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Made available in DSpace on 2020-12-12T02:22:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Skin wound infection requires carefully long-term treatment with an immense financial burden to healthcare systems worldwide. Various strategies such as drug delivery systems using polymer matrix from natural source have been used to enhance wound healing. Natural rubber latex (NRL) from Hevea brasiliensis has shown angiogenic and tissue repair properties. Gentamicin sulfate (GS) is a broad-spectrum antibiotic which inhibits the growth of a wide variety of microorganisms and, because of this, it has also been applied topically for treatment of local infections. The aim of this study was to develop a GS release system using NRL as matrix for Staphylococcus aureus and Escherichia coli infected skin ulcers treatment, without changing drug antibiotic properties. The matrix did not change the GS antimicrobial activity against S. aureus and E. coli strains. Moreover, the NRL-GS biomembrane did not exhibit hemolytic activity, being non-toxic to red blood cells. The eluates of NRL-GS biomembranes and GS solutions did not significantly reduce the survival of Caenorhabditis elegans worms for 24 h at any of the tested concentrations. Thus, these results emphasize that the NRL-GS biomembrane proved to be a promising biomaterial for future studies on the development of dressings for topical uses, inexpensive and practicable, keeping drug antibiotic properties against pathogens and to reduce the side effects. Department of Biotechnology São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biochemistry and Chemical Technology São Paulo State University (UNESP) Institute of Chemistry Department of Biotechnology and Bioprocesses Engineering São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Clinical Analysis São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Clinical Toxicological and Bromatological Analysis São Paulo University (USP) School of Pharmaceutical Sciences Department of Biotechnology São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biochemistry and Chemical Technology São Paulo State University (UNESP) Institute of Chemistry Department of Biotechnology and Bioprocesses Engineering São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Clinical Analysis São Paulo State University (UNESP) School of Pharmaceutical Sciences

Made available in DSpace on 2022-04-29T08:41:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 The number of mammalian animals killed for research purposes has increased significantly in recent years. For this reason, several alternative animal models have been widely used. In this study, two phases of calcium phosphates synthesized by the sol-gel method were added by precipitation to the surface of the natural rubber latex membranes for bone applications. The toxicity eluates from the membrane were evaluated by in vitro assays through cell viability and hemolysis; and by in vivo models using Caenorhabditis elegans worms and zebrafish embryos through survival analysis. Hemolysis levels were less than 3%, which indicates that there was no significant hemolytic activity. It was demonstrated that latex presents enzymes that can dissociate osteoblasts MC3T3, so it was necessary to eliminate these enzymes to obtain a good adhesion and cellular proliferation without morphological alterations. The eluates of membranes did not reduce the survival of Caenorhabditis elegans larvae for 48 h of analysis, except for a hydroxyapatite sample incubated for 5 days. In the zebrafish embryotoxicity assay for 96 hpf of analysis, 60% of embryos survived and there was no evidence of embryos with malformation or developmental delay. In summary, alternative animal models, which are not commonly used to assess biocompatibility, have provided reliable in vivo results that allow suggesting the use of these membranes in the bone biomedical. Department of Biochemistry and Chemical Technology Chemistry Institute UNESP - Univ. Estadual Paulista Department of Chemistry & CESAM Universidade de Aveiro, Campus Universitário de Santiago Department of Biotechnology and Bioprocess Engineering School of Pharmaceutical Sciences UNESP - Univ. Estadual Paulista Department of Clinical Analysis School of Pharmaceutical Sciences UNESP - Univ. Estadual Paulista Area of Exact Sciences and Engineering UCS - University of Caxias do Sul, Rio Grande do Sul Department of Biochemistry and Chemical Technology Chemistry Institute UNESP - Univ. Estadual Paulista Department of Biotechnology and Bioprocess Engineering School of Pharmaceutical Sciences UNESP - Univ. Estadual Paulista Department of Clinical Analysis School of Pharmaceutical Sciences UNESP - Univ. Estadual Paulista

Made available in DSpace on 2022-04-29T08:36:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) In this work we synthesize and characterize AuNPs using two chemical routes: via sodium citrate and via sodium citrate/borohydride. The cytotoxicity of AuNPs was tested by traditional in vitro assays and the in vivo toxicity was evaluated using alternative animals (Galleria mellonella and Caenorhabditis elegans). Avoidance tests with Folsomia candida were carried out to evaluate possible toxic effects in the soil ecosystem. Through Scanning Electron Microscopy and Transmission Electron Microscopy we verified the spherical shape of the AuNPs, with diameters of 34.8 ± 5.5 nm (via sodium citrate) and 7.9 ± 2.2 nm (via sodium citrate/borohydride). The hydrodynamic diameters values were compatible with those obtained microscopically, 30.1 ± 10.1 nm (method A) and 8.3 ± 2.8 nm (method B). Stability of up to 6 months was verified for AuNPs synthesized via sodium citrate, while synthesis via citrate/sodium borohydride was stable for only 1 month. The AuNPs from method A showed better efficacy in the NPs formation with higher absorbance in the plasmonic band at 523 nm, higher concentration and size, spherical shape, homogeneity and stability. The cytotoxicity of the two AuNPs exhibited a slight dose- and size-dependent behavior, with cell viability greater than 80%. The in vivo toxicity in the G. mellonella model showed a survival rate of 100% for 7 days, however, with C. elegans, the percent of survival increased as the concentration of AuNPs decreased. All concentrations of AuNPs, except for the AuNPs synthesized by the citrate pathway (1 mg/kg), showed non-avoidance responses when exposed to contaminated soil. School of Pharmaceutical Sciences Department of Clinical Analysis São Paulo State University (UNESP) Institute of Chemistry Department of Biochemistry & Chemical Technology São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Bioprocesses and Biotechnology Engineering São Paulo State University (UNESP) School of Chemistry and Molecular Biosciences The University of Queensland Postgraduate Program in Agricultural and Natural Ecosystems (PPGEAN) Federal University of Santa Catarina (UFSC) Area of Exact Sciences and Engineering University of Caxias do Sul (UCS) Institute of Exact Sciences Federal University of Southern and Southeastern Pará (UNIFESSPA) School of Pharmaceutical Sciences Department of Clinical Analysis São Paulo State University (UNESP) Institute of Chemistry Department of Biochemistry & Chemical Technology São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Bioprocesses and Biotechnology Engineering São Paulo State University (UNESP) FAPESP: 2016/11836-0 FAPESP: 2017/19603-8 CNPq: 470261/2012-9

Made available in DSpace on 2022-04-29T08:36:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Cryptococcosis is associated with high rates of morbidity and mortality, especially in AIDS patients. Its treatment is carried out by combining amphotericin B and azoles or flucytosine, which causes unavoidable toxicity issues in the host. Thus, the urgency in obtaining new antifungals drives the search for antimicrobial peptides (AMPs). This study aimed to extend the understanding of the mechanism of action of an AMP analog from wasp peptide toxins, MK58911-NH2, on Cryptococcus neoformans. We also evaluated if MK58911-NH2 can act on cryptococcal cells in macrophages, biofilms, and an immersion zebrafish model of infection. Finally, we investigated the structure-antifungal action and the toxicity relationship of MK58911-NH2 fragments and a derivative of this peptide (MH58911-NH2). The results demonstrated that MK58911-NH2 did not alter the fluorescence intensity of the cell wall-binding dye calcofluor white or the capsule-binding dye 18b7 antibody-fluorescein isothiocyanate (FITC) in C. neoformans but rather reduced the number and size of fungal cells. This activity reduced the fungal burden of C. neoformans in both macrophages and zebrafish embryos as well as within biofilms. Three fragments of the MK58911-NH2 peptide showed no activity against Cryptococcus and not toxicity in lung cells. The derivative peptide MH58911-NH2, in which the lysine residues of MK58911-NH2 were replaced by histidines, reduced the activity against extracellular and intracellular C. neoformans. On the other hand, it was active against biofilms and showed reduced toxicity. In summary, these results showed that peptide MK58911-NH2 could be a promising agent against cryptococcosis. This work also opens a perspective for the verification of the antifungal activity of other derivatives. Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP Department of Basic and Applied Biology Institute of Biosciences São Paulo State University-UNESP Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP Department of Basic and Applied Biology Institute of Biosciences São Paulo State University-UNESP

Aim: In this work, mastoparan analog peptides from wasp venom were tested against Candida albicans and safety assays were performed using cell culture and model zebrafish. Materials & methods: Minimal inhibitory concentration was determined and toxicity was performed using human skin keratinocyte and embryo zebrafish. Also, permeation of peptides through embryo chorion was performed. Results: The peptides demonstrated anti- C. albicans activity, with low cytotoxicity and nonteratogenicity in Danio rerio. The compounds had different permeation through chorion, suggesting that this occurs due to modifications in their amino acid sequence. Conclusion: The results showed that the studied peptides can be used as structural study models for novel potential antifungal agents.

The number of animals killed used for research purposes has increased significantly in recent years. For this reason, several alternatives of animal models are widely used. In this study, two phases of calcium phosphates synthesized by the sol-gel method were added by precipitation to the surface of the natural rubber latex membranes for bone applications. Membranes cytotoxicity and toxicity was evaluated by in vitro and in vivo methods. Hemolysis levels were less than 3%, which indicates that there was no significant hemolytic activity. It was demonstrated that latex presents enzymes that can dissociate osteoblasts MC3T3, so it was necessary to eliminate these enzymes to obtain a good adhesion and cellular proliferation without morphological alterations. The in vivo toxicity test with Caenorhabditis elegans worms did not show evidence of toxicity with a sample of hydroxyapatite incubated for 5 days, exhibiting the lowest survival rate of the worms. In the Zebrafish embryotoxicity assay, 60% of embryos survived and there was no evidence of embryos with malformation or developmental delay. In summary, alternative animal models, which are not commonly used to assess biocompatibility, have provided reliable in vivo results that allow suggesting the use of these membranes in the bone biomedical.

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&rsquo, s response could be proposed as new therapeutic and/or diagnostic tools for histoplasmosis.

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.

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.

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.

Made available in DSpace on 2021-06-25T10:37:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-14 The oral cavity is a highly diverse microbial environment in which microorganisms interact with each other, growing as biofilms on biotic and abiotic surfaces. Understanding the interaction among oral microbiota counterparts is pivotal for clarifying the pathogenesis of oral diseases. Candida spp. is one of the most abundant fungi in the oral mycobiome with the ability to cause severe soft tissue lesions under certain conditions. Paracoccidioides spp., the causative agent of paracoccidioidomycosis, may also colonize the oral cavity leading to soft tissue damage. It was hypothesized that both fungi can interact with each other, increasing the growth of the biofilm and its virulence, which in turn can lead to a more aggressive infectivity. Therefore, this study aimed to evaluate the dynamics of mono- and dual-species biofilm growth of Paracoccidioides brasiliensis and Candida albicans and their infectivity using the Galleria mellonella model. Biomass and fungi metabolic activity were determined by the crystal violet and the tetrazolium salt reduction tests (XTT), respectively, and the colony-forming unit (CFU) was obtained by plating. Biofilm structure was characterized by both scanning electronic- and confocal laser scanning- microscopy techniques. Survival analysis of G. mellonella was evaluated to assess infectivity. Our results showed that dual-species biofilm with P. brasiliensis plus C. albicans presented a higher biomass, higher metabolic activity and CFU than their mono-species biofilms. Furthermore, G. mellonella larvae infected with P. brasiliensis plus C. albicans presented a decrease in the survival rate compared to those infected with P. brasiliensis or C. albicans, mainly in the form of biofilms. Our data indicate that P. brasiliensis and C. albicans co-existence is likely to occur on oral mucosal biofilms, as per in vitro and in vivo analysis. These data further widen the knowledge associated with the dynamics of fungal biofilm growth that can potentially lead to the discovery of new therapeutic strategies for these infections. Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP Laboratory of Mycology and Environmental Diagnosis University of Franca Department of Preventive and Community Dentistry Dental School Federal University of Rio Grande do Sul Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP

Skin wound infection requires carefully long-term treatment with an immense financial burden to healthcare systems worldwide. Various strategies such as drug delivery systems using polymer matrix from natural source have been used to enhance wound healing. Natural rubber latex (NRL) from

Aim: Geraniol and linalool are major constituents of the essential oils of medicinal plants. Materials & methods: Antifungal activity of geraniol and linalool were evaluated against five Candida species. The genotoxicity of these compounds was evaluated by the cytokinesis-block micronucleus test, and the embryotoxic assays use zebrafish model. Results: Geraniol and linalool inhibited Candida growth, but geraniol was more effective. The geraniol at concentration of 800 μg/ml and the linalool at concentration of 125 μg/ml significantly increased chromosome damage. Geraniol was more toxic to zebrafish embryo than linalool: LC50 values were 31.3 and 193.3 μg/ml, respectively. Conclusion: Geraniol and linalool have anticandidal activity, but they also exert genotoxic and embryotoxic effects at the highest tested concentrations.

Aim: This work aimed to evaluate the activity of 3′-hydroxychalcone against Cryptococcus gattii in planktonic and biofilm forms and their toxicity using alternative animal models. Materials & methods: Minimum inhibitory concentration and minimum fungicide concentration were determined. Biofilm formation and the susceptibility tests were performed by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]-2H-tetrazolium hydroxide assay. Toxicity and efficacy were checked in Danio rerio and Galleria mellonella models. Results: The compound 3′-hydroxychalcone showed fungicidal activity against C. gattii in both planktonic and biofilm forms. The toxicity in zebrafish embryos revealed a low lethal concentration. In G. mellonella, the compound did not show antifungal activity and larvae toxicity. Conclusion: Because of the activity of 3′-hydroxychalcone against C. gattii in vitro, molecular modifications should be made to improve efficacy and to reduce toxicity in vivo. [Formula: see text]

Aim: Gallic acid and its ester derivatives have shown antifungal activity in vitro. This study was performed to investigate their activity against Candida albicans and their toxicity in the animal models Caenorhabditis elegans and zebrafish embryos. Results: The compounds protected worms from C. albicans infection. The dodecyl gallate was the most effective. In zebrafish embryo, gallic acid and dodecyl gallate were the least toxic. Conclusion: Gallic acid and its ester derivatives have potential for in vivo use against C. albicans infection. The antifungal effects and toxicity of gallate esters in these alternative animal models were dependent on carbon chain length.

Made available in DSpace on 2020-12-12T01:58:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Aim: This study aimed to evaluate the activity of 2′-hydroxychalcone-loaded in nanoemulsion (NLS + 2′chalc), the cytotoxic effect and toxicity against Paracoccidioides brasiliensis and Paracoccidioides lutzii using a zebrafish model. Materials & methods: Preparation and physical-chemical characterization of nanoemulsion (NLS) and NLS + 2′chalc were performed. MIC and minimum fungicide concentration, cytotoxicity and toxicity were also evaluated in the Danio rerio model. Results: NLS + 2′chalc showed fungicidal activity against Paracoccidioides spp. without cytotoxicity in MRC5 and HepG2 lines. It also had high selectivity index values and no toxicity in the zebrafish model based on MIC values. Conclusion: NLS + 2′chalc is a potential new alternative treatment for paracoccidioidomycosis. Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas Araraquara Proteomics Center Mycology Laboratory Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas Araraquara Department of Drugs and Medicines Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas Araraquara Department of Clinical Analysis Cytology Laboratory Institute of Physics of Sao Carlos IFSC/USP Paulista Central University Center UNICEP Universidade Estadual Paulista (UNESP) Instituto de Biociências Letras e Ciências Exatas São José do Rio Preto Department of Chemistry and Environmental Sciences Structural Genomics Consortium Research Institute-UNICAMP Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas Araraquara Proteomics Center Mycology Laboratory Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas Araraquara Department of Drugs and Medicines Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas Araraquara Department of Clinical Analysis Cytology Laboratory Universidade Estadual Paulista (UNESP) Instituto de Biociências Letras e Ciências Exatas São José do Rio Preto Department of Chemistry and Environmental Sciences FAPESP: 2016/05581-0

Made available in DSpace on 2020-12-12T01:51:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-06 Invasive fungal infections, such as cryptococcosis and paracoccidioidomycosis are associated with significant rates of morbidity and mortality. Cryptococcosis, caused by Cryptococcus neoformans, is distributed worldwide and has received much attention as a common complication in patients with HIV. Invasive fungal infections are usually treated with a combination of amphotericin B and azoles. In addition, 5-fluorocytosine (5-FC) is applied in cryptococcosis, specifically to treat central nervous system infection. However, host toxicity, high cost, emerging number of resistant strains, and difficulty in developing new selective antifungals pose challenges. The need for new antifungals has therefore prompted a screen for inhibitory peptides, which have multiple mechanisms of action. The honeycomb moth Galleria mellonella has been widely used as a model system for evaluating efficacy of antifungal agents. In this study, a peptide analog from the mastoparan class of wasps (MK58911) was tested against Cryptococcus spp. and Paracoccidioides spp. In addition, peptide toxicity tests on lung fibroblasts (MRC5) and glioblastoma cells (U87) were performed. Subsequent tests related to drug interaction and mechanism of action were also performed, and efficacy and toxicity of the peptide were evaluated in vivo using the G. mellonella model. Our results reveal promising activity of the peptide, with an MIC in the range of 7.8–31.2 μg/mL, and low toxicity in MRC and U87 cells (IC50 > 500 μg/mL). Taken together, these results demonstrate that MK58911 is highly toxic in fungal cells, but not mammalian cells (SI > 16). The mechanism of toxicity involved disruption of the plasma membrane, leading to death of the fungus mainly by necrosis. In addition, no interaction with the drugs amphotericin B and fluconazole was found either in vitro or in vivo. Finally, the peptide showed no toxic effects on G. mellonella, and significantly enhanced survival rates of larvae infected with C. neoformans. Although not statistically significant, treatment of larvae with all doses of MK58911 showed a similar trend in decreasing the fungal burden of larvae. These effects were independent of any immunomodulatory activity. Overall, these results present a peptide with potential for use as a new antifungal drug to treat systemic mycoses. Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP Department of Biology Center for the Study of Social Insects Institute of Biosciences São Paulo State University-UNESP Department of Clinical Analysis School of Pharmaceutical Sciences São Paulo State University-UNESP Department of Biology Center for the Study of Social Insects Institute of Biosciences São Paulo State University-UNESP

Copyright © Spandidos Publications 2021. All rights reserved., 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., The present study was supported by grants from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Fundação para a Ciência e a Tecnologia (grant no. 345/13), the Conselho Nacional de Pesquisa e Desenvolvimento the Fundação de Amparo à Pesquisa do Estado de São Paulo (grant no. 2014/10446-9) and Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas da UNESP.

Made available in DSpace on 2018-12-11T17:07:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-11-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Faculdade de Ciências Farmacêuticas de Araraquara Universidade Estadual Paulista (UNESP) Departamento de Análises Clínicas Laboratório de Micologia Clínica Instituto de Química Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas de Araraquara Universidade Estadual Paulista (UNESP) Departamento de Análises Clínicas Laboratório de Micologia Clínica Instituto de Química Universidade Estadual Paulista (UNESP) FAPESP: 2013/10917-9 FAPESP: 2014/10446-9 FAPESP: 2015/03700-9 FAPESP: 2015/14023-8

Dimorphic fungi can be found in the yeast form during infection and as hyphae in the environment and are responsible for a large number of infections worldwide. Invertebrate animals have been shown to be convenient models in the study of fungal infections. These models have the advantages of being low cost, have no ethical issues, and an ease of experimentation, time-efficiency, and the possibility of using a large number of animals per experiment compared to mammalian models. Invertebrate animal models such as Galleria mellonella, Caenorhabditis elegans, and Acanthamoeba castellanii have been used to study dimorphic fungal infections in the context of virulence, innate immune response, and the efficacy and toxicity of antifungal agents. In this review, we first summarize the features of these models. In this aspect, the growth temperature, genome sequence, availability of different strains, and body characteristics should be considered in the model choice. Finally, we discuss the contribution and advances of these models, with respect to dimorphic fungi Paracoccidioides spp., Histoplasma capsulatum, Blastomyces dermatitidis, Sporothrix spp., and Talaromyces marneffei (Penicillium marneffei).

Made available in DSpace on 2018-11-26T17:55:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-09-07 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Antimicrobial peptides (AMPs) can be found in various organisms, and could be considered an alternative for pesticides used to control plant pathogens, including those affecting citrus. Brazil is the largest producer and exporter of frozen concentrated orange juice in the world. However, the citrus industry has been affected by several diseases such as citrus canker and huanglongbing (HLB), caused by the bacteria Xanthomonas citri subsp. citri (X. citri) and Candidatus Liberibacter asiaticus (CaLas), respectively. In order to control these pathogens, putative AMPs were prospected in databases containing citrus sequences. Furthermore, AMPs already reported in the literature were also used for in vitro and in vivo assays against X. citri. Since CaLas cannot be cultivated in vitro, surrogates as Sinorhizobium meliloti and Agrobacterium tumefaciens were used. This study reports the evaluation of six AMPs obtained from different sources, two of them from Citrus spp. (citrus-amp1 and citrus-amp2), three from amphibians (Hylin-a1, K-0-W-6-Hy-a1 and Ocellatin 4-analogue) and one from porcine (Tritrpticin). Peptides K-0-W-6-Hy-a1, Ocellatin 4-analogue, and citrus-amp1 showed bactericidal activity against X. citri and S. meliloti and bacteriostatic effect on A. tumefaciens. These results were confirmed for X. citri in planta. In addition cytotoxicity evaluations of these molecules were performed. The AMPs that showed the lowest hemolytic activities were Triptrpticin, citrus-amp1 and citrus-amp2. Citrus-amp1 and citrus-amp2 not presented toxicity in experiments using in vivo model, G. mellonella and U87 MG cells. To verify the interaction of these AMPs with bacteria and erythrocyte cell membranes, vesicles mimicking these cells were built. Citrus-amp1 and Tritrpticin exhibited higher affinity to bacterial membranes, while Ocellatin 4-analogue and Hylin-a1 showed higher affinity to erythrocyte membranes; exclude their use in citrus. This work demonstrates an essential alternative, trough AMPs obtained from Citrus spp., which can be feasibly used to control bacterial pathogens. Inst Agron, Ctr Citricultura Sylvio Moreira, Sao Paulo, Brazil Univ Estadual Campinas, Inst Biol, Sao Paulo, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut, Sao Paulo, Brazil Univ Estadual Paulista, Inst Quim Araraquara, Dept Bioquim & Tecnol Quim, Sao Paulo, Brazil Embrapa Cassava & Fruits, Cruz Das Almas, BA, Brazil Fundo Def Citricultura, FUNDECITRUS, Sao Paulo, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut, Sao Paulo, Brazil Univ Estadual Paulista, Inst Quim Araraquara, Dept Bioquim & Tecnol Quim, Sao Paulo, Brazil FAPESP: 2010/11072-4 FAPESP: 2012/15346-7 FAPESP: 2008/52691-9

Made available in DSpace on 2018-12-11T17:02:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Paracoccidioidomycosis is a systemic mycosis, endemic in Latin America. The etiologic agents of this mycosis are composed of 2 species: Paracoccidioides brasiliensis and P. lutzii. Murine animal models are the gold standard for in vivo studies; however, ethical, economical and logistical considerations limit their use. Galleria mellonella is a suitable model for in vivo studies of fungal infections. In this study, we compared the virulence of P. brasiliensis and P. lutzii in G. mellonella model. The deaths of larvae infected with P. brasiliensis or P. lutzii were similar, and both species were able to reduce the number of hemocytes, which were estimated by microscopy and flow cytometer. Additionally, the phagocytosis percentage was similar for both species, but when we analyze hemocyte-Paracoccidioides spp. interaction using flow cytometer, P. lutzii showed higher interactions with hemocytes. The gene expression of gp43 as well as this protein was higher for P. lutzii, and this expression may contribute to a greater adherence to hemocytes. These results helped us evaluate the behavior of Paracoccidioides spp in G. mellonella, which is a convenient model for investigating the host-Paracoccidioides spp. interaction. © 2015 Taylor & Francis Group, LLC. Departamento de Análises Clínicas e Núcleo de Proteômica Faculdade de Cieˆncias Farmaceˆuticas UNESP-Univ Estadual Paulista Laboratório de Micologia Clínica, Campus Araraquara Departamento de Análises Clínicas e Núcleo de Proteômica Faculdade de Cieˆncias Farmaceˆuticas UNESP-Univ Estadual Paulista Laboratório de Micologia Clínica, Campus Araraquara FAPESP: 2013/10917-9 FAPESP: 2015/03700-9 CNPq: 403586/2012-7

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.

Made available in DSpace on 2018-12-11T17:37:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-25 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, caused by Paracoccidioides spp. A limited number of antifungal agents are available and the search for new compounds has increased. Additionally, nanostructured lipid system (NLS) has emmerged as an interesting strategy to carrier compounds for the treatment of mycosis. In this work, the antifungal efficacy and toxicity of dodecyl gallate (DOD) associated with a NLS was evaluated through in vitro and in vivo tests. DOD showed good in vitro antifungal activity and low toxicity in lung fibroblasts and zebrafish embryos, but no antifungal efficacy in infected mice, which may have been a result of low bioavailability. On the other hand, the association of DOD + NLS was beneficial and resulted in lower toxicity in lung fibroblasts and zebrafish embryos. In addition, NLS + DOD promoted a significant reduction in the fungal burden of mice lungs and could be a potential therapeutic option against PCM. São Paulo State University (UNESP) School of Pharmaceutical Sciences São Paulo State University (UNESP) Institute of Biosciences Letters and Exact Sciences, São José do Rio Preto São Paulo State University (UNESP) School of Pharmaceutical Sciences São Paulo State University (UNESP) Institute of Biosciences Letters and Exact Sciences, São José do Rio Preto FAPESP: 2013/10917-9 FAPESP: 2014/10446-9 FAPESP: 2015/03700-9

Made available in DSpace on 2018-12-11T17:19:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Paracoccidioidomycosis (PCM) is a fungal disease restricted to Latin countries, and its etiologic agents derive from the Paracoccidioides genus. Attenuation or loss of virulence in Paracoccidioidesspp. following successive subculturing has been described. However, virulence can be recovered by passage in mammalian host. In this study, the recovery of adhesion of P. brasiliensis through passage in mice was compared to that in the insect Galleria mellonella. Analysis of in vitro fungal-host cell interaction, gene expression of adhesins, and analysis of the survival curves revealed that Galleria mellonella is useful for the reactivation of P. brasiliensis adhesion. Laboratório de Micologia Clínica Departamento de Análises Clínicas Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas, Rodovia Araraquara-Jaú, Km 1 Laboratório de Micologia Clínica Departamento de Análises Clínicas Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas, Rodovia Araraquara-Jaú, Km 1 FAPESP: 2013/10917-9 FAPESP: 2014/10446-9 FAPESP: 2015/03700-9 FAPESP: 2015/14023-8 FAPESP: 2016/17048-4

Made available in DSpace on 2018-12-11T17:18:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-02-01 Paracoccidioidomycosis is a systemic fungal infection affecting mainly Latin American countries that is caused by Paracoccidioides brasiliensis and Paracoccidioides lutzii. During the study of fungal pathogenesis, in vivo studies are crucial to understand the overall mechanisms involving the infection as well as to search for new therapeutic treatments and diagnosis. Caenorhabditis elegans is described as an infection model for different fungi species and a well-characterized organism to study the innate immune response. This study evaluates C. elegans as an infection model for Paracoccidioides spp. It was observed that both species do not cause infection in C. elegans, as occurs with Candida albicans, and one possible explanation is that the irregular size and shape of Paracoccidioides spp. difficult the ingestion of these fungi by the nematode. Besides this difficulty in the infection, we could observe that the simple exposition of C. elegans to Paracoccidioides species was able to trigger a distinct pattern of expression of antimicrobial peptide genes. The expression of cnc-4, nlpl-27 and nlp-31 was superior after the exposure to P. brasiliensis in comparison to P. lutzii (P < 0.05), and these findings demonstrate important differences regarding innate immune response activation caused by the two species of the Paracoccidioides genus. Laboratório de Micologia Clínica Departamento de Análises Clínicas Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas Unidad de Biología Celular Unidad Funcional de Investigación de Enfermedades Crónicas Instituto de Salud Carlos III Laboratório de Micologia Clínica Departamento de Análises Clínicas Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas

Made available in DSpace on 2018-11-26T17:34:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-06-12 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Programa de Apoio ao Desenvolvimento Cientfico da Faculdade de Ciencias Farmacuticas da UNESP (PADC/ FCF) Dodecyl protocatechuate (dodecyl) is a derivative of protocatechuic acid (3,4dihydroxybenzoic acid) that possesses anti-oxidant and antifungal properties. Nanostructured lipid systems (NLS) can potentiate the action of many antifungal agents, reducing the required dose and side effects by improving their activity. This work aimed to evaluate dodecyl protocatechuate loaded into a NLS (NLS+dodecyl) as a strategy for the treatment of Paracoccidioides brasiliensis and P. lutzii in vitro. Antifungal activity against P. brasiliensis and P. lutzii was evaluated using the microdilution technique. NLS+dodecyl showed high antifungal activity with a minimum inhibitory concentration ranging from 0.06 to 0.03 mu g/mL; 4- to 16-fold higher than that of free dodecyl. NLS+dodecyl was able to inhibit fungal adhesion of the extracellular artificial matrix proteins (laminin and fibronectin), resulting in 82.4 and 81% inhibition, respectively, an increase of 8-17% compared with free dodecyl. These findings corroborate previous results demonstrating 65 and 74% inhibition of fungal adhesion in pulmonary fibroblast cells by dodecyl and NLS+dodecyl, respectively, representing a 9% increase in inhibition for NLS+dodecyl. Subsequently, cytotoxicity was evaluated using the 0.4% sulforhodamine B assay. NLS+dodecyl did not exhibit cytotoxicity in MRC5 (human pneumocyte) and HepG2 (human hepatic carcinoma) cells, thus increasing the selectivity index for NLS+dodecyl. In addition, cytotoxicity was evaluated in vivo using the Caenorhabditis elegans model; neither dodecyl nor NLS+dodecyl exhibited any toxic effects. Taken together, these results suggest that NLS can be used as a strategy to improve the activity of dodecyl against P. brasiliensis and P. lutzii because it improves antifungal activity, increases the inhibition of fungal adhesion in lung cells and the extracellular matrix in vitro, and does not exhibit any toxicity both in vitro and in vivo. Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Clin Anal, Mycol Lab, Araraquara, Brazil Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Clin Anal, Nucleus Prote, Araraquara, Brazil Sao Paulo State Univ, Inst Biosci Humanities & Exact Sci, Dept Chem & Environm Sci, Sao Jose Do Preto, Araraquara, Brazil Sao Paulo State Univ, Inst Chem, Dept Chem, Araraquara, Brazil Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Drugs & Med, Araraquara, Brazil Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Clin Anal, Mycol Lab, Araraquara, Brazil Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Clin Anal, Nucleus Prote, Araraquara, Brazil Sao Paulo State Univ, Inst Biosci Humanities & Exact Sci, Dept Chem & Environm Sci, Sao Jose Do Preto, Araraquara, Brazil Sao Paulo State Univ, Inst Chem, Dept Chem, Araraquara, Brazil Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Drugs & Med, Araraquara, Brazil

Made available in DSpace on 2020-12-12T01:46:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Faculdade de Ciências Farmacêuticas UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Análises Clínicas Laboratório de Micologia Clinica Faculdade de Odontologia UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Fisiologia e Patologia Unidad de Biología Celular y Molecular Corporación para Investigaciones Biológicas (CIB) Faculdade de Ciências Farmacêuticas UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Ciências Biológicas Laboratório de Biologia Molecular e Celular de Microrganismos Grupo de Investigación MICROBA -Escuela de Microbiología Universidad de Antioquia Facultad de Medicina Universidad de Antioquia Instituto Carlos Chagas Fundação Oswaldo Cruz (Fiocruz) Institute of Science and Technology São Paulo State University (UNESP) Faculdade de Ciências Farmacêuticas UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Análises Clínicas Laboratório de Micologia Clinica Faculdade de Odontologia UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Fisiologia e Patologia Faculdade de Ciências Farmacêuticas UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Ciências Biológicas Laboratório de Biologia Molecular e Celular de Microrganismos Institute of Science and Technology São Paulo State University (UNESP)

Made available in DSpace on 2018-12-11T17:27:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-04-01 Denture liners have physical properties that favour plaque accumulation and colonization by Candida species, irritating oral tissues and causing denture stomatitis. To isolate and determine the incidence of oral Candida species in dental prostheses, oral swabs were collected from the dental prostheses of 66 patients. All the strains were screened for their ability to form biofilms; both monospecies and dual-species combinations were tested. Candida albicans (63 %) was the most frequently isolated microorganism; Candida tropicalis (14 %), Candida glabrata (13 %), Candida rugosa (5 %), Candida parapsilosis (3 %), and Candida krusei (2 %) were also detected. The XTT assay showed that C. albicans SC5314 possessed a biofilm-forming ability significantly higher (p < 0.001) than non-albicans Candida strains, after 6 h 37 °C. The total C. albicans CFU from a dual-species biofilm was less than the total CFU of a monospecies C. albicans biofilm. In contrast to the profuse hyphae verified in monospecies C. albicans biofilms, micrographies showed that the C. albicans/non-albicans Candida biofilms consisted of sparse yeast forms and profuse budding yeast cells that generated a network. These results suggested that C. albicans and the tested Candida species could co-exist in biofilms displaying apparent antagonism. The study provide the first description of C. albicans/C. rugosa mixed biofilm. Laboratorio de Pesquisa em Microbiologia Aplicada Universidade de Franca UNIRAN, Av. Dr. Armando Salles de Oliveira, 201, Pq. Universitario Universidade Estadual Paulista Júlio de Mesquita Filho Faculdade de Ciências Farmacêuticas UNESP, Rodovia Ararquara-Jaú, km 1 Universidade Estadual Paulista Júlio de Mesquita Filho Faculdade de Ciências Farmacêuticas UNESP, Rodovia Ararquara-Jaú, km 1

Made available in DSpace on 2018-12-11T17:27:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-02-17 The interaction between the fungal pathogen Paracoccidioides brasiliensis and host cells is usually mediated by specific binding events between adhesins on the fungal surface and receptors on the host extracellular matrix or cell surface. One molecule implicated in the P. brasiliensis-host interaction is the 14-3-3 protein. The 14-3-3 protein belongs to a family of conserved regulatory molecules that are expressed in all eukaryotic cells and are involved in diverse cellular functions. Here, we investigated the relevance of the 14-3-3 protein to the virulence of P. brasiliensis. Using antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated a 14-3-3-silenced strain (expression reduced by ˜55%). This strain allowed us to investigate the interaction between 14-3-3 and the host and to correlate the functions of P. brasiliensis 14-3-3 with cellular features, such as morphological characteristics and virulence, that are important for pathogenesis. UNESP - Univ Estadual Paulista Departamento de Análises Clínicas; Laboratório de Micologia Clínica Unidad de Biología Celular y Molecular Corporación para Investigaciones Biológicas (CIB) - Medellín Escuela de Microbiología Universidad de Antioquia Universidad de Antioquia UNESP - Univ Estadual Paulista Departamento de Análises Clínicas; Laboratório de Micologia Clínica

Paracoccidioides brasiliensis and P. lutzii are etiologic agents of paracoccidioidomycosis (PCM), an important endemic mycosis in Latin America. During its evolution, these fungi have developed characteristics and mechanisms that allow their growth in adverse conditions within their host through which they efficiently cause disease. This process is multi-factorial and involves host–pathogen interactions (adaptation, adhesion, and invasion), as well as fungal virulence and host immune response. In this review, we demonstrated the glycoproteins and polysaccharides network, which composes the cell wall of Paracoccidioides spp. These are important for the change of conidia or mycelial (26°C) to parasitic yeast (37°C). The morphological switch, a mechanism for the pathogen to adapt and thrive inside the host, is obligatory for the establishment of the infection and seems to be related to pathogenicity. For these fungi, one of the most important steps during the interaction with the host is the adhesion. Cell surface proteins called adhesins, responsible for the first contact with host cells, contribute to host colonization and invasion by mediating this process. These fungi also present the capacity to form biofilm and through which they may evade the host’s immune system. During infection, Paracoccidioides spp. can interact with different host cell types and has the ability to modulate the host’s adaptive and/or innate immune response. In addition, it participates and interferes in the coagulation system and phenomena like cytoskeletal rearrangement and apoptosis. In recent years, Paracoccidioides spp. have had their endemic areas expanding in correlation with the expansion of agriculture. In response, several studies were developed to understand the infection using in vitro and in vivo systems, including alternative non-mammal models. Moreover, new advances were made in treating these infections using both well-established and new antifungal agents. These included natural and/or derivate synthetic substances as well as vaccines, peptides, and anti-adhesins sera. Because of all the advances in the PCM study, this review has the objective to summarize all of the recent discoveries on Paracoccidioides-host interaction, with particular emphasis on fungi surface proteins (molecules that play a fundamental role in the adhesion and/or dissemination of the fungi to host-cells), as well as advances in the treatment of PCM with new and well-established antifungal agents and approaches.

A aldosterona participa da progressão das doenças renais em modelos experimentais e ensaios clínicos. Considerando que o modelo de hipertensão renovascular 2 rins-1 clipe (2R-1C) é caracterizado pela atividade elevada do sistema renina-angiotensina-aldosterona, o objetivo desse trabalho foi avaliar o papel da aldosterona na lesão renal presente nesse modelo através do bloqueio do receptor mineralocorticóide (RM) com espironolactona. Ratos Wistar foram submetidos ao procedimento cirúrgico para colocação de um clipe de prata na artéria renal esquerda e após 2 semanas do desenvolvimento da hipertensão renovascular 2R-1C, foram divididos em 3 grupos sendo que para o primeiro grupo nenhuma droga foi administrada (n=8), para o segundo grupo foi administrado por via oral 20 mg/Kg/dia de espironolactona (n=10) e para o terceiro, 7 mg/Kg/dia de amilorida (n=12). O peso e a pressão sistólica foram monitorados semanalmente. Coletas de urina (durante 24 h) e sangue foram realizadas em 3 períodos distintos: antes do procedimento cirúrgico; na 2° semana após a cirurgia (antes do início do tratamento) e na 6° semana após a cirurgia (após o término do tratamento). As amostras foram utilizadas para análise de creatinina, osmolalidade, sódio, potássio e albuminúria. Ao final do experimento, os rins foram perfundidos e pesados. Análise histológica para avaliar a extensão das alterações tubulointersticiais foi realizada. Além disso, marcadores de inflamação (ED-1 e p-JNK), de produção de matriz extracelular (fibronectina), de miofibroblastos (-actina de músculo liso) e de transdiferenciação tubular (vimentina) na região tubulointersticial cortical e de lesão nos podócitos (desmina) foram avaliados. O tratamento com espironolactona foi capaz de atenuar o aumento na excreção de albumina, o aumento na concentração plasmática de creatinina e a redução na depuração de creatinina. No rim clipado dos ratos 2R-1C, as lesões tubulointersticias e podocitárias, demonstradas pelos marcadores estudados, foram discretas e a terapia com espironolactona ou amilorida não foi capaz de minimizá-las. Por outro lado, no rim não clipado, a administração de espironolactona atenuou o aumento de matriz extracelular e a lesão nos podócitos. Os efeitos benéficos da espironolactona ocorreram independentes da redução na pressão sanguínea e podem ser em parte, dependentes do bloqueio do canal epitelial de sódio (ENaC). Tais efeitos trazem perspectiva para espironolactona como uma ferramenta terapêutica a ser explorada em pacientes com estenose de artéria renal. Aldosterone is involved in the progression of renal disease in experimental models and clinical trials. Considering that the model of renovascular hypertension 2 kidney-1 clip (2K-1C) is characterized by a high activity of the renin-angiotensin-aldosterone system, the objective of this study was to assess the role of aldosterone in renal injury in this model by blocking the mineralocorticoid receptor (MR) with spironolactone. Male Wistar rats were submitted to surgery to place a silver clip on the left renal artery and after two weeks of the development of 2K-1C renovascular hypertension they were divided into three groups. The first group was administered no drug (n=8), in the second group spironolactone 20 mg/kg/day was given orally (n=10) and the third group received amiloride 7 mg/kg/day (n=12). Body weight and systolic blood pressure were monitored weekly. Samples of urine (collected for 24 h) and blood were performed in 3 distinct periods: before surgery; 2 weeks after surgery (before treatment) and 6 weeks after surgery (after treatment). The samples were used to analyze creatinine, osmolality, sodium, potassium and albuminuria. At the end of the experiment, the kidneys were perfused and weighed. Histological analysis to assess the extent of tubulointerstitial changes was performed. Additionally, markers of inflammation (ED-1 and p-JNK), production of extracellular matrix (fibronectin), myofibroblasts (-smooth muscle actin) and tubular transdifferentiation (vimentin) in the area of tubulointerstitial cortical and injury in podocytes (desmin) were evaluated. Treatment with spironolactone was able to attenuate the increase in albumin excretion, the increase in plasma concentration of creatinine and the reduction in creatinine clearance. In the clipped kidney, tubulointerstitial and podocytes injuries, demonstrated by markers studied, were discrete and therapy with spironolactone or amiloride was not able to minimize them. However, in the non-clipped kidney, administration of spironolactone attenuated the increase of extracellular matrix and podocyte injury. The beneficial effects of spironolactone occurred independent of the reduction blood pressure and can be partly dependent of epithelial sodium channel (ENaC) blockade. These effects bring perspective to espironolactone as a therapeutic tool to be explored in patients with renal artery stenosis.

Chemical composition of the hexanic fraction, antioxidant activity (DPPH assay) and total phenolic content (Folin-Ciocalteau assay) of aerial parts of sixteen Lippia species were determined. Hexanic fraction was analyzed by gas chromatography-mass spectrometry (GC-MS). The compounds β-myrcene, limonene, γ-terpinene, linalool, β-caryophyllene, germacrene D, α–copaene, β-bourbunene, β-elemene, aloaromadendrene, bicyclogermacrene, and δ-cadinene appeared in the chemical analysis in most of the Lippiahexanic fraction studied. This fraction showed low radical scavenging activity; however, the ethanolic fraction of Lippia species showed higher radical-scavenging activity than the commercial antioxidant butylated hydroxy toluene (BHT). Total phenolic content of the polar fraction, as gallic acid equivalents, ranged from 105.5 mg/g in Lippia pseudo-thea to 255.4 mg/g in Lippia sericea. The presence of phenolic compounds in the ethanolic fraction ofLippia spp. may also be the major cause of its high radical-scavenging activities. Key words: Verbenaceae, gas chromatography-mass spectrometry (GC-MS), phenolic content, antioxidant assay.

The aim of this work was to analyze terpene oil production and terpene synthases (TPS) gene expression from leaves at different developmental stages of different chemotypes of Lippia alba (Mill.) N.E. Br. ex Britton & P. Wilson, Verbenaceae. Hydro-distilled essential oil were used for chemical analysis and gene expression of three monoterpene synthase genes called LaTPS12, LaTPS23 and LaTPS25 were used for analyses of gene expression associated to oil production. The putative genes were associated to TPS-b gene class. Semi-quantitative PCR and quantitative PCR (qPCR) analysis were used to investigate the expression profile of those three putative genes in different leaf stages and different chemotypes. Additionally, total oil production and gene expression of putative TPS genes cloned from L. alba chemotype linalool were evaluated at different stages of leaf development. The expression level of those three genes was higher when the highest oil production was observed, mainly in young leaves at the fourth nodal segment for all evaluated chemotypes. Total oil production was higher at leaves that had unopened trichomes. We also observed that the 1mM of MeJA treatment increased the gene expression in all chemotypes after 24 h elicitation. Made available in DSpace on 2022-09-05T19:20:26Z (GMT). No. of bitstreams: 1 Oil-production-at-different-stages-of-leaf-development-in-Lippia-alba.pdf: 999182 bytes, checksum: ab2936825971b8e4189fb44cbc0f9b70 (MD5) Previous issue date: 2012

A comparison between two extraction approaches of volatiles compounds from six species of Verbenaceae collected at Serra do Cipó, Minas Gerais, Brazil was done. The essential oil and hexanic fraction of leaves from two Lantana and four Lippia species collected in two different seasons were analyzed by GC/MS. Among various identified compounds from both extraction methods the majority of species showed major amounts of β-caryophyllene followed by germacrene D, bicyclogermacrene and α-pinene. Few differences were observed between the composition of essential oil and the hexanic fraction regarding the two studied genera. These results suggest that the analysis of hexanic fraction can be used, as an alternative way, to analyze the volatile compounds of the essential oil. Foi realizada a comparação entre dois métodos de extração dos compostos voláteis obtidos de seis espécies de Verbenaceae, coletadas na Serra do Cipó, Minas Gerais, Brasil. Os óleos essenciais e as frações hexanica obtidos das folhas de duas espécies de Lantana e quatro espécies de Lippia, coletadas em duas diferentes estações do ano, foram analisados por CG/EM. Grande número de constituintes foi identificado nas amostras oriundas dos dois métodos de extração e o componente majoritário para a maior parte das espécies foi o β-cariofileno, seguido pelo germacreno D, biciclogermacreno e α-pineno. Para os dois gêneros estudados, foram observadas pequenas diferenças na composição do óleo essencial e fração hexânica. Esses resultados sugerem que a análise da fração hexânica pode ser usada para identificar os componentes voláteis majoritários dessas espécies, além de ser uma técnica alternativa para a análise dos compostos voláteis presentes no óleo essencial, uma vez que ambos mostraram composição similar.

Antimicrobial peptides (AMPs) are compounds that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. These molecules have become increasingly important as a consequence of remarkable microorganism resistance to common antibiotics. This report shows Escherichia coli expressing the recombinant antimicrobial peptide Pg-AMP1 previously isolated from Psidium guajava seeds. The deduced Pg-AMP1 open reading frame consists in a 168 bp long plus methionine also containing a His6 tag, encoding a predicted 62 amino acid residue peptide with related molecular mass calculated to be 6.98 kDa as a monomer and 13.96 kDa at the dimer form. The recombinant Pg-AMP1 peptide showed inhibitory activity against multiple Gram-negative (E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Staphylococcus epidermides) bacteria. Moreover, theoretical structure analyses were performed in order to understand the functional differences between natural and recombinant Pg-AMP1 forms. Data here reported suggest that Pg-AMP1 is a promising peptide to be used as a biotechnological tool for control of human infectious diseases.