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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV160005	1/2	Homo sapiens	Bronchoalveolar lavage fluid	(d)(U)C Filtration	Héliot A	2016	55%
Study summary Full title Smoker extracellular vesicles influence status of human bronchial epithelial cells. All authors Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ Journal Int J Hyg Environ Health Abstract Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide) EV-METRIC 55% (33rd 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 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 Bronchoalveolar 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (d)(U)C + Filtration Adj. k-factor 126 (pelleting) / 126 (washing) Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Bronchoalveolar lavage fluid Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 126.0 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 126.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81 Characterization: Particle analysis NTA Report type Size range/distribution;Mean Reported size (nm) 138.1±2.2 EV concentration Yes Particle yield 2.48E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200 Extra information UC rotors added to the EV-TRACK entry after publication

	EV160005	2/2	Homo sapiens	Bronchoalveolar lavage fluid	(d)(U)C Filtration	Héliot A	2016	55%
Study summary Full title Smoker extracellular vesicles influence status of human bronchial epithelial cells. All authors Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ Journal Int J Hyg Environ Health Abstract Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide) EV-METRIC 55% (33rd 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 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 Bronchoalveolar lavage fluid Sample origin Smokers Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (d)(U)C + Filtration Adj. k-factor 126 (pelleting) / 126 (washing) Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Bronchoalveolar lavage fluid Sample Condition Smokers Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 126.0 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 126.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81 Characterization: Particle analysis NTA Report type Size range/distribution;Mean Reported size (nm) 139.8±3.3 EV concentration Yes Particle yield 1.94E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200 Extra information UC rotors added to the EV-TRACK entry after publication

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