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.

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 &lt;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 Cell-Permeable, Small-Molecule Activators of the Insulin-Degrading Enzyme

2012 ◽  
Vol 17 (10) ◽  
pp. 1348-1361 ◽  
Author(s):  
Sayali S. Kukday ◽  
Surya P. Manandhar ◽  
Marissa C. Ludley ◽  
Mary E. Burriss ◽  
Benjamin J. Alper ◽  
...  
Keyword(s):  
Small Molecules ◽  
Biologically Active ◽  
Small Peptides ◽  
Insulin Degrading Enzyme ◽  
Potential Therapeutic Target ◽  
Aβ Peptides ◽  
Degrading Enzyme ◽  
Enzyme Substrate ◽  
A Cell

The insulin-degrading enzyme (IDE) cleaves numerous small peptides, including biologically active hormones and disease-related peptides. The propensity of IDE to degrade neurotoxic Aβ peptides marks IDE as a potential therapeutic target for Alzheimer disease. Using a synthetic reporter based on the yeast a-factor mating pheromone precursor, which is cleaved by multiple IDE orthologs, we identified seven small molecules that stimulate rat IDE activity in vitro. Half-maximal activation of IDE by the compounds is observed in vitro in the range of 43 to 198 µM. All compounds decrease the Km of IDE. Four compounds activate IDE in the presence of the competing substrate insulin, which disproportionately inhibits IDE activity. Two compounds stimulate rat IDE activity in a cell-based assay, indicating that they are cell permeable. The compounds demonstrate specificity for rat IDE since they do not enhance the activities of IDE orthologs, including human IDE, and they appear specific for a-factor–based reporters since they do not enhance rat IDE-mediated cleavage of Aβ-based reporters. Our results suggest that IDE activators function in the context of specific enzyme-substrate pairs, indicating that the choice of substrate must be considered in addition to target validation in IDE activator screens.

scholarly journals Extracellular Vesicles from Aspergillus flavus Induce M1 Polarization In Vitro

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Verônica S. Brauer ◽  
André M. Pessoni ◽  
Tamires A. Bitencourt ◽  
Renato G. de Paula ◽  
Liliana de Oliveira Rocha ◽  
...  
Keyword(s):  
Fungal Infection ◽  
Extracellular Vesicles ◽  
Aspergillus Flavus ◽  
Crucial Role ◽  
Galleria Mellonella ◽  
Biologically Active ◽  
Common Cause ◽  
Content Type ◽  
M1 Macrophage

ABSTRACT Aspergillus flavus, a ubiquitous and saprophytic fungus, is the second most common cause of aspergillosis worldwide. Several mechanisms contribute to the establishment of the fungal infection. Extracellular vesicles (EVs) have been described as “virulence factor delivery bags” in several fungal species, demonstrating a crucial role during the infection. In this study, we evaluated production of A. flavus EVs and their immunomodulatory functions. We verified that A. flavus EVs induce macrophages to produce inflammatory mediators, such as nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1β. Furthermore, the A. flavus EVs enhance phagocytosis and killing by macrophages and induce M1 macrophage polarization in vitro. In addition, a prior inoculation of A. flavus EVs in Galleria mellonella larvae resulted in a protective effect against the fungal infection. Our findings suggest that A. flavus EVs are biologically active and affect the interaction between A. flavus and host immune cells, priming the innate immune system to eliminate the fungal infection. Collectively, our results suggest that A. flavus EVs play a crucial role in aspergillosis. IMPORTANCE Immunocompromised patients are susceptible to several fungal infections. The genus Aspergillus can cause increased morbidity and mortality. Developing new therapies is essential to understand the fungal biology mechanisms. Fungal EVs carry important virulence factors, thus playing pivotal roles in fungal pathophysiology. No study to date has reported EV production by Aspergillus flavus, a fungus considered to be the second most common cause of aspergillosis and relevant food contaminator found worldwide. In this study, we produced A. flavus EVs and evaluated the in vitro immunomodulatory effects of EVs on bone marrow-derived macrophages (BMDMs) and in vivo effects in a Galleria mellonella model.

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.

N-doped graphene quantum dots as a novel highly-efficient matrix for the analysis of perfluoroalkyl sulfonates and other small molecules by MALDI-TOF MS

2017 ◽  
Vol 9 (13) ◽  
pp. 2014-2020 ◽  
Author(s):  
Ziyu Rao ◽  
Fanglan Geng ◽  
Yiqi Zhou ◽  
Dong Cao ◽  
Yuehui Kang
Keyword(s):  
Quantum Dots ◽  
Small Molecules ◽  
Small Molecule ◽  
Graphene Quantum Dots ◽  
Maldi Tof Ms ◽  
Maldi Matrix ◽  
Maldi Tof ◽  
Doped Graphene ◽  
Small Molecule Analysis ◽  
Tof Ms

N-doped graphene quantum dots (N-GQDs) were sythesized by a facile method and applied as a MALDI matrix for small-molecule analysis.

scholarly journals DNA display of fragment pairs as a tool for the discovery of novel biologically active small molecules

2015 ◽  
Vol 6 (1) ◽  
pp. 739-744 ◽  
Author(s):  
J.-P. Daguer ◽  
C. Zambaldo ◽  
M. Ciobanu ◽  
P. Morieux ◽  
S. Barluenga ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Biologically Active ◽  
High Affinity ◽  
Affinity Ligand ◽  
High Affinity Ligand

A focused library for Hsp70 was prepared from fragments identified from an array combinatorially pairing two libraries of small molecule fragments. Screening of the focus library yielded high affinity ligand to Hsp70.

scholarly journals Presoaking dried blood spot with water improves efficiency for small-molecule analysis

BioTechniques ◽  
2019 ◽  
Vol 67 (5) ◽  
pp. 219-228
Author(s):  
You-Rim Lee ◽  
Jiyeong Lee ◽  
Hee-Gyoo Kang
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Extraction Efficiency ◽  
Current Method ◽  
Dried Blood Spots ◽  
Extraction Process ◽  
Precipitation Method ◽  
Modified Method ◽  
Small Molecule Analysis ◽  
Method Group

The current method of extracting small molecules from dried blood spots (DBSs) and liquid blood is similar. However, owing to their different physical characteristics, a modification of the extraction process for DBS is required. We propose a modified method involving presoaking in water that results in better extraction efficiency for small-molecule analysis than the conventional protein precipitation method. Using blood and DBSs from eight subjects, the similarities, recovery rates and extraction efficiencies of both methods were compared. Quantitative analysis showed that seven and six out of ten conditions for the modified method group exhibited almost 100% recovery and extraction efficiency rates, respectively, compared with the conventional method group. Taken together, the results suggest that a presoaking step is needed for efficient DBS analysis.

scholarly journals Identification of Biologically Active, HIV TAR RNA-Binding Small Molecules Using Small Molecule Microarrays

2014 ◽  
Vol 136 (23) ◽  
pp. 8402-8410 ◽  
Author(s):  
Joanna Sztuba-Solinska ◽  
Shilpa R. Shenoy ◽  
Peter Gareiss ◽  
Lauren R. H. Krumpe ◽  
Stuart F. J. Le Grice ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Rna Binding ◽  
Biologically Active ◽  
Tar Rna

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.

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