Amphibian skin is a multifunctional organ that plays key roles in defense, breathing, and water balance. In this study, skin secretion samples of the fire salamander (Salamandra salamandra) were separated using RP-HPLC and de novo sequenced using MALDI-TOF MS/MS. Next, we used an in silico platform to screen antioxidant molecules in the framework of density functional theory. One of the identified peptides, salamandrin-I, [M + H]⁺ = 1406.6 Da, was selected for solid-phase synthesis; it showed free radical scavenging activity against DPPH and ABTS radicals. Salamandrin-I did not show antimicrobial activity against Gram-positive and -negative bacteria. In vitro assays using human microglia and red blood cells showed that salamandrin-I has no cytotoxicity up to the concentration of 100 µM. In addition, in vivo toxicity tests on Galleria mellonella larvae resulted in no mortality at 20 and 40 mg/kg. Antioxidant peptides derived from natural sources are increasingly attracting interest. Among several applications, these peptides, such as salamandrin-I, can be used as templates in the design of novel antioxidant molecules that may contribute to devising strategies for more effective control of neurological disease.

Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae–all potential predators of fungi–grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits. Fungi from the genus Paracoccidioides cause paracoccidioidomycosis, a neglected tropical disease that mainly affects poor rural workers in Latin America. Paracoccidioides can live its whole life in the soil, without the need to infect humans or other animals to complete its life cycle. Studies with other such free-living organisms suggest they have acquired the ability to survive and cause disease in humans by interacting with soil predators such as amoebae and worms. In this study we have investigated organisms present in soil in which Paracoccidioides had been previously detected, with a focus on predatory amoebae. Cultures from soil samples showed numerous amoebae, which were isolated and identified using genetic tools. These amoebae were able to ingest and destroy Paracoccidioides yeast cells. More detailed experiments made with a laboratory strain of Acanthamoeba castellanii showed that the interaction with amoebae did increase the fungal ability to survive in and kill not only cells such as amoebae and mouse macrophages, but also whole organisms such as Galleria mellonella larvae and mice. This better understanding of the habitat in which Paracoccidioides lives in nature might lead to new and improved strategies to prevent infection and thus mitigate the burden of this disease.

Candida albicans is one of the agents of invasive candidiasis, a life-threatening disease strongly associated with hospitalization, particularly among patients in intensive care units with central venous catheters. This study aimed to evaluate the synergistic activity of the antifungal peptide ToAP2 combined with fluconazole against C. albicans biofilms grown on various materials. We tested combinations of different concentrations of the peptide ToAP2 with fluconazole on C. albicans biofilms. These biofilms were generated on 96-well plates, intravenous catheters, and infusion tubes in RPMI medium at two maturation stages. Scanning electron microscopy and atomic force microscopy were employed to assess the biofilm structure. We also evaluated the expression of genes previously proven to be involved in C. albicans biofilm formation in planktonic and biofilm cells after treatment with the peptide ToAP2 using qPCR. ToAP2 demonstrated a synergistic effect with fluconazole at concentrations up to 25 µM during both the early and mature stages of biofilm formation in 96-well plates and on medical devices. Combinations of 50, 25, and 12.5 µM of ToAP2 with 52 µM of fluconazole significantly reduced the biofilm viability compared to individual treatments and untreated controls. These results were supported by substantial structural changes in the biofilms observed through both scanning and atomic force microscopy. The gene expression analysis of C. albicans cells treated with 25 µM of ToAP2 revealed a decrease in the expression of genes associated with membrane synthesis, along with an increase in the expression of genes involved in efflux pumps, adhesins, and filamentation. Our results highlight the efficacy of the combined ToAP2 and fluconazole treatment against C. albicans biofilms. This combination not only shows therapeutic potential but also suggests its utility in developing preventive biofilm tools for intravenous catheters.

A qualidade microbiológica do leite cru e do queijo coalho está diretamente relacionada com a produção e comercialização do produto. Esta pesquisa objetivou avaliar as condições higiênico-sanitárias por meio da detecção de microrganismos indicadores de contaminação de leite cru e queijo coalho comercializados em mercados públicos da cidade de Parnaíba, Piauí. Foram coletadas 16 amostras de leite cru e 16 amostras de queijo coalho (8 conservadas por refrigeração e 8 sem conservação) em quatro mercados públicos distintos, no período de abril a outubro de 2012. Na análise do leite cru, 11 das 16 amostras apresentaram números elevados tanto de coliformes totais como termotolerantes. Com relação à análise do queijo coalho, das 16 amostras 12 apresentavam índices elevados de coliformes totais, sendo que 11 estavam em desacordo com os padrões estabelecidos pela lei vigente da ANVISA. A conservação do queijo também foi avaliada como um parâmetro. Os resultados obtidos mostraram que o leite cru e o queijo coalho, comercializados na cidade de Parnaíba, estão sendo manipulados sob condições higiênico-sanitárias insatisfatórias, resultando em um produto final com elevados números de microrganismos indicadores de contaminação, ocasionando um risco para a saúde pública.