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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210441	1/2	Bos taurus	Blood plasma	(d)(U)C Filtration	Zhao G	2018	33%
Study summary Full title Placental exosome-mediated Bta-miR-499-Lin28B/let-7 axis regulates inflammatory bias during early pregnancy. All authors Zhao G, Yang C, Yang J, Liu P, Jiang K, Shaukat A, Wu H, Deng G Journal Cell Death Dis Abstract Abnormal inflammatory bias in the maternal-fetal interface leads to reproductive failure in mammals. (show more...)Abnormal inflammatory bias in the maternal-fetal interface leads to reproductive failure in mammals. Placental exosomes are involved in maternal-fetal communication during pregnancy. However, whether the placenta or fetus is involved in regulating the balance of uterine local inflammation through exosomes remains unclear, and the mechanism must be further explored. Here we demonstrated that placenta-specific exosomes are abundant in the peripheral blood of dairy cows during early pregnancy and selectively load miRNAs, such as bta-miR-499. In vitro, placental exosome-derived bta-miR-499 inhibits the activation of NF-κB via the Lin28B/let-7 axis, thus repressing LPS-induced inflammation in bovine endometrial epithelial (BEND) cells. Subsequently, inhibition of mmu-miR-499 leads to an impaired balance of inflammation at the maternal-fetal interface in vivo, resulting in an increased risk of pregnancy failure due to placental loss and fetal growth restriction. Thus, our data demonstrate that placental exosomal miR-499 may be a critical immune regulator in the regulation of the inflammation balance at the maternal-fetal interface in the early gestation of dairy cows and other mammals. (hide) EV-METRIC 33% (65th 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 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 Filtration Protein markers EV: PLAP/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Bos taurus Sample Type Blood plasma 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: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: time (min) 120 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63 Not detected EV-associated proteins PLAP ELISA Antibody details provided? No Not detected EV-associated proteins Not detected contaminants PLAP Flow cytometry Type of Flow cytometry Accuri C6 f(BD Biosciences) Antibody details provided? No Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Accuri C6 f(BD Biosciences) Hardware adjustment Calibration bead size 0 EM EM-type Transmission-EM Image type Wide-field

	EV210441	2/2	Bos taurus	Blood plasma	(d)(U)C Filtration	Zhao G	2018	22%
Study summary Full title Placental exosome-mediated Bta-miR-499-Lin28B/let-7 axis regulates inflammatory bias during early pregnancy. All authors Zhao G, Yang C, Yang J, Liu P, Jiang K, Shaukat A, Wu H, Deng G Journal Cell Death Dis Abstract Abnormal inflammatory bias in the maternal-fetal interface leads to reproductive failure in mammals. (show more...)Abnormal inflammatory bias in the maternal-fetal interface leads to reproductive failure in mammals. Placental exosomes are involved in maternal-fetal communication during pregnancy. However, whether the placenta or fetus is involved in regulating the balance of uterine local inflammation through exosomes remains unclear, and the mechanism must be further explored. Here we demonstrated that placenta-specific exosomes are abundant in the peripheral blood of dairy cows during early pregnancy and selectively load miRNAs, such as bta-miR-499. In vitro, placental exosome-derived bta-miR-499 inhibits the activation of NF-κB via the Lin28B/let-7 axis, thus repressing LPS-induced inflammation in bovine endometrial epithelial (BEND) cells. Subsequently, inhibition of mmu-miR-499 leads to an impaired balance of inflammation at the maternal-fetal interface in vivo, resulting in an increased risk of pregnancy failure due to placental loss and fetal growth restriction. Thus, our data demonstrate that placental exosomal miR-499 may be a critical immune regulator in the regulation of the inflammation balance at the maternal-fetal interface in the early gestation of dairy cows and other mammals. (hide) EV-METRIC 22% (50th 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 Pregnant 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 Filtration Protein markers EV: PLAP/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Bos taurus Sample Type Blood plasma 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: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: time (min) 120 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ PLAP ELISA Antibody details provided? No Detected EV-associated proteins PLAP Flow cytometry Type of Flow cytometry Accuri C6 f(BD Biosciences) Antibody details provided? No Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Accuri C6 f(BD Biosciences) Hardware adjustment Calibration bead size 0 Report type Not Reported

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EV-TRACK ID	EV210441
species	Bos taurus
sample type	Blood plasma
condition	Control condition	Pregnant
separation protocol	dUC/ Filtration	dUC/ Filtration
Exp. nr.	1	2
EV-METRIC %	33	22