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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210338	1/1	Bos taurus	1% skim milk	(d)(U)C	Fang, Zhou	2022	56%
Study summary Full title Small Extracellular Vesicles in Milk Cross the Blood-Brain Barrier in Murine Cerebral Cortex Endothelial Cells and Promote Dendritic Complexity in the Hippocampus and Brain Function in C57BL/6J Mice All authors Fang Zhou, Pearl Ebea, Ezra Mutai, Haichuan Wang, Sonal Sukreet, Shya Navazesh, Haluk Dogan, Wenhao Li, Juan Cui, Peng Ji, Denise M O Ramirez, Janos Zempleni Journal Frontiers Nutr. Abstract Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, (show more...)Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, whereas infant formulas contain only trace amounts of sEVs and microRNAs. We assessed the transport of sEVs across the blood-brain barrier (BBB) and sEV accumulation in distinct regions of the brain in brain endothelial cells and suckling mice. We further assessed sEV-dependent gene expression profiles and effects on the dendritic complexity of hippocampal granule cells and phenotypes of EV depletion in neonate, juvenile and adult mice. The transfer of sEVs across the BBB was assessed by using fluorophore-labeled bovine sEVs in brain endothelial bEnd.3 monolayers and dual chamber systems, and in wild-type newborn pups fostered to sEV and cargo tracking (ECT) dams that express sEVs labeled with a CD63-eGFP fusion protein for subsequent analysis by serial two-photon tomography and staining with anti-eGFP antibodies. Effects of EVs on gene expression and dendritic architecture of granule cells was analyzed in hippocampi from juvenile mice fed sEV and RNA-depleted (ERD) and sEV and RNA-sufficient (ERS) diets by using RNA-sequencing analysis and Golgi-Cox staining followed by integrated neuronal tracing and morphological analysis of neuronal dendrites, respectively. Spatial learning and severity of kainic acid-induced seizures were assessed in mice fed ERD and ERS diets. bEnd.3 cells internalized sEVs by using a saturable transport mechanism and secreted miR-34a across the basal membrane. sEVs penetrated the entire brain in fostering experiments; major regions of accumulation included the hippocampus, cortex and cerebellum. Two hundred ninety-five genes were differentially expressed in hippocampi from mice fed ERD and ERS diets; high-confidence gene networks included pathways implicated in axon guidance and calcium signaling. Juvenile pups fed the ERD diet had reduced dendritic complexity of dentate granule cells in the hippocampus, scored nine-fold lower in the Barnes maze test of spatial learning and memory, and the severity of seizures was 5-fold higher following kainic acid administration in adult mice fed the ERD diet compared to mice fed the ERS diet. We conclude that sEVs cross the BBB and contribute toward optimal neuronal development, spatial learning and memory, and resistance to kainic acid-induced seizures in mice. (hide) EV-METRIC 56% (16th 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 Protein markers EV: Alix/ CD9/ CD63/ CD81/ Integrin-beta/ Histone H3/ Apolipoprotein B non-EV: None Proteomics no Show all info Study aim Function/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) 50 Wash: time (min) 90 Wash: Rotor Type Fiberlite-F37-8x-100 rotor Wash: speed (g) 120,000 Characterization: Protein analysis Protein Concentration Method BCA/ Qubit Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ CD63/ CD81 Not detected EV-associated proteins Integrin-beta/ Histone H3/ Apolipoprotein B Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 106.6 +/- 6.7 EV concentration Yes EM EM-type Scanning-EM Image type Wide-field

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EV-TRACK ID	EV210338
species	Bos taurus
sample type	1% skim milk
condition	Control condition
separation protocol	dUC
Exp. nr.	1
EV-METRIC %	56