Past and Contemporaneous Otolith Fingerprints Reveal Potential Anthropogenic Interferences and Allows Refinement of the Population Structure of Isopisthus parvipinnis in the South Brazil Bight.

In this study, otolith geochemical signatures (Element:Ca ratios) were used to investigate the long-term spatial shifts of the population structure of Isopisthus parvipinnis, Bigtooth corvina, an economically and ecologically important Brazilian fish species. Two-hundred and ninety-seven juvenile individuals from historical (1975) and contemporary (2018/2019) samples were collected in five subareas [São Paulo: North-NSP, Center-CSP and South-SSP; Paraná (PR) and Santa Catarina (SC)] of the shallow waters off the coast of the South Brazil Bight were analyzed. The main informative single elements were Co:Ca, Cu:Ca, Li:Ca, Mg:Ca, Mn:Ca, Ni:Ca, Na:Ca, and Rb:Ca. Multivariate analysis showed spatial differences in otolith chemical composition over the years. Samples from 1975 presented an overall low reclassification rate (58%), suggesting the existence of two population units: (1) SP + PR; and (2) SC. However, samples from 2018/2019 discriminated four distinct population units with a good overall reclassification (80%): (1) NSP; (2) CSP; (3) SSP + PR; and (4) SC. This spatial differentiation on the geochemical signatures probably reflects the effects of long-term temporal variability in oceanographic conditions, anthropogenic influence, and climate change on this coastal ecosystem. The data also corroborate and refines the population structure scenario of I. parvipinnis recently described using complementary phenotypic tags.

Associação entre o índice de massa corpórea e presença de afecções dermatológicas em escolares / Association between body mass index and presence of skin disorders in schoolchildren

A obesidade é um distúrbio metabólico crônico, multifatorial e complexo, no qual danos orgânicos podem ser evidenciáveis em curto ou longo prazo, destacando-se as afecções dermatológicas. Com o objetivo de verificar a associação entre o índice de massa corpórea e a presença de afecções dermatológicas em estudantes de uma escola pública do sertão Baiano. Tratou-se de um estudo transversal onde foram avaliados 169 escolares. O desfecho foram as afecções dermatológicas e as variáveis independentes o IMC, idade e sexo. Os dados foram analisados com qui-quadrado e α≤0,05. Cerca de 41 estudantes (24,3%) tinham baixo peso, 92 (54,4%) eutróficos e 36 (21,3%) obesos. Dentre as afecções dermatológicas, a estria se destacou com uma prevalência de 58%, seguida da celulite 26% e acne 24,3%. A faixa etária (14-20 anos) esteve significativamente associada com os valores do IMC (p=0,05). Do mesmo modo que a acantose (p=0,05), estrias, hidradenite e celulite também apresentaram relação significativa com o IMC (p=0,01). Houve significância estatística (p=0,01) quando comparada a presença de até seis dermatoses com os grupos baixo peso, eutrófico e obeso. Determinadas afecções dermatológicas estiveram significativamente associadas ao IMC. É necessário que sejam realizadas ações educacionais de conscientização e prevenção direcionadas para estes jovens que levem a uma redução dos casos de obesidade, tendo em vista que neste grupo as alterações dermatológicas foram mais evidentes.

Obesity is a chronic, multifactorial and complex metabolic disorder, in which organic damage can be evident in the short or long term, especially dermatological disorders. With the objective of verifying the association between the body mass index and the presence of dermatological conditions, students at a public school in the backcountry of Bahia were studied. This was a cross-sectional study in which 169 schoolchildren were evaluated. The outcome was skin conditions, and the independent variables were BMI, age, and sex. Data were analyzed with the chi-squared test and an α≤0.05. About 41 students (24.3%) were underweight, 92 (54.4%) were eutrophic, and 36 (21.3%) were obese. Among the dermatological conditions, stretch marks stood out with a prevalence of 58%, followed by cellulite with 26%, and acne with 24.3%. The age group (14-20 years) was significantly associated with BMI values (p=0.05). Similarly, acanthosis (p=0.05), striae, hidradenitis, and cellulite also showed a significant relationship with BMI (p=0.01). There was statistical significance (p=0.01) when the presence of up to six dermatoses was compared with the low weight, eutrophic, and obese groups. Certain skin conditions were significantly associated with BMI. It is necessary to carry out educational awareness and prevention actions aimed at these young people that lead to a reduction in obesity cases, considering that in this group the dermatological changes were more evident.

Elemental impurities in lipsticks: Results from a survey of the Portuguese and Brazilian markets.

For safety reasons, European regulations prohibit the use of a long list of metal(loid)s as ingredients of cosmetic products. However, their presence as impurities in finished products is virtually unavoidable, even under GMP conditions. This study aimed at determining the elemental profile of lipsticks available in the Portuguese and Brazilian markets. A total of 96 lipsticks were purchased in Brazil (n = 53; 9 brands) and Portugal (n = 43; 7 brands) and the content of 44 elements was determined. Results ranged from <1 µg/g to several tens of µg/g (e.g., Sn, Mn, Zn). Significant differences were found between Portuguese and Brazilian products for several elements, particularly for Pb. For the elements of major toxicological concern (Pb, Cd, As, Sb, Hg), mean values were always below the current limits set by the German competent authority. However, a significant percentage of exceedances were observed for Pb (24%) and Cd (21%). A safety assessment was carried out for the toxicologically relevant elements. Results showed that, except for Pb, the systemic exposure resulting from lipstick use represents less than 0.2% (ca. 3% for Pb) of the respective permitted daily exposure even in the worst-case scenario (i.e., ingestion of the total amount of product applied).

Paracoccidioides spp. catalases and their role in antioxidant defense against host defense responses.

Dimorphic human pathogenic fungi interact with host effector cells resisting their microbicidal mechanisms. Yeast cells are able of surviving within the tough environment of the phagolysosome by expressing an antioxidant defense system that provides protection against host-derived reactive oxygen species (ROS). This includes the production of catalases (CATs). Here we identified and analyzed the role of CAT isoforms in Paracoccidioides, the etiological agent of paracoccidioidomycosis. Firstly, we found that one of these isoforms was absent in the closely related dimorphic pathogen Coccidioides and dermatophytes, but all of them were conserved in Paracoccidioides, Histoplasma and Blastomyces species. We probed the contribution of CATs in Paracoccidioides by determining the gene expression levels of each isoform through quantitative RT-qPCR, in both the yeast and mycelia phases, and during the morphological switch (transition and germination), as well as in response to oxidative agents and during interaction with neutrophils. PbCATP was preferentially expressed in the pathogenic yeast phase, and was associated to the response against exogenous H2O2. Therefore, we created and analyzed the virulence defects of a knockdown strain for this isoform, and found that CATP protects yeast cells from H2O2 generated in vitro and is relevant during lung infection. On the other hand, CATA and CATB seem to contribute to ROS homeostasis in Paracoccidioides cells, during endogenous oxidative stress. CAT isoforms in Paracoccidioides might be coordinately regulated during development and dimorphism, and differentially expressed in response to different stresses to control ROS homeostasis during the infectious process, contributing to the virulence of Paracoccidioides.

Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms.

Alternative oxidase plays an important role in Paracoccidioides brasiliensis cellular homeostasis and morphological transition.

Paracoccidioides brasiliensis is the etiologic agent of one of the most common systemic mycoses in Latin America. As a dimorphic fungus, it must adapt to different environments during its life cycle, either in nature or within the host, enduring external stresses such as temperature or host-induced oxidative stress. In this study we addressed the role of alternative oxidase (PbAOX) in cellular homeostasis during batch culture growth and the morphological transition of P. brasiliensis. Using a PbAOX-antisense-RNA (PbAOX-aRNA) strain with a 70% reduction in gene expression, we show that PbAOX is crucial for maintaining cell viability and vitality during batch culture growth of yeast cells, in what appears to be a pH-dependent manner. We also show that silencing of PbAOX drastically reduced expression levels of other detoxifying enzymes (PbY20 and PbMSOD). In addition, our data indicate that PbAOX plays a role during the morphological transition, namely, during the yeast-to-mycelia germination and mycelia/conidia-to-yeast transition, essential events during the establishment of infection by dimorphic fungal pathogens. Altogether, our findings support the hypothesis that PbAOX is important for the maintenance of cellular homeostasis, possibly by assisting redox balancing during cell growth and the morphological switch of P. brasiliensis.

Involvement of the 90 kDa heat shock protein during adaptation of Paracoccidioides brasiliensis to different environmental conditions.

HSP90 is a molecular chaperone that participates in folding, stabilization, activation, and assembly of several proteins, all of which are key regulators in cell signaling. In dimorphic pathogenic fungi such as Paracoccidioides brasiliensis, the adaptation to a higher temperature, acid pH and oxidative stress, is an essential event for fungal survival and also for the establishing of the infectious process. To further understand the role of this protein, we used antisense RNA technology to generate a P. brasiliensis isolate with reduced PbHSP90 gene expression (PbHSP90-aRNA). Reduced expression of HSP90 decreased yeast cell viability during batch culture growth and increased susceptibility to acid pH environments and imposed oxidative stress. Also, PbHSP90-aRNA yeast cells presented reduced viability upon interaction with macrophages. The findings presented here suggest a protective role for HSP90 during adaptation to hostile environments, one that promotes survival of the fungus during host-pathogen interactions.

Morphological heterogeneity of Paracoccidioides brasiliensis: relevance of the Rho-like GTPase PbCDC42.

Paracoccidioides brasiliensis budding pattern and polymorphic growth were previously shown to be closely linked to the expression of PbCDC42 and to influence the pathogenesis of the fungus. In this work we conducted a detailed morphogenetic evaluation of the yeast-forms of 11 different clinical and environmental P. brasiliensis isolates comprising four phylogenetic lineages (S1, PS2, PS3 and Pb01-like), as well as a PbCDC42 knock-down strain. High variations in the shape and size of mother and bud cells of each isolate were observed but we did not find a characteristic morphologic profile for any of the phylogenetic groups. In all isolates studied, the bud size and shape were demonstrated to be highly dependent on the mother cell. Importantly, we found strong correlations between PbCDC42 expression and both the shape of mother and bud cells and the size of the buds in all isolates and the knock-down strain. Our results suggested that PbCDC42 expression can explain approximately 80% of mother and bud cell shape and 19% of bud cell size. This data support PbCDC42 expression level as being a relevant predictor of P. brasiliensis morphology. Altogether, these findings quantitatively describe the polymorphic nature of the P. brasiliensis yeast form and provide additional support for the key role of PbCDC42 expression on yeast cell morphology.

Gene knockdown in Paracoccidioides brasiliensis using antisense RNA.

Paracoccidioides brasiliensis is a thermal dimorphic fungus which in the host environment exhibits a multinucleated and multibudding yeast form. The cellular and molecular mechanisms underlying these phenotypes remain to be clarified, mostly due to the absence of efficient classical genetic and molecular techniques. Here we describe a method for gene expression knockdown in P. brasiliensis by antisense RNA (aRNA) technology taking advantage of an Agrobacterium tumefaciens-mediated transformation (ATMT) system. Together, these techniques represent a reliable toolbox that can be employed for functional genetic analysis of putative virulence factors and morphogenic regulators, aiming to the identification of new potential drug targets.

The hydrolase PbHAD32 participates in the adherence of Paracoccidioides brasiliensis conidia to epithelial lung cells.

Adherence of the dimorphic pathogenic fungus Paracoccidioides brasiliensis to lung epithelial cells is considered an essential event for the establishment of infection. We have previously shown that the PbHAD32 hydrolase is important in this early stage of the host-P. brasiliensis yeast cells interaction. The aim of this study was to further elucidate the role of PbHAD32 in conidial thermodimorphism and their interaction with lung epithelial cells. Analysis of the PbHAD32 gene expression revealed higher mRNA levels during the conidia to mycelia (C-M) germination when compared to the conidia to yeast (C-Y) transition. Moreover, PbHAD32 was consistently expressed at higher levels upon infection of lung epithelial cells, but to a greater extent when conidia germinated to produce mycelia. Interestingly, at this particular transitional stage, more conidia adhered to epithelial cells than when they were transiting to the yeast form. Altogether our data further corroborates the importance of PbHAD32 during initial adherence to host cells and suggest that the 32-KDa hydrolase may also participate at different stages of the C-M and C-Y conversions.

Alternative oxidase mediates pathogen resistance in Paracoccidioides brasiliensis infection.

BACKGROUND: Paracoccidioides brasiliensis is a human thermal dimorphic pathogenic fungus. Survival of P. brasiliensis inside the host depends on the adaptation of this fungal pathogen to different conditions, namely oxidative stress imposed by immune cells. AIMS AND METHODOLOGY: In this study, we evaluated the role of alternative oxidase (AOX), an enzyme involved in the intracellular redox balancing, during host-P. brasiliensis interaction. We generated a mitotically stable P. brasiliensis AOX (PbAOX) antisense RNA (aRNA) strain with a 70% reduction in gene expression. We evaluated the relevance of PbAOX during interaction of conidia and yeast cells with IFN-γ activated alveolar macrophages and in a mouse model of infection. Additionally, we determined the fungal cell's viability and PbAOX in the presence of H2O2. RESULTS: Interaction with IFN-γ activated alveolar macrophages induced higher levels of PbAOX gene expression in PbWt conidia than PbWt yeast cells. PbAOX-aRNA conidia and yeast cells had decreased viability after interaction with macrophages. Moreover, in a mouse model of infection, we showed that absence of wild-type levels of PbAOX in P. brasiliensis results in a reduced fungal burden in lungs at weeks 8 and 24 post-challenge and an increased survival rate. In the presence of H2O2, we observed that PbWt yeast cells increased PbAOX expression and presented a higher viability in comparison with PbAOX-aRNA yeast cells. CONCLUSIONS: These data further support the hypothesis that PbAOX is important in the fungal defense against oxidative stress imposed by immune cells and is relevant in the virulence of P. brasiliensis.

A 32-kilodalton hydrolase plays an important role in Paracoccidioides brasiliensis adherence to host cells and influences pathogenicity.

One of the most crucial events during infection with the dimorphic fungus Paracoccidioides brasiliensis is adhesion to pulmonary epithelial cells, a pivotal step in the establishment of disease. In this study, we have evaluated the relevance of a 32-kDa protein, a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, in the virulence of this fungus. Protein sequence analyses have supported the inclusion of PbHad32p as a hydrolase and have revealed a conserved protein only among fungal dimorphic and filamentous pathogens that are closely phylogenetically related. To evaluate its role during the host-pathogen interaction, we have generated mitotically stable P. brasiliensis HAD32 (PbHAD32) antisense RNA (aRNA) strains with consistently reduced gene expression. Knockdown of PbHAD32 did not alter cell vitality or viability but induced morphological alterations in yeast cells. Moreover, yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to human epithelial cells and presented decreased virulence in a mouse model of infection. These data support the hypothesis that PbHad32p binds to extracellular matrix (ECM) proteins and modulates the initial immune response for evasion of host defenses. Our findings point to PbHAD32 as a novel virulence factor active during the initial interaction with host cells in P. brasiliensis.