Silicone-geranium essential oil blend for long-term antifouling coatings.

This study explores the potential of geranium essential oil as a natural solution for combating marine biofouling, addressing the environmental concerns associated with commercial antifouling coatings. Compounds with bactericidal activities were identified by 13Carbon nuclear magnetic resonance (13C NMR). Thermogravimetric analysis (TGA) revealed minimal impact on film thermal stability, maintaining suitability for antifouling applications. The addition of essential oil induced changes in the morphology of the film and Fourier transform infrared spectroscopy (FTIR) analysis indicated that oil remained within the film. Optical microscopy showed an increase in coating porosity after immersion in a marine environment. A total of 18 bacterial colonies were isolated, with Psychrobacter adeliensis and Shewanella algidipiscicola being the predominant biofilm-forming species. The geranium essential oil-based coating demonstrated the ability to reduce the formation of Psychrobacter adeliensis biofilms and effectively inhibit macrofouling adhesion for a duration of 11 months.

Capsicumicine, a New Bioinspired Peptide from Red Peppers Prevents Staphylococcal Biofilm In Vitro and In Vivo via a Matrix Anti-Assembly Mechanism of Action.

Staphylococci are pathogenic biofilm-forming bacteria and a source of multidrug resistance and/or tolerance causing a broad spectrum of infections. These bacteria are enclosed in a matrix that allows them to colonize medical devices, such as catheters and tissues, and that protects against antibiotics and immune systems. Advances in antibiofilm strategies for targeting this matrix are therefore extremely relevant. Here, we describe the development of the Capsicum pepper bioinspired peptide "capsicumicine." By using microbiological, microscopic, and nuclear magnetic resonance (NMR) approaches, we demonstrate that capsicumicine strongly prevents methicillin-resistant Staphylococcus epidermidis biofilm via an extracellular "matrix anti-assembly" mechanism of action. The results were confirmed in vivo in a translational preclinical model that mimics medical device-related infection. Since capsicumicine is not cytotoxic, it is a promising candidate for complementary treatment of infectious diseases. IMPORTANCE Pathogenic biofilms are a global health care concern, as they can cause extensive antibiotic resistance, morbidity, mortality, and thereby substantial economic loss. So far, no effective treatments targeting the bacteria in biofilms have been developed. Plants are constantly attacked by a wide range of pathogens and have protective factors, such as peptides, to defend themselves. These peptides are common components in Capsicum baccatum (red pepper). Here, we provide insights into an antibiofilm strategy based on the development of capsicumicine, a natural peptide that strongly controls biofilm formation by Staphylococcus epidermidis, the most prevalent pathogen in device-related infections.

Transcriptomic analysis reveals that mTOR pathway can be modulated in macrophage cells by the presence of cryptococcal cells.

Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3ß was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen.

Zrg1, a cryptococcal protein associated with regulation of growth in nutrient deprivation conditions.

Cryptococcus gattii is one of the causes of cryptococcosis, a life-threatening disease generally characterized by pneumonia and/or meningitis. Zinc is an essential element for life, being required for the activity of many proteins with catalytic and structural roles. Here, we characterize ZRG1 (zinc-related gene 1), which codes a product involved in zinc metabolism. Transcriptional profiling revealed that zinc availability regulated the expression of ZRG1, and its null mutants demonstrated impaired growth in zinc- and nitrogen-limiting conditions. Moreover, zrg1 strains displayed alterations in the expression of the zinc homeostasis-related genes ZAP1 and ZIP1. Notably, cryptococcal cells lacking Zrg1 displayed upregulation of autophagy-like phenotypes. Despite no differences were detected in the classical virulence-associated traits; cryptococcal cells lacking ZRG1 displayed decreased capacity for survival inside macrophages and attenuated virulence in an invertebrate model. Together, these results indicate that ZRG1 plays an important role in proper zinc metabolism, and is necessary for cryptococcal fitness and virulence.

A Plumieridine-Rich Fraction From Allamanda polyantha Inhibits Chitinolytic Activity and Exhibits Antifungal Properties Against Cryptococcus neoformans.

Cryptococcosis is a fungal infection caused mainly by the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii. The infection initiates with the inhalation of propagules that are then deposited in the lungs. If not properly treated, cryptococci cells can disseminate and reach the central nervous system. The current recommended treatment for cryptococcosis employs a three-stage regimen, with the administration of amphotericin B, flucytosine and fluconazole. Although effective, these drugs are often unavailable worldwide, can lead to resistance development, and may display toxic effects on the patients. Thus, new drugs for cryptococcosis treatment are needed. Recently, an iridoid named plumieridine was found in Allamanda polyantha seed extract; it exhibited antifungal activity against C. neoformans with a MIC of 250 µg/mL. To address the mode of action of plumieridine, several in silico and in vitro experiments were performed. Through a ligand-based a virtual screening approach, chitinases were identified as potential targets. Confirmatory in vitro assays showed that C. neoformans cell-free supernatant incubated with plumieridine displayed reduced chitinase activity, while chitinolytic activity was not inhibited in the insoluble cell fraction. Additionally, confocal microscopy revealed changes in the distribution of chitooligomers in the cryptococcal cell wall, from a polarized to a diffuse cell pattern state. Remarkably, further assays have shown that plumieridine can also inhibit the chitinolytic activity from the supernatant and cell-free extracts of bacteria, insect and mouse-derived macrophage cells (J774.A1). Together, our results suggest that plumieridine can be a broad-spectrum chitinase inhibitor.

Pseudonajide peptide derived from snake venom alters cell envelope integrity interfering on biofilm formation in Staphylococcus epidermidis.

BACKGROUND: The increase in bacterial resistance phenotype cases is a global health problem. New strategies must be explored by the scientific community in order to create new treatment alternatives. Animal venoms are a good source for antimicrobial peptides (AMPs), which are excellent candidates for new antimicrobial drug development. Cathelicidin-related antimicrobial peptides (CRAMPs) from snake venoms have been studied as a model for the design of new antimicrobial pharmaceuticals against bacterial infections. RESULTS: In this study we present an 11 amino acid-long peptide, named pseudonajide, which is derived from a Pseudonaja textilis venom peptide and has antimicrobial and antibiofilm activity against Staphylococcus epidermidis. Pseudonajide was selected based on the sequence alignments of various snake venom peptides that displayed activity against bacteria. Antibiofilm activity assays with pseudonajide concentrations ranging from 3.12 to 100 µM showed that the lowest concentration to inhibit biofilm formation was 25 µM. Microscopy analysis demonstrated that pseudonajide interacts with the bacterial cell envelope, disrupting the cell walls and membranes, leading to morphological defects in prokaryotes. CONCLUSIONS: Our results suggest that pseudonajide's positives charges interact with negatively charged cell wall components of S. epidermidis, leading to cell damage and inhibiting biofilm formation.

Alterations of zinc homeostasis in response to Cryptococcus neoformans in a murine macrophage cell line.

AIM: To evaluate alterations of zinc homeostasis in macrophages exposed to Cryptococcus neoformans. Materials & methods: Using a fluorescent zinc probe-based flow cytometry and atomic absorption spectrometry, zinc levels were evaluated in J774.A1 cell lines exposed to C. neoformans H99 cells. The transcription profile of macrophage zinc related homeostasis genes - metallothioneins and zinc transporters (ZnTs) of the SLC30 and SLC39 (Zrt-Irt-protein) families - was analyzed by quantitative PCR. RESULTS: Macrophage intracellular labile zinc levels decreased following exposure to C. neoformans. A significant decrease in transcription levels was detected in specific ZnTs from both the Zrt-Irt-protein and ZnT families, especially 24 h after infection. CONCLUSION: These findings suggest that macrophages may exhibit zinc depletion in response to C. neoformans infection.

The Anti-helminthic Compound Mebendazole Has Multiple Antifungal Effects against Cryptococcus neoformans.

Cryptococcus neoformans is the most lethal pathogen of the central nervous system. The gold standard treatment of cryptococcosis, a combination of amphotericin B with 5-fluorocytosine, involves broad toxicity, high costs, low efficacy, and limited worldwide availability. Although the need for new antifungals is clear, drug research and development (R&D) is costly and time-consuming. Thus, drug repurposing is an alternative to R&D and to the currently available tools for treating fungal diseases. Here we screened a collection of compounds approved for use in humans seeking for those with anti-cryptococcal activity. We found that benzimidazoles consist of a broad class of chemicals inhibiting C. neoformans growth. Mebendazole and fenbendazole were the most efficient antifungals showing in vitro fungicidal activity. Since previous studies showed that mebendazole reaches the brain in biologically active concentrations, this compound was selected for further studies. Mebendazole showed antifungal activity against phagocytized C. neoformans, affected cryptococcal biofilms profoundly and caused marked morphological alterations in C. neoformans, including reduction of capsular dimensions. Amphotericin B and mebendazole had additive anti-cryptococcal effects. Mebendazole was also active against the C. neoformans sibling species, C. gattii. To further characterize the effects of the drug a random C. gattii mutant library was screened and indicated that the antifungal activity of mebendazole requires previously unknown cryptococcal targets. Our results indicate that mebendazole is as a promising prototype for the future development of anti-cryptococcal drugs.

Pathogenic diversity amongst serotype C VGIII and VGIV Cryptococcus gattii isolates.

Cryptococcus gattii is one of the causative agents of human cryptococcosis. Highly virulent strains of serotype B C. gattii have been studied in detail, but little information is available on the pathogenic properties of serotype C isolates. In this study, we analyzed pathogenic determinants in three serotype C C. gattii isolates (106.97, ATCC 24066 and WM 779). Isolate ATCC 24066 (molecular type VGIII) differed from isolates WM 779 and 106.97 (both VGIV) in capsule dimensions, expression of CAP genes, chitooligomer distribution, and induction of host chitinase activity. Isolate WM 779 was more efficient than the others in producing pigments and all three isolates had distinct patterns of reactivity with antibodies to glucuronoxylomannan. This great phenotypic diversity reflected in differential pathogenicity. VGIV isolates WM 779 and 106.97 were similar in their ability to cause lethality and produced higher pulmonary fungal burden in a murine model of cryptococcosis, while isolate ATCC 24066 (VGIII) was unable to reach the brain and caused reduced lethality in intranasally infected mice. These results demonstrate a high diversity in the pathogenic potential of isolates of C. gattii belonging to the molecular types VGIII and VGIV.

Effects of zinc transporters on Cryptococcus gattii virulence.

Zinc is an essential nutrient for all living organisms because it is a co-factor of several important proteins. Furthermore, zinc may play an essential role in the infectiousness of microorganisms. Previously, we determined that functional zinc metabolism is associated with Cryptococcus gattii virulence. Here, we characterized the ZIP zinc transporters in this human pathogen. Transcriptional profiling revealed that zinc levels regulated the expression of the ZIP1, ZIP2 and ZIP3 genes, although only the C. gattii zinc transporter Zip1 was required for yeast growth under zinc-limiting conditions. To associate zinc uptake defects with virulence, the most studied cryptococcal virulence factors (i.e., capsule, melanin and growth at 37 °C) were assessed in ZIP mutant strains; however, no differences were detected in these classical virulence-associated traits among the mutant and WT strains. Interestingly, higher levels of reactive oxygen species were detected in the zip1Δ and in the zip1Δ zip2Δ double mutants. In line with these phenotypic alterations, the zip1Δ zip2Δ double mutant displayed attenuated virulence in a murine model of cryptococcosis. Together, these results indicate that adequate zinc uptake is necessary for cryptococcal fitness and virulence.

Zap1 regulates zinc homeostasis and modulates virulence in Cryptococcus gattii.

Zinc homeostasis is essential for fungal growth, as this metal is a critical structural component of several proteins, including transcription factors. The fungal pathogen Cryptococcus gattii obtains zinc from the stringent zinc-limiting milieu of the host during the infection process. To characterize the zinc metabolism in C. gattii and its relationship to fungal virulence, the zinc finger protein Zap1 was functionally characterized. The C. gattii ZAP1 gene is an ortholog of the master regulatory genes zafA and ZAP1 that are found in Aspergillus fumigatus and Saccharomyces cerevisiae, respectively. There is some evidence to support an association between Zap1 and zinc metabolism in C. gattii: (i) ZAP1 expression is highly induced during zinc deprivation, (ii) ZAP1 knockouts demonstrate impaired growth in zinc-limiting conditions, (iii) Zap1 regulates the expression of ZIP zinc transporters and distinct zinc-binding proteins and (iv) Zap1 regulates the labile pool of intracellular zinc. In addition, the deletion of ZAP1 reduces C. gattii virulence in a murine model of cryptococcosis infection. Based on these observations, we postulate that proper zinc metabolism plays a crucial role in cryptococcal virulence.

Tensile bond strength of dual curing resin-based cements to commercially pure titanium.

OBJECTIVES: The aim of this study was to evaluate the tensile bond strength of dual curing luting resin cements to commercially pure titanium at 10 min and 24h after removal of the oxide layer. METHODS: One hundred and twenty titanium discs were obtained by casting and polishing with silicon carbide papers. The titanium discs were sandblasted with 50 microm aluminum oxide, ultrasonic cleaned and bonded in pairs with the resin-based cements Panavia F and Rely X ARC at 10 min and 24h after the sandblasting. The tensile test was performed with a crosshead speed of 0.5mm/min in an Instron Universal testing machine. RESULTS: The Rely X ARC reached the highest tensile strength value at 24h after sandblasting (18.27 MPa), but there was no statistically significant difference between the two dual curing resin cements for both times tested. All specimens showed a mixture of cohesive fracture in the resin cement and adhesive failure. However, the predominant failure mode for Panavia F was cohesive in resin cement, and the Rely X ARC exhibited a greater proportion of specimens with adhesive failure between the alloy and resin luting cement at 10 min and 24h. SIGNIFICANCE: Both cements had, statistically, the same tensile bond strength. But in the fracture mode analysis, the adhesive predominant fracture mode of Rely X ARC cement indicates a premature clinical adhesive failure. On the other hand, the cohesive predominant fracture mode of Panavia F indicates a longer clinical adhesive bond with titanium.

Influência do tratamento superficial na resistência de uniäo dos reparos de resina composta / Influence of superficial treatment on the shear bond strength of composite resin repairs

Este estudo avaliou, in vitro, a resistência de uniäo de seis técnicas diferentes de tratamento superficial em reparos de resina composta. Os maiores valores de resistência de uniäo foram obtidos com o tratamento superficial utilizando-se ácido fosfórico, aplicaçäo de um agente de sinalizaçäo e aplicaçäo do adesivo