Green Chromatographic Methods and in Vitro Pharmacological Analysis of Varronia curassavica Leaf Derivatives.

Varronia curassavica displays anti-inflammatory, antiulcerogenic, and antioxidant activities. Herein, we employed new UHPLC -UV green chromatographic methods for the analysis of in vitro antioxidant and anti-inflammatory activities of V. curassavica and its embryotoxicity in Zebrafish. Cordialin A, brickellin, and artemetin were purified from the ethanol (EtOH) extract of V. Curassavica leaves and identified using spectrometric techniques. In line with Green Analytical Chemistry principles, the proposed UHPLC methods involve the use of ethanol as organic modifier with low mobile phase consumption, and without sample pretreatment (OLE-UHPLC-UV). The application of the Agree and HPLC-EAT tools for greenness assessment yielded this pattern: HPLC-UV (reference)

Differential miRNA Expression in Human Macrophage-Like Cells Infected with Histoplasma capsulatum Yeasts Cultured in Planktonic and Biofilm Forms.

Histoplasma capsulatum affects healthy and immunocompromised individuals, sometimes causing a severe disease. This fungus has two morphotypes, the mycelial (infective) and the yeast (parasitic) phases. MicroRNAs (miRNAs) are small RNAs involved in the regulation of several cellular processes, and their differential expression has been associated with many disease states. To investigate miRNA expression in host cells during H. capsulatum infection, we studied the changes in the miRNA profiles of differentiated human macrophages infected with yeasts from two fungal strains with different virulence, EH-315 (high virulence) and 60I (low virulence) grown in planktonic cultures, and EH-315 grown in biofilm form. MiRNA profiles were evaluated by means of reverse transcription-quantitative polymerase chain reaction using a commercial human miRNome panel. The target genes of the differentially expressed miRNAs and their corresponding signaling pathways were predicted using bioinformatics analyses. Here, we confirmed biofilm structures were present in the EH-315 culture whose conditions facilitated producing insoluble exopolysaccharide and intracellular polysaccharides. In infected macrophages, bioinformatics analyses revealed especially increased (hsa-miR-99b-3p) or decreased (hsa-miR-342-3p) miRNAs expression levels in response to infection with biofilms or both growth forms of H. capsulatum yeasts, respectively. The results of miRNAs suggested that infection by H. capsulatum can affect important biological pathways of the host cell, targeting two genes: one encoding a protein that is important in the cortical cytoskeleton; the other, a protein involved in the formation of stress granules. Expressed miRNAs in the host's response could be proposed as new therapeutic and/or diagnostic tools for histoplasmosis.

In Vitro and In Vivo Effect of Peptides Derived from 14-3-3 Paracoccidioides spp. Protein.

BACKGROUND: Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from Paracoccidioides brasiliensis 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. METHODS: The in vitro antifungal activity and cytotoxicity of the 14-3-3 peptides were evaluated. The influence of the peptides in immunological and survival aspects was evaluated in vivo, using Galleria mellonella and the expression of antimicrobial peptide genes in Caenorhabditis elegans. RESULTS: None of the peptides were toxic to HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, and only P1 exhibited antifungal activity against Paracoccidioides spp. The peptides could induce an immune response in G. mellonella. Moreover, the peptides caused a delay in the death of Paracoccidioides spp. infected larvae. Regarding C. elegans, the three peptides were able to increase the expression of the antimicrobial peptides. These peptides had essential effects on different aspects of Paracoccidioides spp. infection showing potential for a therapeutic vaccine. Future studies using mammalian methods are necessary to validate our findings.

Down-regulation of TUFM impairs host cell interaction and virulence by Paracoccidioides brasiliensis.

The genus Paracoccidioides consist of dimorphic fungi geographically limited to the subtropical regions of Latin America, which are responsible for causing deep systemic mycosis in humans. However, the molecular mechanisms by which Paracoccidioides spp. causes the disease remain poorly understood. Paracoccidioides spp. harbor genes that encode proteins involved in host cell interaction and mitochondrial function, which together are required for pathogenicity and mediate virulence. Previously, we identified TufM (previously known as EF-Tu) in Paracoccidioides brasiliensis (PbTufM) and suggested that it may be involved in the pathogenicity of this fungus. In this study, we examined the effects of downregulating PbTUFM using a silenced strain with a 55% reduction in PbTUFM expression obtained by antisense-RNA (aRNA) technology. Silencing PbTUFM yielded phenotypic differences, such as altered translation elongation, respiratory defects, increased sensitivity of yeast cells to reactive oxygen stress, survival after macrophage phagocytosis, and reduced interaction with pneumocytes. These results were associated with reduced virulence in Galleria mellonella and murine infection models, emphasizing the importance of PbTufM in the full virulence of P. brasiliensis and its potential as a target for antifungal agents against paracoccidioidomycosis.

Antifungal activity of fluconazole-loaded natural rubber latex against Candida albicans.

AIM: This work aimed to produce a membrane based on fluconazole-loaded natural rubber latex (NRL), and study their interaction, drug release and antifungal susceptibility against Candida albicans. MATERIALS & METHODS: Fluconazole-loaded NRL membrane was obtained by casting method. RESULTS: The Fourier Transform Infrared Spectroscopy showed no modifications either in NRL or fluconazole after the incorporation. Mechanical test presented low Young's modulus and high strain, indicating the membranes have sufficient elasticity for biomedical application. The bio-membrane was able to release the drug and inhibit the growth of C. albicans as demonstrated by disk diffusion and macrodilution assays. CONCLUSION: The biomembrane was able to release fluconazole and inhibit the growth of C. albicans, representing a promising biomaterial for skin application.

Preliminary evaluation of circulating microRNAs as potential biomarkers in paracoccidioidomycosis.

MicroRNAs (miRNAs) are small RNAs (length, 19-24 nucleotides) that regulate gene expression by either mRNA degradation or translational inhibition of proteins. Circulating miRNAs, which are extremely stable and protected from RNAse-mediated degradation in body fluids, have appeared as candidate biomarkers for numerous diseases. However, little is known about circulating miRNAs in fungal infections. Paracoccidioidomycosis (PCM) is caused by the Paracoccidioides species, and is endemic in Central and South America, with predominance in adult male workers from rural areas. The current study aimed to identify a serum miRNA expression profile that could serve as a novel diagnostic biomarker for PCM. Total RNA was isolated and the levels of circulating miRNAs were compared between patients with PCM and healthy control subjects using reverse transcription-quantitative polymerase chain reaction. Bioinformatic analysis was used to evaluate the potential roles of these miRNAs in PCM. Eight miRNAs were differentially expressed in serum samples from patients with PCM. These miRNAs are associated with apoptosis and immune response. The identified miRNAs facilitate with understanding the regulatory mechanisms involved in the host-parasite interaction of PCM. Furthermore, considering that the diagnosis of PCM presents difficulties, these miRNAs may serve as novel biomarkers for this disease.

Synergistic effect of pedalitin and amphotericin B against Cryptococcus neoformans by in vitro and in vivo evaluation.

Cryptococcosis is an opportunistic fungal infection responsible for high morbidity and mortality in immunocompromised patients. Combination of antifungal substances is a promising way to increase the percentage of successful treatment. Pedalitin (PED) is a natural substance obtained from Pterogyne nitens. The aim of this study was to verify the efficacy of PED alone and in combination with amphotericin B (AmB) in vitro and in vivo against Cryptococcus spp. In the in vitro assay, minimum inhibitory concentrations (MICs) of 0.125 mg/L for AmB and 3.9 mg/L for PED were found when the substances were tested alone, whilst in the combination treatment the active concentration of both decreased, with MICs of 0.03 mg/L for AmB and 1 mg/L for PED. In the survival assay, fungal burden study and histopathological assays it was possible to study the efficacy of the substances alone and in combination. The efficacy of combination therapy was considered better than monotherapy as evaluated in a Galleria mellonella model and a murine model. Thus, the combination of PED and AmB is an interesting alternative for anticryptococcal fungal treatment. Moreover, a correlation was observed between the invertebrate and murine models for this antifungal treatment combination.

Evaluation of the efficacy of antifungal drugs against Paracoccidioides brasiliensis and Paracoccidioides lutzii in a Galleria mellonella model.

Paracoccidioides brasiliensis and P. lutzii belong to a group of thermodimorphic fungi and cause paracoccidioidomycosis (PCM), which is a human systemic mycosis endemic in South and Central America. Patients with this mycosis are commonly treated with amphotericin B (AmB) and azoles. The study of fungal virulence and the efficacy and toxicity of antifungal drugs has been successfully performed in a Galleria mellonella infection model. In this work, G. mellonella larvae were infected with two Paracoccidioides spp. and the efficacy and toxicity of AmB and itraconazole were evaluated in this model for the first time. AmB and itraconazole treatments were effective in increasing larval survival and reducing the fungal burden. The fungicidal and fungistatic effects of AmB and itraconazole, respectively, were observed in the model. Furthermore, these effects were independent of changes in haemocyte number. G. mellonella can serve as a rapid model for the screening of new antifungal compounds against Paracoccidioides and can contribute to a reduction in experimental animal numbers in the study of PCM.

Searching new antifungals: The use of in vitro and in vivo methods for evaluation of natural compounds.

In the last decades, the increased number of immunocompromised patients has led to the emergence of many forms of fungal infections. Furthermore, there are a restricted arsenal of antifungals available and an increase in the development of resistance to antifungal drugs. Because of these disadvantages, the search for new antifungal agents in natural sources has increased. The development of these new antifungal drugs involves various steps and methodologies. The evaluation of the in vitro antifungal activity and cytotoxicity are the first steps in the screening. There is also the possibility of antifungal combinations to improve the therapy and reduce toxicity. Despite that, the application of the new antifungal candidate could be used in association with photodynamic therapy or using nanotechnology as an ally. In vivo tests can be performed to evaluate the efficacy and toxicity using conventional and alternative animal models. In this work, we review the methods available for the evaluation of the antifungal activity and safety of natural products, as well as the recent advances of new technology in the application of natural products for antifungal therapy.