Isolation of extracellular vesicle from blood plasma using electrophoretic migration through porous membrane

Biomarkers for amyotrophic lateral sclerosis/motor neuron disease (ALS/MND) are currently not clinically available for disease diagnosis or analysis of disease progression. If identified, biomarkers could improve patient outcomes by enabling early intervention and assist in the determination of treatment efficacy. We hypothesized that neural-enriched extracellular vesicles could provide microRNA (miRNA) fingerprints with unequivocal signatures of neurodegeneration. Using blood plasma from ALS/MND patients and controls, we extracted neural-enriched extracellular vesicle fractions and conducted next-generation sequencing and qPCR of miRNA components of the transcriptome. We here report eight miRNA sequences which significantly distinguish ALS/MND patients from controls in a replicated experiment using a second cohort of patients and controls. miRNA sequences from patient blood samples using neural-enriched extracellular vesicles may yield unique insights into mechanisms of neurodegeneration and assist in early diagnosis of ALS/MND.

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Differentially expressed extracellular vesicle, exosome and non-exosome miRNA profile in high and low tick-resistant beef cattle

10.21203/rs.3.rs-929210/v1 ◽

2021 ◽

Author(s):

Pevindu Abeysinghe ◽

Natalie Turner ◽

Hassendrini Peiris ◽

Kanchan Vaswani ◽

Nick Cameron ◽

...

Keyword(s):

Beef Cattle ◽

Blood Plasma ◽

Noncoding Rna ◽

Extracellular Vesicle ◽

Micro Rna ◽

Differentially Expressed ◽

Economic Losses ◽

Transcriptional Level ◽

Beef Industry ◽

Tick Resistance

Abstract Heavy tick burden on beef cattle account for huge economic losses globally, with an estimated value of US$22-30 billion per annum. In Australia, ticks cost the northern beef industry approximately A$170-200 million. Methods to evaluate and predict tick resistance would therefore be of great value to the global cattle trade. Exosomes (EX) are small extracellular vesicles (EVs) of ~30-150nm diameter and have gained popularity for their diagnostic and prognostic potential. EX contain, among other biomolecules, various types of RNA including micro-RNA (miRNA) and long noncoding RNA (lncRNA). MiRNA specifically have been validated as therapeutic biomarkers as they perform regulatory functions at the post-transcriptional level and are differentially expressed between divergent groups. The objective of the present study was to evaluate the miRNA profiles of EV and fractionated exosomal samples of high and low tick-resistant beef cattle to highlight potential miRNA biomarkers of tick resistance. Cows (n = 3/group) were classified into high or low tick resistant groups according to a novel scoring system. EVs and EX were isolated and fractionated from the blood plasma of high and low tick resistant cattle using established isolation and enrichment protocols. The resultant EX and non-EX samples were processed for next generation miRNA sequencing. Offspring of the cows in each high and low tick resistant group underwent the same processing for blood plasma EX, non-EX and miRNA analysis to evaluate the heritability of miRNA associated with tick resistance.A total of 2631 miRNAs were identified in EX and non-EX fractionated samples from high and low tick-resistant beef cattle. MiR-449a was highly expressed in maternal high tick-resistant EX samples. Of these, 174 were novel miRNAs, and 10 were differentially expressed (DE) (FDR < 0.05). These 10 DE miRNAs were also present in EVs, and three miRNAs were highly expressed: miR-2419-3p, miR-7861-3p and miR-2372-5p. Although 196 novel miRNAs were identified in fractionated samples of offspring, no miRNA were differentially expressed in these animals.

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Evaluation of plasmatic extracellular vesicles by size

10.21203/rs.3.pex-1287/v1 ◽

2021 ◽

Author(s):

Laura Cantone ◽

Mirjam Hoxha ◽

Chiara Favero ◽

Luca Ferrari ◽

Valentina Bollati

Keyword(s):

Plasma Membrane ◽

Blood Plasma ◽

Extracellular Vesicles ◽

High Precision ◽

Extracellular Vesicle ◽

Nanoparticle Tracking Analysis ◽

Research Needs ◽

Plasma Samples ◽

Constant Flow ◽

Syringe Pump

Abstract Extracellular vesicles (EVs) play a key role in many physiological and pathological processes [1]. EVs are a heterogeneous group of membrane-confined particles including endosome-derived exosomes and plasma membrane-originated microvesicles. The expanding field of extracellular vesicle research needs reproducible and accurate methods to characterize EVs [2]. EV profiling can be challenging due to the small size and heterogeneity. This protocol aims to provide a method to isolate EVs and facilitate high-precision particle quantitation by Nanoparticle Tracking Analysis (NTA)[3, 4]. NTA is commonly used to determine EV concentration and diameter [5, 6]. The protocol here described refers to the isolation of EVs from blood-plasma samples by using ultracentrifugation and then quantification and sizing of EVs with NTA by NanoSight NS300 system (Malvern Panalytical Ltd., Malvern, UK) provided with a syringe pump module enabling analysis in constant flow for improved sample statistics.

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Differentially Expressed Extracellular Vesicle, Exosome and Non-Exosome miRNA Profile in High and Low Tick-Resistant Beef Cattle

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.780424 ◽

2021 ◽

Vol 11 ◽

Author(s):

Pevindu Abeysinghe ◽

Natalie Turner ◽

Hassendrini Peiris ◽

Kanchan Vaswani ◽

Nick Cameron ◽

...

Keyword(s):

Beef Cattle ◽

Blood Plasma ◽

Noncoding Rna ◽

Extracellular Vesicle ◽

Micro Rna ◽

Differentially Expressed ◽

Economic Losses ◽

Transcriptional Level ◽

Beef Industry ◽

Tick Resistance

Heavy tick burden on beef cattle account for huge economic losses globally, with an estimated value of US$22-30 billion per annum. In Australia, ticks cost the northern beef industry approximately A$170-200 million. Methods to evaluate and predict tick resistance would therefore be of great value to the global cattle trade. Exosomes (EX) are small extracellular vesicles (EVs) of ~30-150nm diameter and have gained popularity for their diagnostic and prognostic potential. EX contain, among other biomolecules, various types of RNA including micro-RNA (miRNA) and long noncoding RNA (lncRNA). MiRNA specifically have been validated as therapeutic biomarkers as they perform regulatory functions at the post-transcriptional level and are differentially expressed between divergent groups. The objective of the present study was to evaluate the miRNA profiles of EV and fractionated exosomal samples of high and low tick-resistant beef cattle to highlight potential miRNA biomarkers of tick resistance. Cows (n = 3/group) were classified into high or low tick resistant groups according to a novel scoring system. EVs and EX were isolated and fractionated from the blood plasma of high and low tick resistant cattle using established isolation and enrichment protocols. The resultant EX and non-EX samples were processed for next generation miRNA sequencing. Offspring of the cows in each high and low tick resistant group underwent the same processing for blood plasma EX, non-EX and miRNA analysis to evaluate the heritability of miRNA associated with tick resistance. A total of 2631 miRNAs were identified in EX and non-EX fractionated samples from high and low tick-resistant beef cattle. MiR-449a was highly expressed in maternal high tick-resistant EX samples. Of these, 174 were novel miRNAs, and 10 were differentially expressed (DE) (FDR < 0.05). These 10 DE miRNAs were also present in EVs, and three miRNAs were highly expressed: miR-2419-3p, miR-7861-3p and miR-2372-5p. Although 196 novel miRNAs were identified in fractionated samples of offspring, no miRNA were differentially expressed in these animals.

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isolation and fractionation of blood plasma extracellular vesicle (EV) subpopulations using size exclusion chromatography

10.17760/d20383680 ◽

2020 ◽

Author(s):

Jacqueline Wood

Keyword(s):

Blood Plasma ◽

Size Exclusion Chromatography ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Exclusion Chromatography

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Abstract P-43: Effect of Glucocerebrosidase Dysfunction on the Pool of Plasma Exosomes of Patients with Gaucher Disease

International Journal of Biomedicine ◽

10.21103/ijbm.11.suppl_1.p43 ◽

2021 ◽

Vol 11 (Suppl_1) ◽

pp. S31-S31

Author(s):

Luiza Garaeva ◽

Roman Kamyshinsky ◽

Darya Kulabukhova ◽

Sergey Landa ◽

Elena Varfolomeeva ◽

...

Keyword(s):

Blood Plasma ◽

Extracellular Vesicles ◽

Gaucher Disease ◽

Biological Fluids ◽

Membrane Vesicles ◽

Extracellular Vesicle ◽

Lysosomal Storage ◽

Lysosomal Dysfunction ◽

Human Disorders ◽

Vesicle Pool

Background: Extracellular vesicles (EVs) are small membrane vesicles released from different types of cells. EVs are found in many human biological fluids. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies with the plasma membrane. This type of vesicles is characterized by specific exosomal markers. Exosomes extracted from peripheral body liquids could have specific properties associated with different physiological conditions as well as human disorders, including neurodegenerative diseases. Gaucher disease (GD) – is the most common form of lysosomal storage disorders caused by mutations in the glucocerebrosidase (GBA) gene. Lysosome functionality is critical for the regulation of extracellular vesicle secretion and content. In model animals, the inhibition of glucocerebrosidase has been shown to increase the secretion of extracellular vesicles in brain tissues. Amount evaluation of EVs and their size in the biological fluids of patients with GD has not been early performed; therefore, it is unknown whether lysosomal dysfunction found in GD patients influences the plasma pool of EVs. The aim of this study was to evaluate the amount of blood plasma EVs in patients with GD and their characterization for morphology and size. Methods: EVs were isolated from the blood plasma of 8 GD patients and 8 controls by ultracentrifugation, and were characterized using cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). Also, the presence of exosomal markers CD9, CD63, CD81, and HSP70 was analyzed by flow cytometry and western blot. Results: Here, it was first shown an increased proportion of exosome fraction in EVs from plasma of GD patients compared to controls by DLS and cryo-EM (p<0.001) that was confirmed by mode size detected by NTA (p<0.02). Moreover, an increased number of double and multilayer vesicles in plasma EVs from GD patients was demonstrated by cryo-EM. We also detected an increase in the expression of exosomal markers on the surface of vesicles from the blood plasma of patients with GD compared to controls. Conclusion: Here, we firstly report that the exosomes obtained from the blood plasma of GD patients have a larger size and altered morphology. Thus, we have shown that lysosomal dysfunction in GD patients leads to a striking alteration of blood plasma extracellular vesicle pool.

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Blood Nanoparticles – Influence on Extracellular Vesicle Isolation and Characterization

Frontiers in Pharmacology ◽

10.3389/fphar.2021.773844 ◽

2021 ◽

Vol 12 ◽

Author(s):

Marija Holcar ◽

Maša Kandušer ◽

Metka Lenassi

Keyword(s):

Blood Plasma ◽

Cell Types ◽

Extracellular Vesicle ◽

Phospholipid Bilayer ◽

Spherical Particles ◽

Cell Of Origin ◽

Unique Challenge ◽

Isolation And Characterization ◽

Downstream Analysis ◽

Molecular Components

Blood is a rich source of disease biomarkers, which include extracellular vesicles (EVs). EVs are nanometer-to micrometer-sized spherical particles that are enclosed by a phospholipid bilayer and are secreted by most cell types. EVs reflect the physiological cell of origin in terms of their molecular composition and biophysical characteristics, and they accumulate in blood even when released from remote organs or tissues, while protecting their cargo from degradation. The molecular components (e.g., proteins, miRNAs) and biophysical characteristics (e.g., size, concentration) of blood EVs have been studied as biomarkers of cancers and neurodegenerative, autoimmune, and cardiovascular diseases. However, most biomarker studies do not address the problem of contaminants in EV isolates from blood plasma, and how these might affect downstream EV analysis. Indeed, nonphysiological EVs, protein aggregates, lipoproteins and viruses share many molecular and/or biophysical characteristics with EVs, and can therefore co-isolate with EVs from blood plasma. Consequently, isolation and downstream analysis of EVs from blood plasma remain a unique challenge, with important impacts on the outcomes of biomarker studies. To help improve rigor, reproducibility, and reliability of EV biomarker studies, we describe here the major contaminants of EV isolates from blood plasma, and we report on how different EV isolation methods affect their levels, and how contaminants that remain can affect the interpretation of downstream EV analysis.

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