Corrected and republished from: "Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence".

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants expected to lack NOP16 expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing potentially altered sugar transport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in G. mellonella. These results connect EV biogenesis, cargo, and cryptococcal virulence.

Comparison of the antifungal activity of the pyrimidine analogs flucytosine and carmofur against human-pathogenic dematiaceous fungi.

Chromoblastomycosis (CBM) and pheohyphomycosis (PHM) are the most common implantation mycoses caused by dematiaceous fungi. In the past, flucytosine (5-FC) has been used to treat CBM, but development of resistance is common. Carmofur belongs to the same class as 5-FC and has in vitro inhibitory activity against the main agents of CBM and PHM. The aim of this study was to compare the action of these two pyrimidine analog drugs against CBM and PHM agents. The minimum inhibitory concentration (MIC) and the selectivity index based on cytotoxicity tests of these two drugs against some agents of these mycoses were determined, with carmofur presenting a higher selectivity index than 5-FC. Carmofur demonstrated here synergistic interactions with itraconazole and amphotericin B against Exophiala heteromorpha, Fonsecaea pedrosoi, Fonsecaea monophora, and Fonsecaea nubica strains. Additionally, carmofur plus itraconazole demonstrated here synergism against a Phialophora verrucosa strain. To evaluate the development of carmofur resistance, passages in culture medium containing subinhibitory concentrations of this pyrimidine analog were carried out, followed by in vitro susceptibility tests. Exophiala dermatitidis quickly developed resistance, whereas F. pedrosoi took seven passages in carmofur-supplemented medium to develop resistance. Moreover, resistance was permanent in E. dermatitidis but transient in F. pedrosoi. Hence, carmofur has exhibited certain advantages, albeit accompanied by limitations such as the development of resistance, which was expected as with 5-FC. This underscores its therapeutic potential in combination with other drugs, emphasizing the need for a meticulous evaluation of its application in the fight against dematiaceous fungi.

A retrospective cohort study of pulp prognosis in luxated permanent teeth: a competing risk analysis.

AIM: The present retrospective cohort study aimed to evaluate luxated permanent teeth for pulp prognosis and risk factors. METHODOLOGY: Case records and radiographs of 224 patients, involving 427 luxated permanent teeth, referred for treatment at the Dental Trauma Center-School of Dentistry, Universidade Federal de Minas Gerais, (DTC-SD-UFMG) from 2014 to 2022, were assessed for pulp prognosis classified as vitality, necrosis and pulp canal obliteration (PCO). A competing risk survival analysis estimated the hazards of the three outcomes, and the effect of demographic, clinical and treatment variables was tested using a cause specific Cox regression model. RESULTS: Pulp vitality was found in163 teeth (38.2%), pulp necrosis in 120 teeth (28.1%) and 55 teeth (12.9%) developed PCO. Pulp vitality decreased in the presence of concomitant crown fractures (HR 0.38 95% CI [0.2-0.8] p = 0.006). The risk of pulp necrosis (HR 0.62 95% CI [0.4-0.96] p = 0.03) was lower in cases with open apices but increased with concomitant crown fractures (HR 4.0 95% CI [2.6-6.1] p = 0.001) and intrusions (HR 2.3 95% CI [1.2-4.1] p = 0.007). Lateral or extrusive luxations (HR 3.0 95% CI [1.3-6.9] p = 0.001) and open apices (HR 2.4 95% CI [1.2-4.7] p = 0.01) showed higher rates of PCO. CONCLUSION: Type of luxation, the diameter of the apical foramen, and the presence of concomitant crown fractures were main determinants of pulp prognosis after luxation injuries in permanent teeth. CLINICAL RELEVANCE: Pulp prognosis after tooth luxation is dependent on the presence and direction of tooth displacement together with infection control. CLINICAL TRIAL REGISTRATION: Not applicable.

A Close Look into the Composition and Functions of Fungal Extracellular Vesicles Produced by Phytopathogens.

Fungal extracellular vesicles (EVs) were first described in human pathogens. In a few years, the field of fungal EVs evolved to include several studies with plant pathogens, in which extracellularly released vesicles play fundamental biological roles. In recent years, solid progress has been made in the determination of the composition of EVs produced by phytopathogens. In addition, EV biomarkers are now known in fungal plant pathogens, and the production of EVs during plant infection has been demonstrated. In this manuscript, we review the recent progress in the field of fungal EVs, with a focus on plant pathogens. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Organoselenium Has a Potent Fungicidal Effect on Cryptococcus neoformans and Inhibits the Virulence Factors.

Cryptococcosis therapy is often limited by toxicity problems, antifungal tolerance, and high costs. Studies approaching chalcogen compounds, especially those containing selenium, have shown promising antifungal activity against pathogenic species. This work aimed to evaluate the in vitro and in vivo antifungal potential of organoselenium compounds against Cryptococcus neoformans. The lead compound LQA_78 had an inhibitory effect on C. neoformans planktonic cells and dispersed cells from mature biofilms at similar concentrations. The fungal growth inhibition led to an increase in budding cells arrested in the G2/M phase, but the compound did not significantly affect structural cell wall components or chitinase activity, an enzyme that regulates the dynamics of the cell wall. The compound also inhibited titan cell (Tc) and enlarged capsule yeast (NcC) growth and reduced the body diameter and capsule thickness associated with increased capsular permeability of both virulent morphotypes. LQA_78 also reduced fungal melanization through laccase activity inhibition. The fungicidal activity was observed at higher concentrations (16 to 64 µg/mL) and may be associated with augmented plasma membrane permeability, ROS production, and loss of mitochondrial membrane potential. While LQA_78 is a nonhemolytic compound, its cytotoxic effects were cell type dependent, exhibiting no toxicity on Galleria mellonella larvae at a dose ≤46.5 mg/kg. LQA_78 treatment of larvae infected with C. neoformans effectively reduced the fungal burden and inhibited virulent morphotype formation. To conclude, LQA_78 displays fungicidal action and inhibits virulence factors of C. neoformans. Our results highlight the potential use of LQA_78 as a lead molecule for developing novel pharmaceuticals for treating cryptococcosis.

Human Exposure to Non-Ionizing Radiation from Indoor Distributed Antenna System: Shopping Mall Measurement Analysis.

It is crucial to monitor the levels of Non-Ionizing Radiation (NIR) to which the general population may be exposed and compare them to the limits defined in the current standards, in view of the rapid rise of communication services and the prospects of a connected society. A high number of people visits shopping malls and since these locations usually have several indoor antennas close to the public, it is therefore a kind of place that must be evaluated. Thus, this work presents measurements of the electric field in a shopping mall located in Natal, Brazil. We proposed a set of six measurement points, following two criteria: places with great the flow of people and the presence of one or more Distributed Antenna System (DAS), co-sited or not with WiFi access points. Results are presented and discussed in terms of the distance to DAS (conditions: near and far) and flow density of people in the mall (scenarios: low and high number of people). The highest peaks of electric field measured were 1.96 and 3.26 V/m, respectively corresponding to 5% and 8% of the limits defined by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) and the Brazilian National Telecommunication Agency (ANATEL).

Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence and Requires the NOP16 Gene.

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants lacking NOP16 expression, we observed that this gene was required for EV production. Analysis of the small molecule composition of EVs produced by wild-type cells and two independent nop16Δ mutants revealed that the deletion of NOP16 resulted not only in a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the nop16Δ mutants were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were coinjected with the nop16Δ cells in G. mellonella. These results reveal a role for NOP16 in EV biogenesis and cargo, and also indicate that the composition of EVs is determinant for cryptococcal virulence.

Current Microscopy Strategies to Image Fungal Vesicles: From the Intracellular Trafficking and Secretion to the Inner Structure of Isolated Vesicles.

Extracellular vesicles (EVs) are nano-sized structures that play important roles in a variety of biological processes among members of the Eukaryota domain. They have been studied since the 1940s and a broader use of different microscopy techniques to image either isolated vesicles or vesicles within the intracellular milieu (trafficking) has been limited by their nanometric size, usually below the resolution limit of most standard light microscopes. The development of genetically encoded fluorescent proteins and fluorescent probes able to switch between "on" and "off" states, as well the improvement in computer-assisted microscopy, photon detector devices, illumination designs, and imaging strategies in the late Twentieth century, boosted the use of light microscopes to provide structural and functional information at the sub-diffraction resolution, taking advantage of a nondestructive analytical probe such light, and opening new possibilities in the study of life at the nanoscale. As well, traditional and novel electron microscopy techniques have been widely used in the characterization of subcellular compartments, either isolated or in situ, providing a comprehensive understanding of their functional role in many cellular processes. Here, we present basic aspects of some of these techniques that have already been applied and their potential application to the study of fungal vesicles.

Interactions of Extracellular Vesicles from Pathogenic Fungi with Innate Leukocytes.

Several studies have shown the immunomodulatory effects of extracellular vesicles (EVs) released by pathogenic fungi. Herein, we discuss the data regarding the immunomodulatory properties of fungal EVs, but also of EVs produced by infected leukocytes. This characterizes a two-way path, in which both host and fungal EVs could coexist and play crucial roles in disease progression or protection in fungal infections. We suggest that EVs can dictate the progress of fungal diseases, and their potential as therapeutic targets.

Biogenesis of Fungal Extracellular Vesicles: What Do We Know?

So far, extracellular vesicles (EVs) have been described in 15 genera of fungi. They carry molecules that contribute to the interaction of fungal cells with the host. Although the number of studies on fungal EVs has increased, the mechanisms involved in their biogenesis are still poorly understood. The current knowledge of EV biogenesis shows us that they can originate both in the cytoplasm and at the plasma membrane. In this chapter, we will focus on these two cellular sites to review what is known about the biogenesis of fungal EVs.

Identification of four compounds from the Pharmakon library with antifungal activity against Candida auris and species of Cryptococcus.

There is an urgent need to develop novel antifungals. In this study, we screened 1600 compounds for antifungal activity against Cryptococcus neoformans and Candida auris. We evaluated 4 promising compounds against 24 additional isolates of Cr. neoformans, Ca. auris, Cr. deuterogattii, and Cr. gattii. The four compounds, dequalinium chloride (DQC), bleomycin sulfate (BMS), pentamidine isethionate salt (PIS), and clioquinol (CLQ), varied in their efficacy against these pathogens but were generally more effective against cryptococci. The compounds exerted their antifungal effect via multiple mechanisms, including interference with the capsule of cryptococci and induction of hyphal-like morphology in Ca. auris. Our results indicate that DQC, BMS, PIS, and CLQ represent potential prototypes for the future development of antifungals. LAY SUMMARY: Fungal infections can be lethal and the options to fight them are scarce. We tested 1600 molecules for their ability to control the growth of two important fungal pathogens, namely Candida auris and species of Cryptococcus. Four of these compounds showed promising antifungal activities.

Article-processing charges as a barrier for science in low-to-medium income regions.

It is widely accepted that science is universal by nature. However, to make science universal, access to research findings is imperative. The open access model of publication of academic articles was established and consolidated during the last two decades. However, most of the open access journals apply article-processing charges (APCs), which can cost more than USD 10,000.00. In regions where support for research is scarce, these funds are usually not available. Similar problems occur in countries with weak economies and, consequently, unfavorable currency conversion rates. This situation reveals a barrier to the alleged universality of science and the access to research findings. In this manuscript, the barriers faced by authors and institutions from low-to-middle income regions to cover APCs and make their science freely available are discussed and illustrated with recent numbers.

Brazilian science: towards extinction?

Brazilian science is under attack. In this manuscript, we will discuss the most recent events that, if not reverted, will make Brazilian science inviable. We urge the scientific community in Brazil and abroad to stand up and resist in defense of more than a century of essential scientific contributions.

Non-Ionizing Radiation Measurements for Trajectography Radars.

This work presents a Non-Ionizing Radiation (NIR) measurement campaign and proposes a specific measurement method for trajectography radars. This kind of radar has a high gain narrow beam antenna and emits a high power signal. Power density measurements from a C-band trajectography radar are carried out using bench equipment and a directional receiving antenna, instead of the commonly used isotropic probe. The measured power density levels are assessed for compliance test via comparison with the occupational and general public exposure limit levels of both the International Commission on Non-Ionizing Radiation Protection (ICNIRP) and the Brazilian National Telecommunication Agency (Anatel). The limit for the occupational public is respected everywhere, evidencing the safe operation of the studied radar. However, the limit for the general public is exceeded at a point next to the radar's antenna, showing that preventive measures are needed.

Integration of heterogeneous photocatalysis (TiO2/UV) and activated sludge system operated in air lift reactor for the treatment of industrial effluent red water.

In the industrial production of the explosive 2,4,6-trinitrotoluene (TNT), purification steps are required to ensure the quality of the product, procedures that generate wastewater of a complex nature and with eco-toxicological potential, called red water, which consists of soluble sulfonates, TNT isomers, and other typical nitro aromatic compounds. The present work aimed to study the effects of integrating heterogeneous photocatalysis based on commercial TiO2, with a biological process, based on activated sludge, for red water treatment. For the photocatalytic treatment, a 72% reduction in the typical absorption of nitro aromatic compounds (the region between 195 - 275 nm), a 36% reduction in chemical oxygen demand (COD), and a 68% reduction in total phenols were obtained. In the biological treatment, there was a 60% reduction in absorbance in the typical nitro aromatics region (NA), 10% reduction in COD, and 36% reduction in total phenols (FT). The integration of photocatalytic and biological treatments showed promising results compared to the individual processes. Having 94% reduction in NA absorbance, 72% reduction in FT, and 89% reduction in COD with an association of photocatalytic pretreatment followed by biological, and reductions of 88% in NA absorbance, 62% in FT, and 87% in COD for a biological pretreatment followed by the photocatalytic process. In general, when comparing the chemical and biological processes, isolated and integrated, both types of integration showed significantly superior results. They were able to remove the main nitro aromatic constituents of the Red Water effluent.

The Benefits of Exporting: Engineered Extracellular Vesicles as Promising Vaccine Candidates against Enteric Fever.

The immunological potential of extracellular vesicles produced by Gram-negative bacteria, the so-called outer-membrane vesicles (OMVs), can be improved by genetic engineering, resulting in vesicles containing multiple immunogens. The potential of this approach for the development of a vaccine candidate for enteric fever was recently demonstrated by G. Gasperini, R. Alfini, V. Arato, F. Mancini, et al. (Infect Immun 89:e00699-20, 2021, https://doi.org/10.1128/IAI.00699-20). This commentary will discuss the use of OMVs to generate vaccines for enteric fever and the promise of this approach for prevention of other infectious diseases.

Monoclonal Antibodies against Cell Wall Chitooligomers as Accessory Tools for the Control of Cryptococcosis.

Therapeutic strategies against systemic mycoses can involve antifungal resistance and significant toxicity. Thus, novel therapeutic approaches to fight fungal infections are urgent. Monoclonal antibodies (MAbs) are promising tools to fight systemic mycoses. In this study, MAbs of the IgM isotype were developed against chitin oligomers. Chitooligomers derive from chitin, an essential component of the fungal cell wall and a promising therapeutic target, as it is not synthesized by humans or animals. Surface plasmon resonance (SPR) assays and cell-binding tests showed that the MAbs recognizing chitooligomers have high affinity and specificity for the chitin derivatives. In vitro tests showed that the chitooligomer MAbs increased the fungicidal capacity of amphotericin B against Cryptococcus neoformans. The chitooligomer-binding MAbs interfered with two essential properties related to cryptococcal pathogenesis: biofilm formation and melanin production. In a murine model of C. neoformans infection, the combined administration of the chitooligomer-binding MAb and subinhibitory doses of amphotericin B promoted disease control. The data obtained in this study support the hypothesis that chitooligomer antibodies have great potential as accessory tools in the control of cryptococcosis.

Antifungal activity of Acylhydrazone derivatives against Sporothrix spp.

Sporotrichosis is an emerging mycosis caused by members of the genus Sporothrix The disease affects humans and animals, particularly cats, which plays an important role in the zoonotic transmission. Feline sporotrichosis treatment options include itraconazole (ITC), potassium iodide and amphotericin B, drugs usually associated with deleterious adverse reactions and refractoriness in cats, especially when using ITC. Thus, affordable, non-toxic and clinically effective anti-Sporothrix agents are needed. Recently, acylhydrazones (AH), molecules targeting vesicular transport and cell cycle progression, exhibited a potent antifungal activity against several fungal species and displayed low toxicity when compared to the current drugs. In this work, the AH derivatives D13 and SB-AF-1002 were tested against Sporothrix schenckii and Sporothrix brasiliensis Minimal inhibitory concentrations of 0.12 - 1 µg/mL were observed for both species in vitro D13 and SB-AF-1002 showed an additive effect with itraconazole. Treatment with D13 promoted yeast disruption with release of intracellular components, as confirmed by transmission electron microscopy of S. brasiliensis exposed to the AH derivatives. AH-treated cells displayed thickening of the cell wall, discontinuity of the cell membrane and an intense cytoplasmic degeneration. In a murine model of sporotrichosis, treatment with AH derivatives was more efficient than ITC, the drug of choice for sporotrichosis. The results of the preliminary clinical study in cats indicate that D13 is safe and has potential to become a therapeutic option for sporotrichosis when associated to ITC. Our results expand the antifungal broadness of AH derivatives and suggest that these drugs could be exploited to combat sporotrichosis.

Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus.

Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum has a complex secretion system that mediates the release of macromolecule-degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non-conventional secretion in both ascomycetes and basidiomycetes. Histoplasma capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In this study, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.

Protective effect of fungal extracellular vesicles against murine candidiasis.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFß, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.