Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles

Noble, Jade M.; Roberts, LaDeidra Monét; Vidavsky, Netta; Chiou, Aaron E.; Fischbach, Claudia; Paszek, Matthew J.; Estroff, Lara A.; Kourkoutis, Lena F.

doi:10.1016/j.jsb.2020.107474

Cited by 68 publications

(64 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The MCF10A cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA; catalog number CRL 10317), maintained at 37°C in 5% CO 2 and cultivated in Dulbecco’s modified Eagle’s medium/F12 (Gibco, Grand Island, NY, USA) containing 20 ng mL −1 Epidermal Growth Factor (Sigma, St. Louis, MO, USA), 0.5 µg mL −1 hydrocortisone (Sigma), 100 ng mL −1 cholera toxin (Sigma) and 10 µg mL −1 insulin (Sigma) supplemented with 5% horse serum (Gibco) as previously described 48 and suggested by ATCC. Human THP‐1 monocytes were obtained from ATCC (catalog number TIB 202) and cultured in RPMI (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 0.05 m m β‐mercaptoethanol (Sigma).…”

Section: Methodsmentioning

confidence: 99%

The use of patient‐derived breast tissue explants to study macrophage polarization and the effects of environmental chemical exposure

et al. 2020

View full text Add to dashboard Cite

Ex vivo mammary explant systems are an excellent model to study interactions between epithelium and stromal cell types because they contain physiologically relevant heterotypic interactions in the background of genetically diverse patients. The intact human mammary tissue, termed patient-derived explant (PDE), can be used to investigate cellular responses to a wide variety of external stimuli in situ. For this study, we examined the impact of cytokines or environmental chemicals on macrophage phenotypes. We demonstrate that we can polarize macrophages within human breast tissue PDEs toward M1 or M2 through the addition of interferon-c (IFNc) + lipopolysaccharide (LPS) or interleukin (IL)-4 + IL-13, respectively. Elevated expression levels of M(IFNc + LPS) markers (HLADRA and CXCL10) or M(IL-4 + IL-13) markers (CD209 and CCL18) were observed in cytokine-treated tissues. We also examined the impact of the endocrine-disrupting chemical, benzophenone-3, on PDEs and measured significant, yet varying effects on macrophage polarization. Furthermore, a subset of the PDEs respond to IL-4 + IL-13 through downregulation of E-cadherin and upregulation of vimentin which is reminiscent of epithelial-to-mesenchymal transition (EMT) changes. Finally, we were able to show immortalized nonmalignant breast epithelial cells can exhibit EMT characteristics when exposed to growth factors secreted by M(IL-4 + IL-13) macrophages. Taken together, the PDE model system is an outstanding preclinical model to study early tissue-resident immune responses and effects on epithelial and stromal responses to stimuli found both endogenously in the breast and exogenously as a result of exposures.

show abstract

Section: Methodsmentioning

confidence: 99%

The use of patient‐derived breast tissue explants to study macrophage polarization and the effects of environmental chemical exposure

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Additionally, so far Cryptococcus EV proteomics identified only 92 and 202 proteins, with very poor overlap and no evaluation of their abundance or enrichment (Rodrigues et al 2008, Wolf et al 2014. Finally, although the current model of fungal EV structure contains proteins located on the vesicular surface (by analogy with the mammalian EV structures (Emelyanov et al 2020, Noble et al 2020), more experimental evidence is necessary to identify these putative membrane-associated molecules in fungal EVs.…”

Section: Introductionmentioning

confidence: 99%

“…However, the only structural analyses of EVs from this organism are now very outdated and technologies used have shown tremendous improvements since then (Emelyanov et al 2020, Noble et al 2020. Additionally, so far Cryptococcus EV proteomics identified only 92 and 202 proteins, with very poor overlap and no evaluation of their abundance or enrichment (Rodrigues et al 2008, Wolf et al 2014.…”

Section: Introductionmentioning

confidence: 99%

RevisitingCryptococcusextracellular vesicles properties and their use as vaccine platforms

Rizzo

Wong

Gazi

et al. 2020

Preprint

View full text Add to dashboard Cite

Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs from the pathogenic yeast C. neoformans. By using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and nanoscale flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by a 16 nm thick mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analyzing EV protein cargo from three cryptococcal species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response and significantly prolonged survival of mice upon C. neoformans infection.

show abstract

“…EVs are endogenous delivery systems with a diameter range of~50 nm to 1 µm [40]. EVs are formed in endosomal compartments and secreted after fusion with the plasma membrane by most cell types [41,42]. They can mediate and transmit a variety of intercellular signaling molecules packaging biological cargo, including nucleic acids, small RNAs (sRNAs), proteins, and lipids altering the gene expression, proliferation, and differentiation of recipient cells during physiological and pathological conditions [43].…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, several groups have reported the presence of DNA species, including ssDNA, dsDNA, and mitochondrial (mt) DNA encompassed in EVs from various sources [48][49][50]. The ability of EVs to carry genetic material mark their potential role in the transfer of exogenous genetic materials into the human genome [41]; additionally, EVs are used to deliver their cargo, crossing biological barriers, such as the blood-brain barrier (BBB) [51]. Several published studies report that plasmid RNA successfully loaded into EVs [52]; however, the potential use of EVs to load plasmid DNA for delivery applications remains scarcely described.…”

Section: Introductionmentioning

confidence: 99%

Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid

Orefice

2020

Pharmaceutics

View full text Add to dashboard Cite

Gene therapy is a therapeutic strategy of delivering foreign genetic material (encoding for an important protein) into a patient’s target cell to replace a defective gene. Nucleic acids are embedded within the adeno-associated virus (AAVs) vectors; however, preexisting immunity to AAVs remains a significant concern that impairs their clinical application. Extracellular vesicles (EVs) hold great potential for therapeutic applications as vectors of nucleic acids due to their endogenous intercellular communication functions through their cargo delivery, including lipids and proteins. So far, small RNAs (siRNA and micro (mi)RNA) have been mainly loaded into EVs to treat several diseases, but the potential use of EVs to load and deliver exogenous plasmid DNA has not been thoroughly described. This review provides a comprehensive overview of the principal methodologies currently employed to load foreign genetic material into EVs, highlighting the need to find the most effective strategies for their successful clinical translation.

show abstract

Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles

Cited by 68 publications

References 32 publications

The use of patient‐derived breast tissue explants to study macrophage polarization and the effects of environmental chemical exposure

The use of patient‐derived breast tissue explants to study macrophage polarization and the effects of environmental chemical exposure

RevisitingCryptococcusextracellular vesicles properties and their use as vaccine platforms

Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid

Contact Info

Product

Resources

About