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Mucormycosis is a severe fungal infection that demands immediate and decisive intervention upon suspicion. The causative agents of mucormycosis exhibit inherent resistance to echinocandins and voriconazole, and their in vitro susceptibility to terbinafine is highly variable and species-specific. Considering these factors and the limitations of currently available antifungal therapies, the identification of novel antifungals with potent activity against mucormycosis is of paramount importance. This study aims to identify compounds from the MMV Pathogen Box® presenting antifungal activity against selected mucormycosis agents and to evaluate their potential synergistic effects when combined with antifungal drugs. A screening of the Pathogen Box® compounds was conducted, isolated or in combination with sub-inhibitory concentrations of amphotericin B, isavuconazole or posaconazole, against a Rhizopus oryzae strain. Hits from the screenings were further evaluated against eight Mucoralean strains for minimal inhibitory and fungicidal concentration determinations and to confirm synergistic interactions using the checkerboard method. Ultrastructural studies were performed using scanning electron microscopy. MMV675968 exhibited fungicidal activity against a R. oryzae strain. All but one Rhizopus spp. strains presented MIC ≤ 1 µg/mL, with a geometric mean of 0.78 µg/mL observed across all isolates for this compound, which did not change significantly the cellular structure of this fungus. The combination screening with antifungal drugs revealed six additional compounds potentially active against the R. oryzae strain, two of them demonstrated proven synergism through the checkerboard assay. This first study with the MMV Pathogen Box® and Zigomycetes highlights promising new treatment options for mucormycosis in the future.

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Candida albicans is associated with serious infections in immunocompromised patients. Terpenes are natural-product derivatives, widely studied as antifungal alternatives. In a previous study reported by our group, the antifungal activity of α-pinene against C. albicans was verified; α-pinene presented an MIC between 128-512 µg/mL. In this study, we evaluate time-kill, a mechanism of action using in silico and in vitro tests, anti-biofilm activity against the Candida albicans, and toxicity against human cells (HaCaT). Results from the molecular-docking simulation demonstrated that thymidylate synthase (-52 kcal mol-1), and δ-14-sterol reductase (-44 kcal mol-1) presented the best interactions. Our in vitro results suggest that α-pinene's antifungal activity involves binding to ergosterol in the cellular membrane. In the time-kill assay, the antifungal activity was not time-dependent, and also inhibited biofilm formation, while rupturing up to 88% of existing biofilm. It was non-cytotoxic to human keratinocytes. Our study supports α-pinene as a candidate to treat fungal infections caused by C. albicans.

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Sporotrichosis is a subcutaneous mycosis and is distributed throughout the world, although most cases belong to endemic regions with a warmer climate such as tropical and subtropical areas. The infection occurs mainly by traumatic inoculation of propagules. Similarly, to other organisms, Sporothrix brasiliensis display many biological features that aid in its ability to infect the host, such as extracellular vesicles, bilayered biological structures that provides communication between host cells and between fungi cells themselves. Recently, research on Sporothrix complex have been focused on finding new molecules and components with potential for therapeutic approaches. Here, we study the relationship among EVs and the host's macrophages as well as their role during infection to assess whether these vesicles are helping the fungi or inducing a protective effect on mice during the infection. We found that after cocultivation with different concentrations of purified yeasts EVs from Sb, J774 macrophages displayed an increased fungicidal activity (Phagocytic Index) resulting in lower colony-forming units the more EVs were added, without jeopardizing the viability of the macrophages. Interleukins IL-6, IL-10, and IL-12 were measured during the infection period, showing elevated levels of IL-12 and IL-6 in a dose-dependent manner, but no significant change for IL-10. We also assessed the expression of important molecules in the immune response, such as MHC class II and the immunoglobulin CD86. Both these molecules were overexpressed in Sb yeasts infected mice. Our results indicate that EVs play a protective role during Sporothrix brasiliensis infections.

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Prostaglandin E2 (PGE2) suppresses macrophage effector mechanisms; however, little is known about the function of PGD2 in infected alveolar macrophages (AMs). Using serum-opsonized Histoplasma capsulatum (Ops-H. capsulatum) in vitro, we demonstrated that AMs produced PGE2 and PGD2 in a time-dependent manner, with PGE2 levels exceeding those of PGD2 by 48 h postinfection. Comparison of the effects of both exogenous PGs on AMs revealed that PGD2 increased phagocytosis and killing through the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes receptor, whereas PGE2 had opposite effects, through E prostanoid (EP) receptor 2 (EP2)/EP4-dependent mechanisms. Moreover, PGD2 inhibited phospholipase C-γ (PLC-γ) phosphorylation, reduced IL-10 production, and increased leukotriene B4 receptor expression. In contrast, exogenous PGE2 treatment reduced PLC-γ phosphorylation, p38 and nuclear factor κB activation, TNF-α, H2O2, and leukotriene B4, but increased IL-1ß production. Using specific compounds to inhibit the synthesis of each PG in vitro and in vivo, we found that endogenous PGD2 contributed to fungicidal mechanisms and controlled inflammation, whereas endogenous PGE2 decreased phagocytosis and killing of the fungus and induced inflammation. These findings demonstrate that, although PGD2 acts as an immunostimulatory mediator to control H. capsulatum infection, PGE2 has immunosuppressive effects, and the balance between these two PGs may limit collateral immune damage at the expense of microbial containment.

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ABSTRACT In order to reduce the excessive reliance on the toxic chemical fungicides, the present study aimed to isolate the total potato glycoalkaloids (TPAs), and the two steroidal alkaloids α-chaconine and α-solanine from potatoes, Solanum tuberosum L. Their structures were characterized using physical and spectroscopic methods including (UV, IR, 1H, 13C--NMR, 2D 1H-1H COSY, HMBC and NOESY). Silver nanoparticles (AgNPs) were prepared from potato alkaloids through a green synthesis approach. Potato alkaloids and their nanoparticles inhibited mycelial growth of the phytopathogenic fungi Alternaria alternate, Rhizoctonia solani, Botrytis cinerea and Fusarium oxysporum f. sp. lycopersici with low minimal inhibitory and minimal fungicidal concentrations. R. solani was the most susceptible, while F. oxysporum was the most resistant. TPAs was the most fungitoxic (EC50's were 19.8, 22.5, 26.5 and 32.3 µg/ml against R. solani, A. alternate, B. cinerea and F. oxysporum respectively). A mixture of α-solanine and α-chaconine (1:1) showed a marked antifungal activity. AgNPs (size 39.5-80.3 diameter) from alkaloids showed improved fungitoxic activity (EC50's of TPAs nanoparticles ranged between 10.9 and 16.1 µg/ml). Alkaloids exhibited no or a slight phytotoxicity against wheat and radish. Results recommend the potential of using potato alkaloids and their nanoparticles as biorational alternatives to conventional fungicides.

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Humic acids (HAs) are macromolecules of undefined compositions that vary with origin, the process by which they are obtained and functional groups present in their structure, such as quinones, phenols, and carboxylic acids. In addition to agriculture, there is an increased interest in HAs due to their important pharmacological effects. However, HAs are not readily soluble in water at physiological pH, which may limit their bioavailability. Although primary aggregation forms non-uniform pseudo-micelles, the presence of ionisable groups in the HA molecule makes pH an environmental stimulus for controlled aggregation and precipitation. The aim of this work was to induce HA deprotonation and protonation, without compromising their colloidal dispersion, by means of pH changes as a strategy to produce nanoparticles. Deprotonation and protonation were achieved by treating HAs with sodium hydroxide and acetic acid, respectively, at various concentrations. Non pH-treated HAs at the same concentrations were used as control. The evolution of the treatments was monitored by pH changes in bulk solutions as a function of time. At equilibrium, the conformation of the colloidal structures was characterised by the predominant mean diameter, polydispersity index and absorbance of the solutions. The zeta potential was also measured in protonation assays. Moreover, the fungicidal activity of the nanoparticles was evaluated on the mycelial growth of three fungal genera. The results showed the pH decrease or increment as a function of the balance between hydroxyl and carboxyl groups and of the diffusion rate inside the structures. Deprotonation followed by protonation produced nanosized (100-200nm), electrostatically stable (-30mV) and pH-responsive particles with a polydispersity index <0.5. The protonated nanoparticles significantly inhibited (P≤0.05) the mycelial growth of Candida albicans in vitro, when compared with control, and the fungicidal activity was dose-dependent. No activity was observed for the deprotonated HAs nanoparticles. These results show that deprotonation followed by protonation is an easy and useful strategy for the controlled production of HA nanoparticles, which exhibit a tendency to elicit fungicidal effects, with potential to develop new classes of cosmetics and pharmaceuticals.

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The development of antibacterial and antifungal drugs has been the target of several pharmaceutical and chemical industries mainly due to the lack of effective drugs with low or no side effect. In this work, studies were conducted both in vitro and in vivo with 8-oxyquinolinepropoxycalix[4]arene (A) and 5-Cl-8-oxyquinolinepropoxycalix[4]arene (B) ligands, showing fairly good results. Cytotoxicity and fungicidal actions of compounds A and B were determined in Wistar male rats and peritoneal macrophages of mice. A slight change in the total of leukocytes and erythrocytes was observed on the hematologic assays, showing almost no inflammation after using both compounds in Wistar male rats. We have also noted some, but not significant, alteration in liver enzymes representing modest hepatotoxicity. Cytotoxicity of peritoneal macrophages, in the presence of compound A or B, showed 50% of survival of macrophages. On the other hand, macrophages previously infected with Candida albicans and treated with substance A or B exhibited an increased cytokine IL-10 at 24h incubation. By checking the effect of substance A or B on growing C. albicans, the results pointed that these substances revealed antifungal activity against C. albicans, in 24h culture with a reduction of yeast cells.

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Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

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The objective of present paper was to determine the antifungal activity of the Eucalyptus tereticornis (Myrtaceae) essential oil and two fractions on the Fusarium oxysporum mushroom, a pathogen with clinical and agricultural significance. The total citronelal (44.8 percent) and geraniol (9.78 percent) essential oil had a fungicidal effect at a 3 g/L concentration and a fungicidal activity at small concentrations. The A and B fractions composed most of p-mentane-3,8-diol (18.95 percent) and geraniol acetate (24.34 percent), respectively were more active than the total extract. The observations at microscopic level showed damages and changes in hyphae and chlamydospores, as well as a decrease in the number of conidia. The observed fungicidal activity and the morphologic damages were dependent on the concentration.

El objetivo de este trabajo fue determinar la actividad antifúngica del aceite esencial de Eucalyptus tereticornis (Myrtaceae) y 2 fracciones sobre el hongo Fusarium oxysporum, patógeno de importancia tanto clínica como agrícola. El aceite esencial total, compuesto principalmente por citronelal (44,8 por ciento), citronelol (9,78 por ciento) presentó un efecto fungicida a una concentración de 3 g/L y actividad fungistática a concentraciones menores. La fracciones A y B compuestas en su mayoría por p-mentano-3,8-diol (18,95 por ciento) y acetato de citronelol (24,34 por ciento) respectivamente fueron más activas que el extracto total. Las observaciones a nivel microscópico mostraron daños y cambios en hifas y clamidosporas, así como disminución en el número de conidias. La actividad fungistática observada y los daños morfológicos fueron dependientes de la concentración.

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The chemical composition of the essential oil from the leaves of Pelargonium odoratissimum (L.) L'Hér., Geraniaceae, was determined and the antimicrobial activities against the Aspergillus flavus CML 1816, Aspergillus carbonarius CML1815 and Aspergillus parasiticus CMLA 817 fungi, as well the Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25 992 bacteria were evaluated. The essential oil was isolated by steam distillation using a modified Clevenger apparatus, and its constituents were identified and quantified by GC/MS and GC-FID analyses. In vitro bioanalytical testing was performed using a completely randomized design. The concentrations of essential oil employed ranged from 0.1 to 2 μL.mL-1 (in dimethyl sulfoxide) for the fungus species and from 1 to 500 μL.mL-1 for the bacteria. The diameters of the inhibition zones formed for bacteria and the mean diameters of mycelial growth in perpendicular directions for fungi were measured, followed by calculation of the percentage of inhibition. The essential oil from the leaves of P. odoratissimum furnished methyleugenol (96.80 percent), a phenylpropanoid. This essential oil inhibited the growth of fungi (100 percent inhibition) and exhibited a small effect on the bacteria at the concentrations tested.

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The reported effects of different families of fatty acids (FA; SFA, MUFA, n-3 and n-6 PUFA) on human health and the importance of macrophagerespiratory burst and cytokine release to immune defence led us to examine the influence of palmitic acid (PA), oleic acid (OA),linoleic acid, arachidonic acid, EPA and DHA on macrophage function. We determined fungicidal activity, reactive oxygen species(ROS) and cytokine production after the treatment of J774 cells with non-toxic concentrations of the FA. PA had a late and discrete stimulatingeffect on ROS production, which may be associated with the reduced fungicidal activity of the cells after treatment with this FA.OA presented a sustained stimulatory effect on ROS production and increased fungicidal activity of the cells, suggesting that enrichmentof diets with OA may be beneficial for pathogen elimination. The effects of PUFA on ROS production were time- and dose-dependentlyregulated, with no evident differences between n-3 and n-6 PUFA. It was worth noting that most changes induced after stimulation of thecells with lipopolysaccharide were suppressed by the FA. The present results suggest that supplementation of the diet with specific FA, notclasses of FA, might enable an improvement in host defence mechanisms or a reduction in adverse immunological reactions.

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Interleukin (IL)-15 is a pleiotropic cytokine that regulates the proliferation and survival of many cell types. IL-15 is produced by monocytes and macrophages against infectious agents and plays a pivotal role in innate and adaptive immune responses. This study analyzed the effect of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with Paracoccidioides brasiliensis (Pb18), the agent of paracoccidioidomycosis. Peripheral blood monocytes were pre-incubated with IL-15 and then challenged with Pb18. Fungicidal activity was assessed by viable fungi recovery from cultures after plating on brain-heart infusion-agar. Superoxide anion (O2-), hydrogen peroxide (H2O2), tumour necrosis factor-alpha (TNF-α), IL-6, IL-15 and IL-10 production by monocytes were also determined. IL-15 enhanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by addition of anti-IL-15 monoclonal antibody. A significant stimulatory effect of IL-15 on O2- and H2O2 release suggests that fungicidal activity was dependent on the activation of oxidative metabolism. Pre-treatment of monocytes with IL-15 induced significantly higher levels of TNF-α, IL-10 and IL-15 production by cells challenged with the fungus. These results suggest a modulatory effect of IL-15 on pro and anti-inflammatory cytokine production, oxidative metabolism and fungicidal activity of monocytes during Pb18 infection.

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The chemical composition of ethanol extracts from samples of Brazilian propolis (EEPs) determined by HPLC and their activity against Trypanosoma cruzi, Staphylococcus aureus, Streptococcus pneumoniae, Klebisiella pneumoniae, Candida albicans, Sporothrix schenckii and Paracoccidioides brasiliensis were determined. Based on the predominant botanical origin in the region of samples' collection, the 10 extracts were separated into three groups: A (B. dracunculifolia + Auraucaria spp), B (B. dracunculifolia) and C (Araucaria spp). Analysis by the multiple regression of all the extracts together showed a positive correlation, higher concentrations leading to higher biological effect, of S. aureus with p-coumaric acid (PCUM) and 3-(4-hydroxy-3-(oxo-butenyl)-phenylacrylic acid (DHCA1) and of trypomastigotes of T. cruzi with 3,5-diprenyl-4-hydroxycinnamic acid derivative 4 (DHCA4) and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran (DCBEN). When the same approach was employed for each group, due to the small number of observations, the statistical test gave unreliable results. However, an overall analysis revealed for group A an association of S. aureus with caffeic acid (CAF) and dicaffeoylquinic acid 3 (CAFQ3), of S. pneumoniae with CAFQ3 and monocaffeoylquinic acid 2 (CAFQ2) and of T. cruzi also with CAFQ3. For group B, a higher activity against S. pneumoniae was associated DCBEN and for T. cruzi with CAF. For group C no association was observed between the anitmicrobial effect and any component of the extracts. The present study reinforces the relevance of PCUM and derivatives, especially prenylated ones and also of caffeolyquinic acids, on the biological activity of Brazilian propolis.