scholarly journals A novel protocol for the isolation of fungal extracellular vesicles reveals the participation of a putative scramblase in polysaccharide export and capsule construction in Cryptococcus gattii.

2019 ◽  
Author(s):  
Flavia C. G. Reis ◽  
Beatriz S. Borges ◽  
Luísa J. Jozefowicz ◽  
Bianca A. G. Sena ◽  
Ane W. A. Garcia ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Histoplasma Capsulatum ◽  
Chemical Properties ◽  
Pathogenic Fungi ◽  
Cryptococcus Gattii ◽  
Biologically Active ◽  
Capsular Polysaccharides ◽  
Solid Media ◽  
Transmission Electron ◽  
Prion Transmission

AbstractRegular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and C. neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. These results were reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25, CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release.IMPORTANCEExtracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii. Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid translocating enzyme, in secretory processes of C. gattii.

scholarly journals A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in Cryptococcus gattii

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Flavia C. G. Reis ◽  
Beatriz S. Borges ◽  
Luísa J. Jozefowicz ◽  
Bianca A. G. Sena ◽  
Ane W. A. Garcia ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Histoplasma Capsulatum ◽  
Chemical Properties ◽  
Pathogenic Fungi ◽  
Cryptococcus Gattii ◽  
Biologically Active ◽  
Content Type ◽  
Solid Media ◽  
Transmission Electron ◽  
Prion Transmission

ABSTRACT Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release. IMPORTANCE Extracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii. Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid-translocating enzyme, in secretory processes of C. gattii.

scholarly journals Extracellular Vesicle-Mediated RNA Release in Histoplasma capsulatum

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Lysangela R. Alves ◽  
Roberta Peres da Silva ◽  
David A. Sanchez ◽  
Daniel Zamith-Miranda ◽  
Marcio L. Rodrigues ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Stress Responses ◽  
Histoplasma Capsulatum ◽  
Pathogenic Fungi ◽  
Extracellular Vesicle ◽  
Fungal Species ◽  
Cellular Communication ◽  
Content Type ◽  
Rna Molecules ◽  
Rna Content

ABSTRACT Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNAs with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains of Histoplasma capsulatum, an important human-pathogenic fungal pathogen. A total of 124 mRNAs were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, whereas 31 were enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25 to 40 nucleotides in length) that were strain specific, with a greater number identified in EVs produced by the G217B strain. Remarkably, the most highly enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of microRNA (miRNA)-like molecules in fungal EVs. We also compared the transcriptome profiles of H. capsulatum with the RNA composition of EVs, and no correlation was observed. Taking the results together, our study provided information about the RNA molecules present in H. capsulatum EVs and about the differences in composition between the strains. In addition, we found no correlation between the most highly expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism. IMPORTANCE Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.

scholarly journals Extracellular vesicle-mediated RNA release inHistoplasma capsulatum

2019 ◽  
Author(s):  
Lysangela R. Alves ◽  
Roberta Peres da Silva ◽  
David A. Sanchez ◽  
Daniel Zamith-Miranda ◽  
Marcio L. Rodrigues ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Stress Responses ◽  
Histoplasma Capsulatum ◽  
Pathogenic Fungi ◽  
Extracellular Vesicle ◽  
Fungal Species ◽  
Cellular Communication ◽  
Rna Molecules ◽  
Rna Content ◽  
Reverse Strand

AbstractEukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNA classes with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains ofHistoplasma capsulatum, an important human fungal pathogen. A total of 124 mRNA were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, while 31 enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25-40 long) that were strain-specific, with a greater number of them identified in EVs produced by the G217B strain. Remarkably, the most enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of miRNA-like molecules in fungal EVs. We also compared the transcriptome profiles ofH. capsulatumwith the RNA composition of EVs and no correlation was observed. Altogether, our study provided information about the RNA molecules present inH. capsulatumEVs, and the differences in composition between the G186AR and G217B strains. In addition, we showed that the correlation between the most expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism.ImportanceExtracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. In pathogenic fungi, EV-associated RNA classes have recently been described; however, only a few studies describing the RNA in fungal EVs are available. An improved knowledge on EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates ofHistoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.

scholarly journals Small Molecule Analysis of Extracellular Vesicles Produced by Cryptococcus gattii: Identification of a Tripeptide Controlling Cryptococcal Infection in an Invertebrate Host Model

2021 ◽  
Vol 12 ◽  
Author(s):  
Flavia C. G. Reis ◽  
Jonas H. Costa ◽  
Leandro Honorato ◽  
Leonardo Nimrichter ◽  
Taícia P. Fill ◽  
...  
Keyword(s):  
Small Molecules ◽  
Extracellular Vesicles ◽  
Small Molecule ◽  
Galleria Mellonella ◽  
Pathogenic Fungus ◽  
Cryptococcus Gattii ◽  
Biologically Active ◽  
Small Peptides ◽  
Cryptococcal Infection ◽  
Small Molecule Analysis

The small molecule (molecular mass <900 Daltons) composition of extracellular vesicles (EVs) produced by the pathogenic fungus Cryptococcus gattii is unknown, which limits the understanding of the functions of cryptococcal EVs. In this study, we analyzed the composition of small molecules in samples obtained from solid cultures of C. gattii by a combination of chromatographic and spectrometric approaches, and untargeted metabolomics. This analysis revealed previously unknown components of EVs, including small peptides with known biological functions in other models. The peptides found in C. gattii EVs had their chemical structure validated by chemical approaches and comparison with authentic standards, and their functions tested in a Galleria mellonella model of cryptococcal infection. One of the vesicular peptides (isoleucine-proline-isoleucine, Ile-Pro-Ile) improved the survival of G. mellonella lethally infected with C. gattii or C. neoformans. These results indicate that small molecules exported in EVs are biologically active in Cryptococcus. Our study is the first to characterize a fungal EV molecule inducing protection, pointing to an immunological potential of extracellular peptides produced by C. gattii.

scholarly journals Small molecule analysis of extracellular vesicles produced by Cryptococcus gattii: identification of a tripeptide controlling cryptococcal infection in an invertebrate host model

2021 ◽  
Author(s):  
Flavia C. G. Reis ◽  
Jonas H. Costa ◽  
Leandro Honorato ◽  
Leonardo Nimrichter ◽  
Taícia P. Fill ◽  
...  
Keyword(s):  
Small Molecules ◽  
Extracellular Vesicles ◽  
Small Molecule ◽  
Galleria Mellonella ◽  
Pathogenic Fungus ◽  
Cryptococcus Gattii ◽  
Biologically Active ◽  
Small Peptides ◽  
Cryptococcal Infection ◽  
Small Molecule Analysis

AbstractThe small molecule (molecular mass < 900 Daltons) composition of extracellular vesicles (EVs) produced by the pathogenic fungus Cryptococcus gattii is unknown, which limits the understanding of the functions of cryptococcal EVs. In this study, we analyzed the composition of small molecules in samples obtained from solid cultures of C. gattii by a combination of chromatographic and spectrometric approaches, and untargeted metabolomics. This analysis revealed previously unknown components of EVs, including small peptides with known biological functions in other models. The peptides found in C. gattii EVs had their chemical structure validated by chemical approaches and comparison with authentic standards, and their functions tested in a Galleria mellonella model of cryptococcal infection. One of the vesicular peptides (isoleucine-proline-isoleucine, Ile-Pro-Ile) improved the survival of G. mellonella lethally infected with C. gattii or C. neoformans. These results indicate that small molecules exported in EVs are biologically active in Cryptococcus. Our study is the first to characterize a fungal EV molecule inducing protection, pointing to an immunological potential of extracellular peptides produced by C. gattii.

The microstructure of a TiB2/Ti-47 Al composite material

1989 ◽  
Vol 47 ◽  
pp. 674-675
Author(s):  
O. Popoola ◽  
A.H. Heuer ◽  
P. Pirouz
Keyword(s):  
Composite Material ◽  
Ion Beam ◽  
Chemical Properties ◽  
Intermetallic Alloys ◽  
Transmission Electron ◽  
Diamond Polishing ◽  
Al Composite ◽  
The Matrix ◽  
Argon Ion ◽  
And Chemical Properties

The addition of fibres or particles (TiB2, SiC etc.) into TiAl intermetallic alloys could increase their toughness without compromising their good high temperature mechanical and chemical properties. This paper briefly discribes the microstructure developed by a TiAl/TiB2 composite material fabricated with the XD™ process and forged at 960°C.The specimens for transmission electron microscopy (TEM) were prepared in the usual way (i.e. diamond polishing and argon ion beam thinning) and examined on a JEOL 4000EX for microstucture and on a Philips 400T equipped with a SiLi detector for microanalyses.The matrix was predominantly γ (TiAl with L10 structure) and α2(TisAl with DO 19 structure) phases with various morphologies shown in figure 1.

TEM processing procedure for a bacillus organism grown on solid media

1990 ◽  
Vol 48 (3) ◽  
pp. 624-625
Author(s):  
Jane Payne ◽  
Philip Coudron
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Fume Hood ◽  
Vacuum Aspiration ◽  
Solid Media ◽  
Transmission Electron ◽  
Solid Agar ◽  
Processing Procedure ◽  
Agar Media ◽  
Rat Bone

This transmission electron microscopy (TEM) procedure was designed to examine a gram positive spore-forming bacillus in colony on various solid agar media with minimal artifact. Cellular morphology and organization of colonies embedded in Poly/Bed 812 resin (P/B) were studied. It is a modification of procedures used for undecalcified rat bone and Stomatococcus mucilaginosus.Cultures were fixed and processed at room temperature (RT) under a fume hood. Solutions were added with a Pasteur pipet and removed by gentle vacuum aspiration. Other equipment used is shown in Figure 3. Cultures were fixed for 17-18 h in 10-20 ml of RT 2% phosphate buffered glutaraldehyde (422 mosm/KgH2O) within 5 m after removal from the incubator. After 3 (30 m) changes in 0.15 M phosphate buffer (PB = 209-213 mosm/KgH2O, pH 7.39-7.41), colony cut-outs (CCO) were made with a scalpel.

The size of extracellular vesicles secreted by different types of stem cells

2018 ◽  
pp. 38-42
Author(s):  
И.Б. Алчинова ◽  
М.В. Полякова ◽  
И.Н. Сабурина ◽  
М.Ю. Карганов
Keyword(s):  
Stem Cells ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Living Organism ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Study Results ◽  
Multipotent Mesenchymal Stem Cells ◽  
Rat Adipose Tissue ◽  
Almost All

Механизм терапевтического действия мультипотентных мезенхимных стволовых клеток (ММСК) на облученный организм в последнее время вызывает повышенный интерес исследователей. В качестве активного участника паракринного механизма реализации этого эффекта предлагают рассматривать внеклеточные везикулы, секретируемые практически всеми клетками живого организма. Цель работы: выделить и охарактеризовать внеклеточные везикулы, продуцируемые стволовыми клетками различной природы. Материалы и методы. Суспензии внеклеточных везикул, выделенных по модифицированному протоколу дифференциального центрифугирования из культуральных жидкостей от культур ММСК костного мозга человека 2-го пассажа и ММСК жировой ткани крысы 4-го пассажа, были проанализированы методом просвечивающей электронной микроскопии и методом анализа траекторий наночастиц. Результаты. Исследование показало наличие в обоих образцах микрочастиц размерами до и около 100 нм, однако процентное содержание частиц разных размеров в суспензии различалось для двух анализируемых типов клеток. Заключение. Полученные результаты могут свидетельствовать о специфике секреции, обусловленной клеточным типом. A mechanism of the therapeutic effect of multipotent mesenchymal stem cells (MMSC) on irradiated body has recently arisen much interest of researchers. Extracellular vesicles (EVs) secreted by almost all cells of a living organism were suggested to actively contribute to the paracrine mechanism of this effect. The aim of the study was isolation and characterization of extracellular vesicles produced by various types of stem cells. Materials and methods. Suspensions of EVs were isolated from culture media of passage 2 human bone marrow-derived MMSC and passage 4 rat adipose tissue-derived MMSC using a modified protocol of differential centrifugation and then studied using transmission electron microscopy and nanoparticle tracking analysis. Results. The study showed the presence of microparticles with a size of >100 nm in the examined samples. However, the percent content of particles with different sizes in the suspension was different in two analyzed types of cell culture. Conclusion. The study results might reflect a specificity of secretion determined by the cell type.

Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

2020 ◽  
Author(s):  
Dario Brambilla ◽  
Laura Sola ◽  
Elisa Chiodi ◽  
Natasa Zarovni ◽  
Diogo Fortunato ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Disease Diagnosis ◽  
Cell Culture Supernatant ◽  
Transmission Electron ◽  
Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

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