scholarly journals Isolation of Small Extracellular Vesicles from Human Sera

2021 ◽  
Vol 22 (9) ◽  
pp. 4653
Author(s):  
Małgorzata S. Małys ◽  
Christof Aigner ◽  
Stefan M. Schulz ◽  
Helga Schachner ◽  
Andrew J. Rees ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Human Serum ◽  
Extracellular Vesicles ◽  
Plasma Lipids ◽  
Surface Proteins ◽  
Nanoparticle Tracking Analysis ◽  
Disease Biomarkers ◽  
Exclusion Chromatography ◽  
Human Sera ◽  
Specific Proteins

Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are needed before their potential as disease biomarkers can be realized. Here, we compared isolation of sEV from serum by differential ultracentrifugation (DUC) and by exclusion chromatography using commercially available Exo-spin™ columns. We show that sEV can be purified by both methods but Exo-spin™ columns contain copious additional particles recorded by nanoparticle tracking analysis, invalidating its use for quantifying yields. DUC samples contained higher concentrations of exosome specific proteins CD9, CD63 and CD81 and electron microscopy confirmed that most particles in DUC preparations were sEV, whereas Exo-spin™ samples also contained copious co-purified plasma lipids. MACSPlex bead analysis identified multiple exosome surface proteins, with stronger signals in DUC samples, enabling detection of 21 of 37, compared to only 10 in Exo-spin™ samples. Nevertheless, the pattern of expression was consistent in both preparations, indicating that lipids do not interfere with bead-based technologies. Thus, both DUC and Exo-spin™ can be used to isolate sEV from human serum and what is most appropriate depends on the subsequent use of sEV. In summary, Exo-spin™ enables isolation of sEV from blood with vesicle populations similar to the ones recovered by DUC, but with lower concentrations.

scholarly journals Tell me what type of extracellular vesicles you secrete, and I will tell you who you are: yeast or hypha

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Raquel Martínez-López ◽  
Catarina Vaz ◽  
Esther Redondo ◽  
Guillermo Calvo ◽  
María Luisa Hernáez ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Ribosomal Proteins ◽  
Culture Medium ◽  
Surface Proteins ◽  
Cellular Transport ◽  
Tem Analysis ◽  
Host Interaction ◽  
Human Sera ◽  
Great Relevance ◽  
Shed Light

The transition between yeast and hyphal morphologies plays a crucial role in the pathogenicity of Candida albicans. Recent studies have pointed out the great relevance of extracellular vesicles (EVs) secreted by microorganisms in a wide variety of biological processes including interaction with the host. Therefore, the main objective of this work was to compare the EVs secreted by yeast and hyphal forms to shed light on C. albicans-host interaction. EVs were obtained by ultracentrifugation of the culture medium supernatant and analysed by mass spectrometry. They were characterized by transmission electronic microscopy (TEM) and dynamic light scattering (DLS). DLS and TEM analysis showed that yeast EVs were significantly bigger than hyphal EVs, being most of them in the range between 400 to 500nm while hyphal EVs were ranged mostly around 100-200nm. Proteomic analysis showed greater protein diversity in hyphal EVs when compared to yeast EVs (up to 1700 different proteins identified versus 300), although less amount of total protein was obtained. Gene Ontology (GO) analysis showed that yeast EVs were enriched in surface proteins while hyphal EVs, although containing also most of these surface proteins, were also significantly and exclusively enriched in proteins involved in protein metabolism (ribosomal proteins, many aminoacid-pathway enzymes and proteasome) and cellular transport. The differences between YEVs and HEVs also prompted a different immune host response, as tested with macrophage cell cultures and human sera from patients with invasive candidiasis. All these differences point out a possible different biogenesis and roles of EVs secreted by both morphologies.

scholarly journals Small extracellular vesicles secreted by Candida albicans hyphae have highly diverse protein cargoes that include virulence factors and stimulate macrophages

2020 ◽  
Author(s):  
Raquel Martínez-López ◽  
Maria Luisa Hernáez ◽  
Esther Redondo ◽  
Guillermo Calvo ◽  
Sonja Radau ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Extracellular Vesicles ◽  
Virulence Factors ◽  
Ribosomal Proteins ◽  
Protein Transport ◽  
Surface Proteins ◽  
Immune Reactivity ◽  
Exosome Biogenesis ◽  
Virulence Trait ◽  
Human Sera

ABSTRACTExtracellular vesicles (EVs) have been described as mediators of microorganism survival and interaction with the host. In Candida albicans, a relevant commensal fungal pathogen, the dimorphic transition is an important virulence trait in candidiasis. We have analyzed EVs secreted by yeast (YEVs) or hyphal cells (HEVs) from C. albicans, finding interesting differences in both size distribution and protein loading. In general, HEVs were smaller and carried a much more diverse protein cargo than YEVs, including most of the proteins identified in YEVs, which were mainly cell surface proteins. Virulence factors such as phospholipases, aspartic proteases (Saps), adhesins and invasins, and the precursor protein of candidalysin toxin Ece1p were also detected. HEVs were also enriched in proteasomal and ribosomal proteins, and in enzymes from amino acid biosynthetic pathways, all involved in protein metabolism, as well as proteins related to intracellular protein transport and components of the ESCRT pathway related to exosome biogenesis. Both types of EV presented immune reactivity with human sera from patients suffering invasive candidiasis. In our conditions, only HEVs could elicit the release of TNFα by activated macrophages. This first analysis of C. albicans HEVs shows their relevance to pathogenesis and possible new diagnostics or treatments.

52 Blocking of embryonic development by nanoparticles derived from endometrial and oviductal cells isolated with an Amicon filter system

2020 ◽  
Vol 32 (2) ◽  
pp. 151
Author(s):  
J. Cabezas ◽  
D. Rojas ◽  
B. Melo-Baez ◽  
M. Gutierrez ◽  
F. Castro ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Embryonic Development ◽  
Embryo Development ◽  
Extracellular Vesicles ◽  
Primary Cultures ◽  
Nanoparticle Tracking Analysis ◽  
Endometrial Cells ◽  
Filter System ◽  
Transmission Electron

The success of development of invitro embryo production needs to mimic culture conditions in the maternal environment. Recently, it has been seen that extracellular vesicles (EVs) secreted by oviducal or endometrial cells may improve development and quality of embryos produced invitro. Extracellular vesicles are a mechanism of cellular communication; they carry molecules that are delivered into the target cells changing gene expression and function. Due to the size range and characteristics of EVs, they require specific methods for purification and characterisation. However, the possible contamination with other nanoparticles and their effect on embryo development have not been considered. Based on that, the goal of this work was to evaluate the effect on invitro bovine embryo development, of the addition to culture medium EVs secreted by oviducal and endometrial cells and isolated by centrifugation and concentrates with Amicon filters. For this purpose, cells were isolated from bovine oviduct and endometrium collected in local abattoir and primary cultures of epithelial and stromal cells were derived. The primary cultures from both sources were exposed or not to progesterone (P4; 15ngmL−1) for 4 days and then cultured for 24h in EV depleted media. The supernatant was harvested and EVs were isolated by serial centrifugations and subsequently concentrated by a 100 kDa Amicon filter system. The isolated EVs were characterised by transmission electron microscopy, nanoparticle tracking analysis, and flow cytometry. Oocytes were obtained from ovaries collected in the abattoir. The cumulus-oocyte complexes were matured invitro for 22h and subsequently fertilised for 18h. Presumptive zygotes were invitro cultured in synthetic oviducal fluid with EVs (1000ngmL−1 of total proteins) or not according to experimental group (1: EVs− (control); 2: EVs−OP4+; 3: EVs−OP4−; 4: EVs−EP4+ and 4: EVs−EP4−). Embryos were cultured for 7 days in 5% CO2, 5% O2, and 90% N2 (25 embryos/well in 4-well plates). At Day 7, embryo development was evaluated considering the blastocyst yield. Transmission electron microscopy showed typical structures and morphology of EVs and they were positive for CD9, CD63, and CD81 markers, and negative for CD40. According to nanoparticle tracking analysis, the mean size of EVs was 160±62nm and concentration of 3.29×1011 particlesmL−1 for oviducal and endometrial cells, respectively. A significant reduction of blastocyst rate was observed when embryos were cultured with cell-derived EVs; control: 152/44 (28.9%) vs. treatments with EVs; OP4+: 74/3 (4.1%), OP4−:76/2 (2.6%), EP4+: 74/6 (8.1%), and EP4−: 73/2 (2.7%) (P ≤ 0.01). Our results indicate that the use of nanoparticles, including EVs, isolated from cells of oviduct or endometrium, has a blocking effect on embryonic development and compromises the performance of blastocysts on Day 7 when used at concentrations of 1000ngmL−1 total protein, independent of the use or not of P4 and the source. These data provide insights regarding the use and protocols of acquiring exosomes for embryo supplementation. This research was supported by FONDECYT, Chile-1170310.

scholarly journals Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components

Proteomes ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 18 ◽  
Author(s):  
Mateusz Smolarz ◽  
Monika Pietrowska ◽  
Natalia Matysiak ◽  
Łukasz Mielańczyk ◽  
Piotr Widłak
Keyword(s):  
Human Serum ◽  
Serum Proteins ◽  
Size Exclusion ◽  
Simple Method ◽  
Exclusion Chromatography ◽  
Specific Proteins ◽  
Bona Fide ◽  
Shotgun Approach ◽  
Related Proteins ◽  
Serum Components

Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC–MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC–MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term “extracellular exosome”. Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available.

scholarly journals Methods for Separation and Characterization of Extracellular Vesicles: Results of a Worldwide Survey Performed by the ISEV Rigor and Standardization Subcommittee

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1955 ◽  
Author(s):  
Felix Royo ◽  
Clotilde Théry ◽  
Juan M. Falcón-Pérez ◽  
Rienk Nieuwland ◽  
Kenneth W. Witwer
Keyword(s):  
Extracellular Vesicles ◽  
Initial Response ◽  
Size Exclusion ◽  
Disease Biomarkers ◽  
Quality Controls ◽  
Isolation And Characterization ◽  
Measurement Results ◽  
Exclusion Chromatography ◽  
Important Objective

Research on extracellular vesicles (EVs) is growing exponentially due to an increasing appreciation of EVs as disease biomarkers and therapeutics, an expanding number of EV-containing materials under study, and application of new preparation, detection, and cargo analysis methods. Diversity of both sources and methodologies imposes challenges on the comparison of measurement results between studies and laboratories. While reference guidelines and minimal requirements for EV research have achieved the important objective of assembling community consensus, it is also essential to understand which methodologies and quality controls are currently being applied, and how usage trends are evolving. As an initial response to this need, the International Society for Extracellular Vesicles (ISEV) performed a worldwide survey in 2015 on “Techniques used for the isolation and characterization of extracellular vesicles” and published the results from this survey in 2016. In 2019, a new survey was performed to assess the changing state of the field. The questionnaire received more than 600 full or partial responses, and the present manuscript summarizes the results of this second worldwide survey. The results emphasize that separation methods such as ultracentrifugation and density gradients are still the most commonly used methods, the use of size exclusion chromatography has increased, and techniques based on tangential flow and microfluidics are now being used by more than 10% of respondents. The survey also reveals that most EV researchers still do not perform sample quality controls before or after isolation of EVs. Finally, the majority of EV researchers emphasize that separation and characterization of EVs should receive more attention.

scholarly journals Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed

2021 ◽  
Vol 22 (20) ◽  
pp. 11144
Author(s):  
Mikel Azkargorta ◽  
Ibon Iloro ◽  
Iraide Escobes ◽  
Diana Cabrera ◽  
Juan M. Falcon-Perez ◽  
...  
Keyword(s):  
Human Serum ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Membrane Attack Complex ◽  
Protein Detection ◽  
Size Exclusion ◽  
Blood Proteins ◽  
Proteomic Profiling ◽  
Serum Samples ◽  
Exclusion Chromatography

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (<100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.

STUDIES ON SULFATION FACTOR (SF) ACTIVITY OF HUMAN SERUM

1960 ◽  
Vol XXXV (III) ◽  
pp. 381-396 ◽  
Author(s):  
Sven Almqvist
Keyword(s):  
Human Serum ◽  
Normal Serum ◽  
Treatment Level ◽  
Response Curves ◽  
Normal Children ◽  
Significant Difference ◽  
Human Sera ◽  
Normal Humans ◽  
Pre Treatment ◽  
Dose Levels

ABSTRACT The sulfation factor (SF) activity of human sera has been estimated using a modification of the method of Daughaday et al. (1959). Each assay was statistically evaluated. The method had a mean precision of 0.14 and, used as an assay of GH of human serum, a sensitivity in three pituitary dwarfs of 0.1 to 0.6 μg of HGH/ml of serum. SF activity was found at all ages between 1 month and 75 years. There was a significantly lower mean SF activity below the age of half a year. Three cases of pituitary dwarfism had significantly low SF activities of sera. There was no significant difference between the SF activities of sera from untreated pituitary dwarfs and the sera from normal children below half a year of age. Dose-response curves with large volumes of sera from pituitary dwarfs and small volumes of sera from normal humans had the same slopes. Four mg of HGH prepared according to the method of Li & Papkoff (1956) resulted in a normal serum SF activity in each of the three dwarfs. A significant (P < 0.01) linear relationship was found between the concentration of SF activity of sera from these subjects and the logarithm of the dose of HGH given with dose levels of 1, 2 and 4 mg daily for three days. The decline of serum SF activity to the pre-treatment level following HGH in one dwarf suggested a half life not different from that indicated by others for growth hormone.

scholarly journals Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

2021 ◽  
Vol 10 (2) ◽  
pp. 319
Author(s):  
Hee Cheol Yang ◽  
Won Jong Rhee
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Molecular Beacon ◽  
Disease Diagnosis ◽  
Single Step ◽  
Flow Cytometer ◽  
Surface Proteins ◽  
Biomarker Detection ◽  
In Situ Detection

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

scholarly journals Detection of Exosomes Using Total Internal Reflected Imaging Ellipsometry

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 164
Author(s):  
Haoyu Liu ◽  
Wei Liu ◽  
Gang Jin
Keyword(s):  
Limit Of Detection ◽  
Biological Fluids ◽  
Surface Proteins ◽  
Surface Markers ◽  
Functional Roles ◽  
Disease Biomarkers ◽  
First Order ◽  
Imaging Ellipsometry ◽  
Interaction Kinetics ◽  
Membrane Bound

Exosomes are a kind of membrane-bound phospholipid nanovesicle that are secreted extensively in a variety of biological fluids. Accumulating evidence has indicated that exosomes not only communicate with cells, but also perform functional roles in physiology and pathology. In addition, exosomes have also elicited a great deal of excitement due to their potential as disease biomarkers. Therefore, requirements for sensitive methods capable of precisely and specifically determining exosomes were needed. Herein, we not only develop a sensing surface to capture exosomes but also compare two surface proteins on exosomes, which are appropriate for detecting exosome surface markers by total internal reflected imaging ellipsometry (TIRIE). Protein G and antibody were immobilized on a thin layer of golden substrate to form the biosensing surface. The bio-interaction between antibodies and exosomes was recorded by the TIRIE in real time. The distance between exosomes adhered on a surface was 44 nm ± 0.5 nm. The KD  of anti-CD9 and exosome was lower than anti-CD63 and exosome by introducing pseudo-first-order interaction kinetics, which suggested that CD9 is more suitable for exosome surface markers than CD63. The limit of detection (LOD) of TIRIE was 0.4 μg/mL. In conclusion, we have proposed a surface for the detection of exosomes based on TIRIE, which can make the detection of exosomes convenient and efficient.

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