Neurological aspects of HTLV-1 infection: symptoms in apparently asymptomatic carriers.

Human T-lymphotropic virus type 1 (HTLV-1) is classically associated with the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), although the mechanisms of this neurological disorder remain unclear. In addition, some patients who develop "minor" neurological signs that do not meet diagnostic criteria for HAM/TSP are classified as asymptomatic carriers. This study aims to demonstrate the neurological symptoms of Brazilian patients living with HTLV-1 classified as not-HAM.TSP. This observational study evaluated patients treated in an HTLV reference center in Bahia, Brazil, between February 2022 and July 2023. The data were obtained through the analysis of medical records and neurological consultation. Those individuals classified as HAM/ TSP were excluded from this study. 74 patients were submitted to a careful neurological evaluation: 23 HAM/TSP, 22 were classified with intermediate syndrome (IS), and 29 were oligosymptomatic. Self-reported symptoms were significantly more common in the IS group, including urinary symptoms such as nocturia, urgency, incontinence, dysuria, weakness, paresthesia, lumbar pain, xerostomia, and xerophthalmia. Physical examination findings consistent with reduced vibratory and tactile sensitivity were more common in the IS group (p = 0.017 and p = 0.013). Alterations in the V and VIII cranial nerves were present in both groups. HTLV-1 can lead to the development of important neurological signs and symptoms in apparently asymptomatic individuals. This data highlights the need for more research into the neurological aspects of HTLV-1 infection and emphasizes the importance of early diagnosis, treatment, and support for individuals living with this virus.

Skene's Gland Derivatives in the Female Genital Tract and Cervical Adenoid Basal Carcinoma are Consistently Positive With Prostatic Marker NKX3.1.

Cervical ectopic prostatic tissue and vaginal tubulosquamous polyp are rare lesions which exhibit variable, and often focal, immunohistochemical expression with traditional prostatic markers [prostate-specific antigen and prostatic acid phosphatase (PSAP)]. These lesions are thought to arise from periurethral Skene's glands, the female equivalent of prostatic glands in the male. Adenoid basal carcinoma is a rare and indolent cervical neoplasm. Expression of the prostatic marker NKX3.1 in ectopic prostatic tissue and tubulosquamous polyp has been reported but no studies have examined immunoreactivity with this marker in adenoid basal carcinoma. We stained 19 cases [adenoid basal carcinoma (n=6), cervical ectopic prostatic tissue (n=11), and vaginal tubulosquamous polyp (n=3); 1 case contained both adenoid basal carcinoma and ectopic prostatic tissue] with NKX3.1. In all cases, the glandular component of these lesions exhibited diffuse nuclear immunoreactivity while normal endocervical glands were negative. Prostate-specific antigen was positive in 4 of 9 and 0 of 3 cases of ectopic prostatic tissue and tubulosquamous polyp, respectively, while PSAP was positive in 3 of 4 and 2 of 2 cases of ectopic prostatic tissue and tubulosquamous polyp respectively; 3 of 5 cases of adenoid basal carcinoma tested were focally positive with PSAP and all 5 were negative with prostate-specific antigen. While the specificity of NKX3.1 should be investigated in future studies, positivity with this marker may be useful in diagnosing these uncommon lesions. NKX3.1 appears a more sensitive marker of ectopic prostatic tissue and tubulosquamous polyp than traditional prostatic markers and positive staining provides further support that these lesions exhibit "prostatic" differentiation and are of Skene's gland origin. NKX3.1 and PSAP positivity in adenoid basal carcinoma raises the possibility of an association with benign glandular lesions exhibiting prostatic differentiation and we critically discuss the possible association.

Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans.

Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.

Exploring the Brazilian diversity of Aspergillus sp. strains for lovastatin and itaconic acid production.

Filamentous fungi are well known for producing secondary metabolites applied in various industrial segments. Among these, lovastatin and itaconic acid, produced by Aspergillus terreus, have applications in the pharmaceutical and chemical industries. Lovastatin is primarily used for the control of hypercholesterolemia, while itaconic acid is a building block for the production of synthetic fibers, coating adhesives, among others. In this study, for the first time, 35 strains of Aspergillus sp. from four Brazilian culture collections were evaluated for lovastatin and itaconic acid production and compared to a reference strain, ATCC 20542. From an initial screening, the strains ATCC 20542, URM 224, URM1876, URM 5061, URM 5254, URM 5256, URM 5650, and URM 5961 were selected for genomic comparison. Among tested strains, the locus corresponding to the lovastatin genomic cluster was assembled, showing that all genes essential for lovastatin biosynthesis were present in producing URM 5961 and URM 5650 strains, with 100% and 98.5% similarity to ATCC 20542, respectively. However, in the no producing URM 1876, URM 224, URM 5254, URM 5061, and URM 5256 strains, this cluster was either fragmented or missing. Among the 35 strains evaluated for itaconic acid production in this study, only three strains had titers above 0.5 g/L, 16 strains had production below 0.5 g/L, and the remaining 18 strains had no production, with the highest production of itaconic acid observed in the URM 5254 strain with 2.2 g/L. The essential genes for itaconic acid production, mttA, cadA msfA were also mapped, where all three genes linked to itaconic acid production were found in a single contig in the assembly of each strain. In contrast to lovastatin loci, there is no correlation between the level of itaconic acid production and genetic polymorphisms in the genes associated with its biosynthesis.

Low-dose naltrexone: a novel adjunctive treatment in symptomatic alopecias?

Naltrexone is a competitive antagonist of µ, κ and γ opioid receptors, used for treatment of alcoholism and opioid addiction. Low-dose naltrexone (LDN) is defined as daily doses ranging from 1mg to 5mg. This is purported to have a paradoxical effect that leads to an increase in endogenous opioids, including beta-endorphins, which have anti-inflammatory properties. Theses mechanisms may also justify their possible role in the treatment of inflammatory conditions. The aim of this article is to discuss the use of LDN as an adjuvant therapeutic option in symptomatic alopecias presenting with trichodynia. Trichodynia is defined as scalp discomfort of variable intensity presenting as diffuse or localized dysesthesia and may be described by patients as pain, pruritus, or burning. These are common symptoms in patients with hair loss that negatively impacts quality of life. Scalp discomfort may be refractory to conventional therapies and does not yet have a specific therapeutic guideline. For these cases, LDN would be a possible alternative to be added to the therapeutic arsenal owing to its anti-inflammatory properties, analgesic potential, low cost, and few adverse effects described. Further studies are needed to standardize dosing, better understand its mechanism of action, and evaluate its potential therapeutic indications.

Transcriptomic reprogramming of genus Paracoccidioides in dimorphism and host niches.

The thermodimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic systemic mycosis in Latin America. Paracoccidioides grows as saprophytic mycelia that produce infective conidia propagules, which are inhaled into the lungs where the fungus converts to the pathogenic yeast form. From the lungs, Paracoccidioides may disseminate through blood and lymphatics to several other organs and tissues. During the last decade we have witnessed the generation of a large amount of transcriptomic data regarding the events leading to the morphological transition and host niche adaptation. In this review we summarize those findings and discuss the consequence of gene expression plasticity in the persistence and survival of this pathogen. In addition, we discuss the future trends on the host-pathogen studies and how new molecular strategies, such as RNA-seq, dual RNA-seq and Chip-Seq can be powerful tools to improve our understanding on the pathobiology of this systemic mycosis in Latin America.

Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis.

BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.

The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence.

Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37°C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. Among several mutants unable to grow at 37°C, we explored one bearing an interruption in the URA4 gene. This gene encodes dihydroorotase (DHOase) that is involved in the de novo synthesis of pyrimidine ribonucleotides. Loss of the C. neoformans Ura4 protein, by targeted gene interruption, resulted in an expected uracil/uridine auxotrophy and an unexpected high temperature growth defect. In addition, the ura4 mutant displayed phenotypic defects in other prominent virulence factors (melanin, capsule and phospholipase) and reduced stress response compared to wild type and reconstituted strains. Accordingly, this mutant had a decreased survival rate in macrophages and attenuated virulence in a murine model of cryptococcal infection. Quantitative PCR analysis suggests that this biosynthetic pathway is induced during the transition from 30°C to 37°C, and that transcriptional regulation of de novo and salvage pyrimidine pathway are under the control of the Ura4 protein.

The transcriptional response of Cryptococcus neoformans to ingestion by Acanthamoeba castellanii and macrophages provides insights into the evolutionary adaptation to the mammalian host.

Virulence of Cryptococcus neoformans for mammals, and in particular its intracellular style, was proposed to emerge from evolutionary pressures on its natural environment by protozoan predation, which promoted the selection of strategies that allow intracellular survival in macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then can replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least 2-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes that were found in both groups, we focused on open reading frame (ORF) CNAG_05662, which was potentially related to sugar transport but had no determined biological function. To characterize its function, we constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (polyol transporter protein 1), is involved in the transport of 5- and 6-carbon polyols such as mannitol and sorbitol, but its presence or absence had no effect on cryptococcal virulence for mice or moth larvae. Overall, these results are consistent with the hypothesis that the capacity for mammalian virulence originated from fungus-protozoan interactions in the environment and provide a better understanding of how C. neoformans adapts to the mammalian host.

Resazurin to determine the minimum inhibitory concentration on antifungal susceptibility assays for Fonsecaea sp. using a modified EUCAST protocol.

Chromoblastomycosis is a fungal chronic disease, which affects humans, especially in cutaneous and subcutaneous tissues. There is no standard treatment for Chromoblastomycosis, and it is a therapeutic challenge, due natural resistance of their causative agents, inadequate response of patients and common cases of relapse. Protocols for determination of antifungal drugs susceptibility are not standardized for chromoblastomycosis agents and endpoint definition is usually based on visual inspection, which depends on the analyst, making it sometimes inaccurate. We presented a colorimetric and quantitative methodology based on resazurin reduction to resofurin to determine the metabolic status of viable cells of Fonsecaea sp. Performing antifungal susceptibility assay by a modified EUCAST protocol allied to resazurin, we validated the method to identify the minimum inhibitory concentrations of itraconazole, fluconazole, amphotericin B, and terbinafine for eight Fonsecaea clinical isolates. According to our data, resazurin is a good indicator of metabolic status of viable cells, including those exposed to antifungal drugs. This work aimed to test resazurin as an indicator of the metabolic activity of Fonsecaea species in susceptibility assays to antifungal drugs. Species of this genus are the main causative agents of Chromoblastomycosis, which affects humans.

Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene.

Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB -which converts chorismate to trp-was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp- phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents.

[Consumption of non-standard medication in indigenous health: rational usage?] / Consumo de medicamentos não padronizados na saúde indígena: uso racional?

The study analyzed the consumption of non-standard medication in the health of indigenous peoples, emphasizing the rationality of pharmacotherapy, by conducting a cross-sectional study of secondary data from 2018 and 2019 in the Minas Gerais/Espírito Santo Special Sanitary Indigenous Health Districts. These medicines were classified by Anatomical Therapeutic Chemical Classification. Non-parametric tests were applied to compare the origin of prescription and the form of acquisition, assessing access to medication. Rationality was verified through the consumption profile and the therapeutic option in the list of standardized medicines. A total of 104,928 pharmaceutical presentations were consumed, 66,967 (66%) for the alimentary tract and metabolism, 17,705 (17%) for the nervous system, and 12,961 (12%) for the cardiovascular system. With respect to medicines consumed per region, 171 (90%) out of 190 had a therapeutic option. Prescriptions were more from the SUS. Differences were found in the way the medicines were acquired. The study pointed to significant consumption of non-standard medicines, and there may be failings in therapeutic rationality. In indigenous health, ethnocultural and social issues are challenges to access to medicines with rational use.

O estudo analisou o consumo de medicamentos não padronizados na saúde indígena, enfatizando a racionalidade da farmacoterapia, por meio de um estudo transversal dos dados secundários, de 2018 e 2019, no Distrito Especial Sanitário de Saúde Indígena Minas Gerais/Espírito Santo. Esses medicamentos foram classificados pela Anatomical Therapeutic Chemical Classification. Para a comparação da origem de prescrição e da forma de aquisição, empregaram-se testes não paramétricos, avaliando o acesso a medicamentos. Verificou-se a racionalidade por meio do perfil de consumo e da opção terapêutica na lista de medicamentos padronizados. Foram consumidas 104.928 apresentações farmacêuticas, 66.967 (66%) eram para o trato alimentar e o metabolismo; 17.705 (17%) para o sistema nervoso; 12.961 (12%) para o sistema cardiovascular. Quanto aos medicamentos mais consumidos por regiões, 171 (90%) dos 190 apresentavam opção terapêutica. As prescrições foram mais provenientes do SUS. Encontraram-se diferenças na forma de aquisição dos medicamentos. O estudo apontou importante consumo de medicamentos não padronizados, podendo existir falhas na racionalidade terapêutica. Na saúde indígena, questões etnoculturais e sociais constituem desafios para o acesso aos medicamentos com uso racional.

Role of the Microbiome in Aedes spp. Vector Competence: What Do We Know?

Aedes aegypti and Aedes albopictus are the vectors of important arboviruses: dengue fever, chikungunya, Zika, and yellow fever. Female mosquitoes acquire arboviruses by feeding on the infected host blood, thus being able to transmit it to their offspring. The intrinsic ability of a vector to infect itself and transmit a pathogen is known as vector competence. Several factors influence the susceptibility of these females to be infected by these arboviruses, such as the activation of the innate immune system through the Toll, immunodeficiency (Imd), JAK-STAT pathways, and the interference of specific antiviral response pathways of RNAi. It is also believed that the presence of non-pathogenic microorganisms in the microbiota of these arthropods could influence this immune response, as it provides a baseline activation of the innate immune system, which may generate resistance against arboviruses. In addition, this microbiome has direct action against arboviruses, mainly due to the ability of Wolbachia spp. to block viral genome replication, added to the competition for resources within the mosquito organism. Despite major advances in the area, studies are still needed to evaluate the microbiota profiles of Aedes spp. and their vector competence, as well as further exploration of the individual roles of microbiome components in activating the innate immune system.

Aerobic exercise training mitigates tumor growth and cancer-induced splenomegaly through modulation of non-platelet platelet factor 4 expression.

Exercise training reduces the incidence of several cancers, but the mechanisms underlying these effects are not fully understood. Exercise training can affect the spleen function, which controls the hematopoiesis and immune response. Analyzing different cancer models, we identified that 4T1, LLC, and CT26 tumor-bearing mice displayed enlarged spleen (splenomegaly), and exercise training reduced spleen mass toward control levels in two of these models (LLC and CT26). Exercise training also slowed tumor growth in melanoma B16F10, colon tumor 26 (CT26), and Lewis lung carcinoma (LLC) tumor-bearing mice, with minor effects in mammary carcinoma 4T1, MDA-MB-231, and MMTV-PyMT mice. In silico analyses using transcriptome profiles derived from these models revealed that platelet factor 4 (Pf4) is one of the main upregulated genes associated with splenomegaly during cancer progression. To understand whether exercise training would modulate the expression of these genes in the tumor and spleen, we investigated particularly the CT26 model, which displayed splenomegaly and had a clear response to the exercise training effects. RT-qPCR analysis confirmed that trained CT26 tumor-bearing mice had decreased Pf4 mRNA levels in both the tumor and spleen when compared to untrained CT26 tumor-bearing mice. Furthermore, exercise training specifically decreased Pf4 mRNA levels in the CT26 tumor cells. Aspirin treatment did not change tumor growth, splenomegaly, and tumor Pf4 mRNA levels, confirming that exercise decreased non-platelet Pf4 mRNA levels. Finally, tumor Pf4 mRNA levels are deregulated in The Cancer Genome Atlas Program (TCGA) samples and predict survival in multiple cancer types. This highlights the potential therapeutic value of exercise as a complementary approach to cancer treatment and underscores the importance of understanding the exercise-induced transcriptional changes in the spleen for the development of novel cancer therapies.

Bone mineral density, trabecular bone score and muscle strength in transgender men receiving testosterone therapy versus cisgender men.

INTRODUCTION: Data on body composition, bone mineral density (BMD) and microarchitecture between cis and trans men are scarce. Few studies have reported trabecular bone score (TBS) data for transgender men using testosterone. EXPERIMENTAL: We studied 19 transgender men and 19 cisgender men (mean age 23.6 ± 3.4 years, p = 0.539) paired by age and body mass index (BMI). They underwent clinical and hormonal evaluation, body composition measurement, and evaluation BMD, the TBS, grip strength, the level of physical activity (IPAQ) and physical performance (SPPB). RESULTS: Median serum testosterone levels were similar between the cisgender and transgender groups (638 vs. 685 ng/dl; p = 0.863). Mean serum estradiol levels were slightly higher in the transgender men (51.95 ± 44.26 vs. 32.26 ± 8.40 pg/ml, p = 0.005), and the median testosterone use duration in the transgender group was 24 months. Total muscle mass (44.09 ± 6.27 vs. 55.71 ± 7.28 kg, p < 0.001), and hand grip strength (28.82 ± 5.42 vs. 40.34 ± 8.03 kg, p < 0.001) were considerably lower in the transgender men. Total body BMD (1.208 ± 0.132 vs. 1.271 ± 0.081 g/cm2, p = 0.008) and femoral neck BMD (1.019 ± 0.163 vs. 1.137 ± 0.166 g/cm2, p = 0.016) were lower in the transgender group. The TBS was similar between groups. Thus, our data demonstrated that despite similar serum testosterone levels, transgender men undergoing testosterone therapy had lower muscle strength, muscle mass, and total body and femoral neck BMD values than cisgender men.

Identification of early biomarkers in saliva in genetically engineered mouse model C(3)1-TAg of breast cancer.

Breast cancer is one of leading causes of death worldwide in the female population. Deaths from breast cancer could be reduced significantly through earlier and more efficient detection of the disease. Saliva, an oral fluid that contains an abundance of protein biomarkers, has been recognized as a promising diagnostic biofluid that is easy to isolate through non-invasive techniques. Assays on saliva can be performed rapidly and are cost-effective. Therefore, our work aimed to identify salivary biomarkers present in the initial stages of breast cancer, where cell alterations are not yet detectable by histopathological analysis. Using state-of-the-art techniques, we employed a transgenic mouse model of mammary cancer to identify molecular changes in precancerous stage breast cancer through protein analysis in saliva. Through corroborative molecular approaches, we established that proteins related to metabolic changes, inflammatory process and cell matrix degradation are detected in saliva at the onset of tumor development. Our work demonstrated that salivary protein profiles can be used to identify cellular changes associated with precancerous stage breast cancer through non-invasive means even prior to biopsy-evident disease.

Pathogenicity and Growth Conditions Modulate Fonsecaea Extracellular Vesicles' Ability to Interact With Macrophages.

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous mycosis caused by black, dimorphic, and filamentous fungi of the Herpothrichiellaceae family, such as species of the genus Fonsecaea. These fungi can switch between the saprophytic forms (conidia and hyphae) and the pathogenic form, the muriform cells (MCs), which is considered an essential mechanism for fungal virulence. Nearly all types of cells can produce membranous structures formed by a lipid bilayer that communicate extracellularly with other cells, known as "extracellular vesicles" (EVs), which may act as virulence factors, as observed for several species of pathogenic fungi. Our findings demonstrated for the first time that F. pedrosoi, F. nubica, and F. erecta produce EVs in response to nutritional conditions. The EVs varied in sterol and protein contents, size, and morphology. Moreover, the EVs induced different cytokine and nitric oxide release patterns by bone marrow-derived macrophages (BMDMs). The EVs activated IL-1ß production, possibly acting as the first signal in inflammasome activation. Unlike the pathogenic species, the EVs isolated from F. erecta did not significantly stimulate TNF and IL-10 production in general. Overall, these results demonstrated that different species of Fonsecaea produce EVs capable of modulating pro- and anti-inflammatory cytokine and nitric oxide production by BMDMs and that growth conditions affected the immunomodulatory capacities of the EVs as well as their size, content, and morphology.

Salivary Proteome, Inflammatory, and NETosis Biomarkers in Older Adult Practitioners and Nonpractitioners of Physical Exercise.

Since aging has been associated with increased production of inflammatory biomarkers, the ability to monitor older adults repeatedly is highly desirable, and saliva is an interesting biofluid for the search of biomarkers, as it is easily accessible in a noninvasive manner. However, given the incipient knowledge of salivary biomarkers in aging and its relationship to physical exercise, the present study is aimed at evaluating the protein expression and the levels of inflammatory and NETosis biomarkers in the saliva of practitioners (PE) and nonpractitioners (NPE) of physical exercise older adults. Six (6) practitioner and 4 nonpractitioner older adults were enrolled in this study. Unstimulated whole saliva was collected for analysis of the proteome by label-free mass spectrometry, as well as of the inflammatory status by evaluation of C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and cytokines (TNF-α, interleukin- (IL-) 1ß, and IL-8), while NETosis was assessed by myeloperoxidase (MPO) and neutrophil elastase. Regarding oral health, the decayed, missing, and filled teeth (DMF-T) index, bleeding on probing, suppuration, and probing depth measurement (mm) were evaluated. In addition, functional capacity was investigated using the General Physical Fitness Index (GPFI). In relation to the proteome analysis, 93 and 143 proteins were found exclusively in the PE and NPE groups, respectively; 224 proteins were common to both groups. Among these proteins, 10 proteins showed statistical difference (p < 0.05) between the groups: alpha-2-macroglobulin, component 3 of the complement, serotransferrin, and protein soluble in brain acid 1 were less expressed, while lactotransferrin, alpha-amylase 1, S100-A8, S100-A9, lactoperoxidase, and galectin-3 binding protein were more expressed in the PE group. No differences between groups were observed in the analysis of inflammatory and NETosis biomarkers. This study shows the potential utility of saliva for detecting protein biomarkers in a noninvasive biological sample of the elderly population.

Engineered Fluorescent Strains of Cryptococcus neoformans: a Versatile Toolbox for Studies of Host-Pathogen Interactions and Fungal Biology, Including the Viable but Nonculturable State.

Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans. IMPORTANCE Cryptococcus neoformans is a human-pathogenic yeast that can undergo a dormant state and is responsible for over 180,000 deaths annually worldwide. We engineered a set of fluorescent transformants to aid in research on C. neoformans. A mutant with GFP-tagged Pab1 improved fluorescence-based techniques used in host interaction studies. Moreover, this mutant induced a viable but nonculturable phenotype and uncovered posttranscriptional mechanisms associated with dormant C. neoformans. The experimental use of fluorescent mutants may shed light on C. neoformans-host interactions and fungal biology, including dormant phenotypes.

Characterisation of the heat shock factor of the human thermodimorphic pathogen Paracoccidioides lutzii.

Thermodimorphic fungi include most causative agents of systemic mycoses, but the molecular mechanisms that underlie their defining trait, i.e. the ability to shift between mould and yeast on temperature change alone, remain poorly understood. We hypothesised that the heat shock factor (Hsf), a protein that evolved to sense thermal stimuli quickly, might play a role in this process in addition to the known regulator Drk1 and the Ryp proteins. To test this hypothesis, we characterised the Hsf from the thermodimorph Paracoccidioides lutzii (formerly Paracoccidioides brasiliensis isolate 01). We show in the present work that PlHsf possesses regulatory domains that are exclusive of the Eurotiomycetidae family, suggesting evolutionary specialisation; that it can successfully rescue the otherwise lethal loss of the native protein of Saccharomyces cerevisiae; and that its DNA-binding domain is able to recognise regulatory elements from the promoters of both Drk1 and Ryp1. An in silico screening of all 1 kb sequences upstream of P. lutzii ORFs revealed that 7% of them possess a heat shock element. This is the first description of a heat shock factor in a thermodimorphic fungus.