Differentially expressed proteins in the interaction of Paracoccidioides lutzii with human monocytes.

BACKGROUND: Fungi of the genus Paracoccidioides are the etiological agents of paracoccidioidomycosis, a highly prevalent mycosis in Latin America. Infection in humans occurs by the inhalation of conidia, which later revert to the form of yeast. In this context, macrophages are positioned as an important line of defense, assisting in the recognition and presentation of antigens, as well as producing reactive oxygen species that inhibit fungal spreading. AIMS: The objective of this study was to identify differentially expressed proteins during the interaction between Paracoccidioides lutzii Pb01 strain and human U937 monocytes. METHODS: Two-dimensional electrophoresis, combined with mass spectrometry, was used to evaluate the differential proteomic profiles of the fungus P. lutzii (Pb01) interacting with U937 monocytes. RESULTS: It was possible to identify 25 proteins differentially expressed by Pb01 alone and after interacting with U937 monocytes. Most of these proteins are directly associated with fungal metabolism for energy generation, such as glyceraldehyde-3-phosphate dehydrogenase, and intracellular adaptation to monocytes. Antioxidant proteins involved in the response to oxidative stress, such as peroxiredoxin, cytochrome, and peroxidase, were expressed in greater quantity in the interaction with monocytes, suggesting their association with survival mechanisms inside phagocytic cells. We also identified 12 proteins differentially expressed in monocytes before and after the interaction with the fungus; proteins involved in the reorganization of the cytoskeleton, such as vimentin, and proteins involved in the response to oxidative stress, such as glioxalase 1, were identified. CONCLUSIONS: The results of this proteomic study of a P. lutzii isolate are novel, mimicking in vitro what occurs in human infections. In addition, the proteins identified may aid to understand fungal-monocyte interactions and the pathogenesis of paracoccidioidomycosis.

Alternative workflow for COVID-19 diagnosis using direct RT-PCR screening

Objective: COVID-19 is presently the most serious public health concern and diagnosis is a principal tool for controlling and monitoring the spread of the disease. This study aimed to evaluate the efficiency of direct RT-PCR (dRT-PCR) for detection of SARS-CoV-2. Methods: Twenty-seven nasopharyngeal swabs from symptomatic individuals were evaluated. Standard RT-PCR was conducted, and for dRT-PCR the samples were preheated before amplification. Results: Positive agreement was 63.2% and negative agreement was 100%, being moderately in accord. Conclusion: dRT-PCR may be an alternative for screening symptomatic patients and a reliable option during an eventual shortage of viral RNA purification kits.

Objetivo: A COVID-19 é atualmente um sério problema de saúde pública e o diagnóstico é a principal ferramenta para controlar e monitorar a propagação da doença. Este estudo teve como objetivo avaliar a eficiência da RT-PCR direta (dRT-PCR) para detecção do SARS-CoV-2. Métodos: Vinte e sete amostras de swab nasofaríngeo de indivíduos sintomáticos foram avaliados. A RT-PCR padrão foi realizada e para a dRT-PCR as amostras foram pré-aquecidas antes da amplificação. Resultados: A concordância positiva foi de 63,2% e a concordância negativa foi de 100%, sendo moderadamente concordante. Conclusão: A dRT-PCR pode ser uma alternativa para a triagem de pacientes sintomáticos e uma opção confiável durante uma eventual escassez de kits de purificação de RNA viral.

Evaluation of alternative RNA extraction methods for detection of SARS-CoV-2 in nasopharyngeal samples using the recommended CDC primer-probe set.

Background: The efficiency of isolation and purification of the viral genome is a critical step to the accuracy and reliability of RT-qPCR to detect SARS-CoV-2. However, COVID-19 testing laboratories were overwhelmed by a surge in diagnostic demand that affected supply chains especially in low and middle-income facilities. Objectives: Thus, this study compares the performance of alternative methods to extraction and purification of viral RNA in samples of patients diagnosed with COVID-19. Study design: Nasopharyngeal swabs were submitted to three in-house protocols and three commercial methods; viral genome was detected using the primer-probe (N1 and N2) described by CDC and viral load of samples were determined. Results: The in-house protocols resulted in detection of virus in 82.4 to 86.3% of samples and commercial methods in 94.1 to 98%. The disagreement results were observed in samples with low viral load or below the estimated limit of detection of RT-qPCR. Conclusion: The simplified methods proposed might be less reliable for patients with low viral load and alternative commercial methods showed comparable performance.

In Vitro Activity of Lactobacilli with Probiotic Potential Isolated from Cocoa Fermentation against Gardnerella vaginalis.

Study of the probiotic potential of microorganisms isolated from fermented foods has been increasing, especially studies related to lactobacilli. In intestinal models, lactobacilli have demonstrated beneficial properties, such as anti-inflammatory activity and increased antibody production, but the molecular mechanisms involving probiotic and antagonistic action as well as their effect on human vaginal cells have not yet been fully elucidated. The aim of this study was to evaluate the functional and antagonistic properties of three strains of lactobacilli isolated from cocoa fermentation (Lactobacillus fermentum 5.2, L. plantarum 6.2, and L. plantarum 7.1) against Gardnerella vaginalis. Our results show that the lactobacilli have potential use as probiotics, since they have high hydrophobicity and autoaggregation properties and effectively adhere to vaginal cells. Metabolites secreted into the culture medium and whole cells of the strains under study are capable of interfering with the growth of G. vaginalis to different degrees. The elucidation of the antagonistic mechanisms as well as their effect on human cells may be useful in the development of a product containing such microorganisms or products secreted by them.

Analysis of Protein Composition and Bioactivity of Neoponera villosa Venom (Hymenoptera: Formicidae).

Ants cause a series of accidents involving humans. Such accidents generate different reactions in the body, ranging from a mild irritation at the bite site to anaphylactic shock, and these reactions depend on the mechanism of action of the venom. The study of animal venom is a science known as venomics. Through venomics, the composition of the venom of several ant species has already been characterized and their biological activities described. Thus, the aim of this study was to evaluate the protein composition and biological activities (hemolytic and immunostimulatory) of the venom of Neoponera villosa (N. villosa), an ant widely distributed in South America. The protein composition was evaluated by proteomic techniques, such as two-dimensional electrophoresis. To assess the biological activity, hemolysis assay was carried out and cytokines were quantified after exposure of macrophages to the venom. The venom of N. villosa has a profile composed of 145 proteins, including structural and metabolic components (e.g., tubulin and ATPase), allergenic and immunomodulatory proteins (arginine kinase and heat shock proteins (HSPs)), protective proteins of venom (superoxide dismutase (SOD) and catalase) and tissue degradation proteins (hyaluronidase and phospholipase A2). The venom was able to induce hemolysis in human erythrocytes and also induced release of both pro-inflammatory cytokines, as the anti-inflammatory cytokine release by murine macrophages. These results allow better understanding of the composition and complexity of N. villosa venom in the human body, as well as the possible mechanisms of action after the bite.

Detection of Perkinsus marinus in the oyster Crassostrea rhizophorae in southern Bahia by proteomic analysis / Detecção de Perkinsus marinus na ostra Crassostrea rhizophorae do sul da Bahia por análise proteômica

This study reports the presence of the pathogen Perkinsus marinus, notifiable to the World Organization for Animal Health (Office International des Èpizooties = OIE) in the oyster Crassostrea rhizophorae in southern Bahia via proteomic analysis. We analyzed Crassostrea brasiliana from a long-line cultivation system and C. rhizophorae from an adjacent mangrove in Porto do Campo, Camamu Bay, Bahia, Brazil. The collections (n = 100) were performed in October 2012. In the laboratory, the oysters were measured and opened to remove the meat, which was steeped in dry ice. For extraction of proteins, adaptation of a protocol used for mussels was used, after which separation in the first dimension was taken by isoelectric focusing (IEF). The peptides were transferred to a Mass Spectrometer. The obtained spectra were analyzed with the ProteinLynx Global Server 4.2 software tool and also by MASCOT (Matrix Science) and compared to the databases of the SWISSPROT and NCBI, respectively. The identification was evidenced by beta-tubulin, Perkinsus marinus ATCC 50983 and protein homology code in the database NCBI = gi | 294889481. This is the first record of P. marinus in Bahia and the fourth in Brazil.(AU)

Este estudo relata a presença do patógeno Perkinsus marinus, de notificação obrigatória à Organização Internacional de Epizootias (OIE) na ostra Crassostrea rhizophorae no sul da Bahia, via análise proteômica. Foram analisadas as ostras Crassostrea brasiliana de um cultivo em espinhel e C. rhizophorae de um manguezal adjacente, na localidade de Porto do Campo, Baía de Camamu, Bahia. As coletas (n = 100) foram efetuadas em outubro de 2012. Em laboratório, as ostras foram medidas e abertas para a retirada da carne, que foi macerada em gelo seco. Para a extração das proteínas, foi adotada a adaptação de um protocolo utilizado para mexilhões, após o que foi realizada a separação na primeira dimensão, por focalização isoelétrica (IEF). Os peptídeos foram transferidos para um Espectrômetro de Massas. Os espectros obtidos foram analisados no software ProteinLynx Global Server 4.2 e também pela ferramenta MASCOT (Matrix Science) e comparados com os bancos de dados do SWISSPROT e do NCBI, respectivamente. A identificação foi evidenciada por meio da beta-tubulina, homologia Perkinsus marinus ATCC 50983 e código da proteína no banco de dados NCBI = gi|294889481. Este é o primeiro registro de P. marinus na Bahia e o quarto no Brasil.(AU)