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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	XL5296IL	1/2	Macaca mulatta	Cervicovaginal lavage fluid	(d)(U)C	Zhao, Zezhou	2020	66%
Study summary Full title miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR-186-5p as a potential antiretroviral factor in macrophages All authors Zezhou Zhao, Dillon C Muth, Kathleen Mulka, Zhaohao Liao, Bonita H Powell, Grace V Hancock, Kelly A Metcalf Pate, Kenneth W Witwer Journal FEBS Open Bio Abstract Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage ( (show more...)Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage (CVL). CVL constituents have utility as biomarkers and play protective roles in wound healing and against HIV-1 infection. However, several components of cervicovaginal fluids are less well understood, such as extracellular RNAs and their carriers, for example, extracellular vesicles (EVs). EVs comprise a wide array of double-leaflet membrane extracellular particles and range in diameter from 30 nm to over one micron. The aim of this study was to determine whether differentially regulated CVL microRNAs (miRNAs) might influence retrovirus replication. To this end, we characterized EVs and miRNAs of primate CVL during the menstrual cycle and simian immunodeficiency virus (SIV) infection of macaques. EVs were enriched by stepped ultracentrifugation, and miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform. Whereas hormone cycling was abnormal in infected subjects, EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs changed during the menstrual cycle or SIV infection, for example, miR-186-5p, which was depleted in retroviral infection. This miRNA inhibited HIV replication in infected macrophages in vitro. In silico target prediction and pathway enrichment analyses shed light on the probable functions of miR-186-5p in hindering HIV infections via immunoregulation, T-cell regulation, disruption of viral pathways, etc. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract. (hide) EV-METRIC 66% (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 Cervicovaginal lavage fluid 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 (d)(U)C Adj. k-factor 113.6 (pelleting) Protein markers EV: CD81/ CD63 non-EV: calnexin Proteomics no Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Macaca mulatta Sample Type Cervicovaginal lavage fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type AH-650 Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 113.6 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81 Not detected contaminants calnexin Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) xx-xxx EV concentration Yes Particle yield 2.5E7-3.5E9 EM EM-type Transmission-EM Image type Close-up Report size (nm) up to 200 Extra information Sample volumes and yield limited the isolation and characterization steps we were able to perform.

	XL5296IL	2/2	Macaca mulatta	Cervicovaginal lavage fluid	(d)(U)C	Zhao, Zezhou	2020	66%
Study summary Full title miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR-186-5p as a potential antiretroviral factor in macrophages All authors Zezhou Zhao, Dillon C Muth, Kathleen Mulka, Zhaohao Liao, Bonita H Powell, Grace V Hancock, Kelly A Metcalf Pate, Kenneth W Witwer Journal FEBS Open Bio Abstract Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage ( (show more...)Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage (CVL). CVL constituents have utility as biomarkers and play protective roles in wound healing and against HIV-1 infection. However, several components of cervicovaginal fluids are less well understood, such as extracellular RNAs and their carriers, for example, extracellular vesicles (EVs). EVs comprise a wide array of double-leaflet membrane extracellular particles and range in diameter from 30 nm to over one micron. The aim of this study was to determine whether differentially regulated CVL microRNAs (miRNAs) might influence retrovirus replication. To this end, we characterized EVs and miRNAs of primate CVL during the menstrual cycle and simian immunodeficiency virus (SIV) infection of macaques. EVs were enriched by stepped ultracentrifugation, and miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform. Whereas hormone cycling was abnormal in infected subjects, EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs changed during the menstrual cycle or SIV infection, for example, miR-186-5p, which was depleted in retroviral infection. This miRNA inhibited HIV replication in infected macrophages in vitro. In silico target prediction and pathway enrichment analyses shed light on the probable functions of miR-186-5p in hindering HIV infections via immunoregulation, T-cell regulation, disruption of viral pathways, etc. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract. (hide) EV-METRIC 66% (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 Cervicovaginal lavage fluid Sample origin SIVmac251-infected 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 (d)(U)C Adj. k-factor 113.6 (pelleting) Protein markers EV: CD81/ CD63 non-EV: calnexin Proteomics no Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Macaca mulatta Sample Type Cervicovaginal lavage fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type AH-650 Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 113.6 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81 Not detected contaminants calnexin Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) xx-xxx EV concentration Yes Particle yield 1.75E8-2.15E9 EM EM-type Transmission-EM Image type Close-up Report size (nm) up to 200 nm Extra information Samples from SIV Infected animals were pooled for WB analysis

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EV-TRACK ID	XL5296IL
species	Macaca mulatta
sample type	Cervicovaginal lavage fluid
condition	Control condition	SIVmac251-infected
separation protocol	(d)(U)C	(d)(U)C
Exp. nr.	1	2
EV-METRIC %	66	66