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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230334	1/2	Cystobacter ferrugineus	Cbfe23	(d)(U)C SEC (non-commercial) Filtration	Goes A	2020	57%
Study summary Full title Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections. All authors Goes A, Lapuhs P, Kuhn T, Schulz E, Richter R, Panter F, Dahlem C, Koch M, Garcia R, Kiemer AK, Müller R, Fuhrmann G Journal Cells Abstract In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annual (show more...)In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is , a bacterium that can invade and survive within mammalian cells. intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria strain Cbv34 and strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular . We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular . In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections. (hide) EV-METRIC 57% (91st 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 Cell culture supernatant Sample origin Control condition Focus vesicles Other/ Outer membrane vesicles 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 Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Cystobacter ferrugineus Sample Type Cell culture supernatant EV-producing cells Cbfe23 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Filtration steps NS Size-exclusion chromatography Total column volume (mL) 60 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 100-350 NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV230334	2/2	Cystobacter velatus	Cbv34	(d)(U)C SEC (non-commercial) Filtration	Goes A	2020	43%
Study summary Full title Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections. All authors Goes A, Lapuhs P, Kuhn T, Schulz E, Richter R, Panter F, Dahlem C, Koch M, Garcia R, Kiemer AK, Müller R, Fuhrmann G Journal Cells Abstract In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annual (show more...)In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is , a bacterium that can invade and survive within mammalian cells. intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria strain Cbv34 and strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular . We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular . In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections. (hide) EV-METRIC 43% (81st 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 Cell culture supernatant Sample origin Control condition Focus vesicles Other/ Outer membrane vesicles 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 Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Cystobacter velatus Sample Type Cell culture supernatant EV-producing cells Cbv34 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Filtration steps NS Size-exclusion chromatography Total column volume (mL) 60 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-300 EV concentration Yes EM EM-type Cryo-EM Image type Close-up

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EV-TRACK ID	EV230334
species	Cystobacter ferrugineus	Cystobacter velatus
sample type	Cell culture	Cell culture
cell type	Cbfe23	Cbv34
condition	Control condition	Control condition
separation protocol	dUC/ Size-exclusion chromatography (non-commercial)/ Filtration	dUC/ Size-exclusion chromatography (non-commercial)/ Filtration
Exp. nr.	1	2
EV-METRIC %	57	43