannes R Hov, Susanne Dam Nielsen, Reidun Øvstebø, Marius Trøseid Journal Scientific Reports Abstract HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotox (show more...)HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1β, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets. (hide) EV-METRIC 75% (96th 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 Blood plasma Sample origin Control condition 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 Commercial method Ultrafiltration Protein markers EV: HSP70/ CD9 non-EV: ApoA1/ ApoB/ calnexin Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ HSP70 Not detected contaminants Calnexin ELISA Detected contaminants ApoA1/ ApoB Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 146 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200050	2/4	Homo sapiens	Blood plasma	qEV UF	Vestad, Beate	2021	75%
Study summary Full title Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk All authors Beate Vestad, Tuula A Nyman, Malene Hove-Skovsgaard, Maria Stensland, Hedda Hoel, Anne-Marie Siebke Trøseid, Trude Aspelin, Hans Christian D Aass, Maija Puhka, Johannes R Hov, Susanne Dam Nielsen, Reidun Øvstebø, Marius Trøseid Journal Scientific Reports Abstract HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotox (show more...)HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1β, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets. (hide) EV-METRIC 75% (96th 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 Blood plasma Sample origin type 2 diabetes 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 Commercial method Ultrafiltration Protein markers EV: HSP70/ CD9 non-EV: ApoA1/ ApoB/ calnexin Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ HSP70 Not detected contaminants calnexin ELISA Detected contaminants ApoA1/ ApoB Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 141 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200050	3/4	Homo sapiens	Blood plasma	qEV UF	Vestad, Beate	2021	75%
Study summary Full title Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk All authors Beate Vestad, Tuula A Nyman, Malene Hove-Skovsgaard, Maria Stensland, Hedda Hoel, Anne-Marie Siebke Trøseid, Trude Aspelin, Hans Christian D Aass, Maija Puhka, Johannes R Hov, Susanne Dam Nielsen, Reidun Øvstebø, Marius Trøseid Journal Scientific Reports Abstract HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotox (show more...)HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1β, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets. (hide) EV-METRIC 75% (96th 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 Blood plasma Sample origin HIV 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 Commercial method Ultrafiltration Protein markers EV: HSP70/ CD9 non-EV: ApoA1/ ApoB/ calnexin Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ HSP70 Not detected contaminants calnexin ELISA Detected contaminants ApoA1/ ApoB Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 154 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200050	4/4	Homo sapiens	Blood plasma	qEV UF	Vestad, Beate	2021	75%
Study summary Full title Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk All authors Beate Vestad, Tuula A Nyman, Malene Hove-Skovsgaard, Maria Stensland, Hedda Hoel, Anne-Marie Siebke Trøseid, Trude Aspelin, Hans Christian D Aass, Maija Puhka, Johannes R Hov, Susanne Dam Nielsen, Reidun Øvstebø, Marius Trøseid Journal Scientific Reports Abstract HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotox (show more...)HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1β, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets. (hide) EV-METRIC 75% (96th 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 Blood plasma Sample origin HIV and type 2 diabetes 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 Commercial method Ultrafiltration Protein markers EV: HSP70/ CD9 non-EV: ApoA1/ ApoB/ calnexin Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ HSP70 Not detected contaminants calnexin ELISA Detected contaminants ApoA1/ ApoB Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 146 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV200050
species	Homo sapiens
sample type	Blood plasma
condition	Control condition	type 2 diabetes	HIV	HIV and type 2 diabetes
separation protocol	qEV Ultrafiltration	qEV Ultrafiltration	qEV Ultrafiltration	qEV Ultrafiltration
Exp. nr.	1	2	3	4
EV-METRIC %	75	75	75	75