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OBJECTIVE: To evaluate the association between type of menopause (spontaneous or surgical) and mild cognitive impairment (MCI). STUDY DESIGN: This study was a cross-sectional, observational, and sub-analytical investigation conducted within gynecological consultations across nine Latin American countries. METHOD: We assessed sociodemographic, clinical, and anthropometric data, family history of dementia, and the presence of MCI using the Montreal Cognitive Assessment (MoCA) tool. RESULTS: The study involved 1185 postmenopausal women with a mean age of 55.3 years and a body mass index of 26.4 kg/m2. They had an average of 13.3 years of education, and 37 % were homemakers. Three hundred ninety-nine experienced menopause before 40, including 136 with surgical menopause (bilateral oophorectomy). Out of the 786 women who experienced menopause at 40 or more years, 110 did so due to bilateral oophorectomy. There were no differences in MoCA scores among women who experienced menopause before or after the age of 40. However, lower MoCA scores were observed in women with surgical menopause than in those with spontaneous menopause (23.8 ± 4.9 vs. 25.0 ± 4.3 points, respectively, p < 0.001). Our logistic regression model with clustering of patients within countries found a significant association between MCI and surgical menopause (OR 1.47, 95 % CI: 1.01-2.16), use (ever) of menopausal hormone therapy (OR 0.33, 95 % CI: 0.21-0.50), and having >12 years of education (OR 0.21, 95 % CI: 0.14-0.30). CONCLUSION: When comparing women who experience spontaneous menopause over the age of 40 with those who undergo it before this age, there was no observed increased risk of developing MCI, while those with surgical menopause, independent of age, are more prone to cognitive decline. Women who have ever used menopausal hormone therapy have a lower MCI risk. Further research is warranted to delve deeper into this topic.

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OBJECTIVE: To evaluate the association between menopausal symptoms and cognitive decline in postmenopausal women. METHODS: This was a subanalysis of a cross-sectional, observational study conducted among women attending gynecological consultations across nine Latin American countries. The survey involved late postmenopausal women who were asked to complete a general questionnaire and the Menopause Rating Scale (MRS) to assess menopausal symptoms, with the Montreal Cognitive Assessment used to evaluate cognitive function as an outcome. A Montreal Cognitive Assessment score of less than 21 was used to define women with mild cognitive impairment (MCI). RESULTS: The study included 1,287 postmenopausal women with a mean age of 55.5 years and a mean body mass index of 26.3 kg/m2. On average, participants had 13.8 years of education and 2.3 ± 1.8 children, with 72.8% reporting having a partner. Additionally, 36.7% ever used menopausal hormone therapy. Regarding lifestyle factors, 50.3% engaged in a sedentary lifestyle, whereas 70.5% had never smoked. 15.3% of women had MCI exhibited significantly more intense menopausal symptoms compared with those without MCI (MRS total score 15.24 ± 12.58 vs 10.53 ± 8.84, respectively, P < 0.001). Logistic regression analysis revealed a significant association between severe menopausal symptoms (MRS total score ≥14 points) and MCI (odds ratio [OR], 1.74; 95% CI, 1.25-2.42). Conversely, a lower body mass index (OR, 0.96; 95% CI, 0.95-0.98), sexual activity (OR, 0.70; 95% CI, 0.51-0.96), physical exercise (OR, 0.55; 95% CI, 0.39-0.76), menopausal hormone therapy use (OR, 0.36; 95% CI, 0.24-0.55), and higher educational level (OR, 0.31; 95% CI, 0.21-0.46) were associated with lower odds for MCI. CONCLUSION: Severe menopausal symptoms in postmenopausal women were associated with cognitive impairment. This study highlights the intricate interplay between hormonal, lifestyle, and sociodemographic factors and cognitive health.

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Oral administration of medicines has been the most common route for drug delivery as it is cost-effective and is convenient for patients [...].

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Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of ß-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.

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Emergomyces africanus is a highly fatal fungal pathogen affecting individuals with advanced HIV disease. Molecular patterns and ultrastructural aspects of E. africanus are unknown, and pathogenic models have not been investigated in detail. Since the cell wall of fungi is a determinant for interaction with the host and antifungal development, we characterized the ultrastructural aspects of E. africanus and the general properties of cell wall components under different conditions of growth in vitro and in vivo. We also tested the pathogenic potential of E. africanus in a Galleria mellonella model of infection. Transmission electron microscopy revealed the common intracellular, ultrastructural features of fungi in association with a thick cell wall. Scanning electron microscopy revealed a smooth cell surface, with no apparent decorative structures. Yeast cultures of E. africanus showed the distribution of chitin, chitooligomers, and mannoproteins commonly observed in fungi. However, in mixed microenvironments containing yeast and filamenting forms of E. africanus, the detection of chitooligomers was increased in comparison with isolated yeast cells, while the detection of these components in filamenting forms was markedly reduced. These observations were suggestive of the ability of E. africanus to change its cell wall composition in response to different microenvironments. Although E. africanus was unable to kill G. mellonella, this infection model allowed us to isolate infected hemocytes for further analysis of mannoproteins, chitin, and chitooligomers. Once again, the detection of E. africanus chitooligomers was markedly increased. These results reveal previously unknown ultrastructural features of E. africanus and suggest a high plasticity in the cell wall of this lethal pathogen. IMPORTANCE: The epidemiology of fungal infections is very dynamic, and novel health emergencies are hard to predict. New fungal pathogens have been continuously emerging for the last few decades, and Emergomyces africanus is one of these threats to human health. This complex scenario points to the need for generating knowledge about emerging pathogens so that new therapeutic strategies can be designed. In this study, we characterized the general cellular and pathogenic properties of the emerging fungal pathogen E. africanus. Our results reveal that E. africanus manifests some of the typical properties of fungal cells but also exhibits some unique characteristics that might be helpful for the future development of therapeutic strategies.

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PURPOSE: Vitamin D deficiency has been associated with higher rates of autoimmune disease, including noninfectious uveitis. This PRISMA-compliant review and meta-analysis aimed to analyze the correlation between noninfectious uveitis and vitamin D levels. METHODS: We searched PubMed, Embase, Cochrane, and Web of Science databases for studies, published in English, assessing vitamin D levels in patients diagnosed with noninfectious uveitis. The outcomes of interest were vitamin D deficiency, vitamin D mean level, vitamin D supplementation, and smoking rates. A subgroup analysis of inactive uveitis and active uveitis was performed. The heterogeneity was assessed with Cochrane Q-test and I2 statistics; p > 0.10 and I2 > 50% were considered significant for heterogeneity. Statistical analysis was conducted using Review Manager 5.3. RESULTS: 9 studies were included in the meta-analysis comprising a total of 10 711 patients, of whom 1,368 were diagnosed with noninfectious uveitis. Patients with noninfectious uveitis had worse results regarding vitamin D deficiency when compared with the control group (OR 0.58; CI 95% 0.44 to 0.77; p = 0.0002; I2 = 61%). Patients with inactive uveitis had better results towards vitamin D deficiency when compared with active uveitis (OR 5.00; CI 95% 2.84 to 8.81; p < 0.001; I2 = 0%). CONCLUSION: Our research supports the increasing evidence that associates vitamin D deficiency with noninfectious uveitis and its activity. Further investigation into the efficacy of vitamin D screening and supplementation in reducing the recurrence of uveitis is necessary.

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We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba-related diseases.

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Women, who represent approximately half of the global population according to estimates as of January 2024, may experience signs and symptoms of menopause for at least one-third of their lives, during which they have a higher risk of cardiovascular morbidity and mortality. The effects of menopausal hormone therapy (MHT) on the progression of atherosclerosis and cardiovascular disease (CVD) events vary depending on the age at which MHT is initiated and the time since menopause until its initiation. Beneficial effects on CVD outcomes and all-cause mortality have been observed when MHT was initiated before the age of 60 or within 10 years after menopause. The decision regarding the initiation, dose, regimen, and duration of MHT should be made individually after discussing the benefits and risks with each patient. For primary prevention of postmenopausal chronic conditions, the combined use of estrogen and progestogen is not recommended in asymptomatic women, nor is the use of estrogen alone in hysterectomized women. Hormone-dependent neoplasms contraindicate MHT. For the treatment of genitourinary syndrome of menopause, vaginal estrogen therapy may be used in patients with known cardiovascular risk factors or established CVD. For women with contraindications to MHT or who refuse it, non-hormonal therapies with proven efficacy (antidepressants, gabapentin, and fezolinetant) may improve vasomotor symptoms. Compounded hormonal implants, or "bioidentical" and "compounded" hormones, and "hormone modulation" are not recommended due to lack of scientific evidence of their effectiveness and safety.

Mujeres, que representan aproximadamente la mitad de la población mundial según estimaciones de enero de 2024, pueden experimentar signos y síntomas de la menopausia durante al menos un tercio de sus vidas, durante los cuales tienen un mayor riesgo de morbilidad y mortalidad cardiovascular. Los efectos de la terapia hormonal de la menopausia (THM) en la progresión de la aterosclerosis y los eventos de enfermedad cardiovascular (ECV) varían según la edad en que se inicia la THM y el tiempo transcurrido desde la menopausia hasta su inicio. Se han observado efectos beneficiosos en los resultados de ECV y la mortalidad por todas las causas cuando la THM se inició antes de los 60 años o dentro de los 10 años posteriores a la menopausia. La decisión sobre la iniciación, dosis, régimen y duración de la THM debe tomarse individualmente después de discutir los beneficios y riesgos con cada paciente. Para la prevención primaria de condiciones crónicas en la posmenopausia, no se recomienda el uso combinado de estrógeno y progestágeno en mujeres asintomáticas, ni el uso de estrógeno solo en mujeres histerectomizadas. Las neoplasias dependientes de hormonas contraindican la THM. Para el tratamiento del síndrome genitourinario de la menopausia, se puede usar terapia estrogénica vaginal en pacientes con factores de riesgo cardiovascular conocidos o ECV establecida. Para mujeres con contraindicaciones a la THM o que la rechazan, las terapias no hormonales con eficacia demostrada (antidepresivos, gabapentina y fezolinetant) pueden mejorar los síntomas vasomotores. Los implantes hormonales compuestos, o hormonas "bioidénticas" y "compuestas", y la "modulación hormonal" no se recomiendan debido a la falta de evidencia científica sobre su efectividad y seguridad.

As mulheres, que representam cerca de metade da população mundial segundo estimativas de janeiro de 2024, podem sofrer com sinais e sintomas da menopausa durante pelo menos um terço de suas vidas, quando apresentam maiores risco e morbimortalidade cardiovasculares. Os efeitos da terapia hormonal da menopausa (THM) na progressão de eventos de aterosclerose e doença cardiovascular (DCV) variam de acordo com a idade em que a THM é iniciada e o tempo desde a menopausa até esse início. Efeitos benéficos nos resultados de DCV e na mortalidade por todas as causas ocorreram quando a THM foi iniciada antes dos 60 anos de idade ou nos 10 anos que se seguiram à menopausa. A decisão sobre o início, a dose, o regime e a duração da THM deve ser tomada individualmente após discussão sobre benefícios e riscos com cada paciente. Para a prevenção primária de condições crônicas na pós-menopausa, não se recomendam o uso combinado de estrogênio e progestagênio em mulheres assintomáticas nem o uso de estrogênio sozinho em mulheres histerectomizadas. Neoplasias hormônio-dependentes contraindicam a THM. Para tratamento da síndrome geniturinária da menopausa, pode-se utilizar terapia estrogênica por via vaginal em pacientes com fatores de risco cardiovascular conhecidos ou DCV estabelecida. Para mulheres com contraindicação à THM ou que a recusam, terapias não hormonais com eficácia comprovada (antidepressivos, gabapentina e fezolinetante) podem melhorar os sintomas vasomotores. Os implantes hormonais manipulados, ou hormônios "bioidênticos" "manipulados", e a 'modulação hormonal' não são recomendados pela falta de evidência científica de sua eficácia e segurança.

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Extracellular vesicles (EVs) are produced by all domains of life. In fungal pathogens, they participate in virulence mechanisms and/or induce protective immunity, depending on the pathogenic species. EVs produced by pathogenic members of the Cryptococcus genus mediate virulence, antifungal resistance, as well as humoral and cell-mediated immunity. The isolation of cryptococcal EVs has been laborious and time-consuming for years. In this chapter, we detail a fast protocol for the isolation and analysis of EVs produced by members of the Cryptococcus genus.

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Glucuronoxylomannan (GXM) is the principal capsular component in the Cryptococcus genus. This complex polysaccharide participates in numerous events related to the physiology and pathogenesis of Cryptococcus, which highlights the importance of establishing methods for its isolation and analysis. Conventional methods for GXM isolation have been extensively discussed in the literature. In this chapter, we describe two fast methods for obtaining extracellular fractions enriched with cryptococcal GXM.

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INTRODUCTION: Low-Level Red-Light (LLRL) Therapy is a safe and natural way to promote healing and reduce inflammation in the body. When it comes to treating myopia in children, LLRL therapy is recent, and its efficacy and safety still are not clear. METHODS: A systematic review and meta-analysis of the literature for LLRL was conducted in accordance with the PRISMA guidelines on November 5, 2022. Databases, including PUBMED, Cochrane Library, Web of Science, and Embase were queried. A meta-analysis of random effects was conducted. Inclusion criteria included Randomized Controlled Trials (RCTs) or observational studies where LLRL therapy was used in children (3‒15 years old) with myopia. Exclusion criteria were studies with other ocular abnormalities. Efficacy was evaluated through the mean change in Axial Length (AL) and cycloplegic Spherical Equivalent Error (SER), while safety was evaluated by monitoring adverse effects. RESULTS: A total of 5 final studies were included (4 RCTs, and 1 observational), in which 685 total patients were analyzed. The mean age was 9.7 ± 0.66 years, with 48,2% female patients. The number of eyes in the LRLL arm is 714 and, in the control, arm is 656. LLRL showed better results in SER and AL mean change (OR = 0.58; 95% CI 0.33 to 0.83; p < 0.00001, and MD -0.33; 95% CI -0.52 to -0.13; p = 0.001, respectively), in comparison to the control group. There was no significant difference in adverse effects between groups (MD = 5.76; 95% CI 0.66 to 50.14; p = 0.11). CONCLUSION: LLRL therapy is a non-invasive, effective, and safe short-term treatment option; however, long-term evaluation, particularly in comparison to other therapies, requires additional investigation.

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The use of lignocellulosic residues, originating from sawdust, in composting sewage sludge for organic fertilizer production, is a practice of growing interest. However, few studies have explored the effect of the proportion of sawdust and sewage sludge raw materials on composting performance in the humification process. This study assessed the addition of sawdust in the sewage sludge composting process, regarding carbon content, presence of heavy metals, and humification of the organic compost. The experimental design employed was a randomized complete block design with five treatments featuring different proportions of organic residues to achieve C/N ratios between 30-1 (T1: 100% sewage sludge and 0% sawdust, T2: 86% sewage sludge and 14.0% sawdust, T3: 67% sewage sludge and 33% sawdust, T4: 55% sewage sludge and 45% sawdust, and T5: 46.5% sewage sludge and 53.5% sawdust) and five replications, totaling 25 experimental units. The addition of lignocellulosic residue in sewage sludge composting increased the levels of TOC and the C/N ratio, reduced the levels of pH, P, N, Na, Ba, and Cr, and did not interfere with the levels of K, Ca, Mg, S, CEC, labile carbon, and metals Fe, Zn, Cu, Mn, Ni, and Pb. The increase in the proportion of sawdust residue favored the degradation of aliphatic groups, increasing the presence of aromatic structures and reducing humification at the end of composting. The use of sawdust as a lignocellulosic residue in sewage sludge composting is a viable and efficient alternative to produce high-quality organomineral fertilizers.

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Extracellular vesicles (EVs) have been identified in diverse fungi, including human pathogens. In this protocol, we present two techniques for isolating and analyzing fungal EVs. The first is for high-throughput screening, and the second is for yielding concentrated samples suitable for centrifugation-based density gradients. We describe steps for analytical assays such as nano-flow cytometry and nanoparticle tracking analysis to measure EV dimensions and concentration. EV suspensions can serve diverse assays, including electron microscopy, compositional determination, and cell-to-cell communication assays. For complete details on the use and execution of this protocol, please refer to Rizzo et al.,1 Rizzo et al.,2 Reis et al.,3 and Reis et al.4.

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Currently, a clear interest has been given to berries due to their richness in active metabolites, including anthocyanins and non-coloured phenolics. Therefore, the main aim of the present work is to investigate the phenolic profile, antioxidant abilities, and antiproliferative effects on normal human dermal fibroblasts (NHDF) and human colon carcinoma cell line (Caco-2) cells of phenolic-rich extracts from three red fruits highly appreciated by consumers: two species of blackberries (Rubus fruticosus and Rubus ulmifolius) and one species of mulberry (Morus nigra). A total of 19 different phenolics were identified and quantified by HPLC-DAD-ESI/MSn and HPLC-DAD, respectively. Focusing on the biological potential of the phenolic-rich extracts, all of them revealed notable scavenging abilities. Concerning the antiproliferative properties, R. fruticosus presented a cytotoxic selectivity for Caco-2 cells compared to NHDF cells. To deeper explore the biological potential, combinations with positive controls (ascorbic acid and 5-fluorouracil) were also conducted. Finally, the obtained data are another piece of evidence that the combination of phenolic-rich extracts from natural plants with positive controls may reduce clinical therapy costs and the possible toxicity of chemical drugs.

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OBJECTIVE: Musculoskeletal disorders frequently affect postmenopausal women. This study aims to compare muscle disorders between women according to the type of experienced menopause: premature (PM) or normal age of menopause (NAM). METHODS: This was a cross-sectional study conducted in nine Latin American countries in which late postmenopausal women (55 to 70 years) were surveyed with a general questionnaire, the Menopause Rating Scale (MRS: item #4 exploring musculoskeletal discomfort), and strength, assistance with walking, rising from a chair, climbing stairs, and falling questionnaire (risk of sarcopenia). RESULTS: A total of 644 women were included: 468 who had NAM, and 176 who had PM (116 spontaneous and 60 surgical). The overall mean age of the participants was 60.9 ± 4.2 years. Women who had PM experienced more musculoskeletal discomfort (33.5% vs 20.9%, P < 0.001) and a higher likelihood of sarcopenia (35.2% vs 19.9%, P < 0.001) than women who had a NAM. Women who had surgical PM exhibited a higher prevalence of severe musculoskeletal discomfort (46.7% vs 29.3%, P < 0.02) and a higher likelihood of sarcopenia (45.0% vs 27.6%, P < 0.02) than women who had a NAM. After adjusting for covariates (age, body mass index, menopausal hormone therapy use, physical activity, education, cigarette consumption, use of antidepressants, sexual activity, comorbidities, and having a partner), our logistic regression model determined that spontaneous PM was not associated with higher odds of musculoskeletal discomfort and higher odds of sarcopenia. On the other hand, women who had surgical PM were more likely to experience musculoskeletal discomforts (odds ratio: 2.26; 95% confidence interval: 1.22-4.17) and higher odds for sarcopenia (odds ratio: 2.05; 95% confidence interval: 1.16-3.65) as compared to women who experienced a NAM. CONCLUSIONS: Women experiencing surgical PM have a higher likelihood of developing muscle disorders. This underscores the potential significance of hormonal levels in influencing musculoskeletal health during postmenopause.

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The release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs' protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.

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Chromoblastomycosis (CBM) and pheohyphomycosis (PHM) are the most common implantation mycoses caused by dematiaceous fungi. In the past, flucytosine (5-FC) has been used to treat CBM, but development of resistance is common. Carmofur belongs to the same class as 5-FC and has in vitro inhibitory activity against the main agents of CBM and PHM. The aim of this study was to compare the action of these two pyrimidine analog drugs against CBM and PHM agents. The minimum inhibitory concentration (MIC) and the selectivity index based on cytotoxicity tests of these two drugs against some agents of these mycoses were determined, with carmofur presenting a higher selectivity index than 5-FC. Carmofur demonstrated here synergistic interactions with itraconazole and amphotericin B against Exophiala heteromorpha, Fonsecaea pedrosoi, Fonsecaea monophora, and Fonsecaea nubica strains. Additionally, carmofur plus itraconazole demonstrated here synergism against a Phialophora verrucosa strain. To evaluate the development of carmofur resistance, passages in culture medium containing subinhibitory concentrations of this pyrimidine analog were carried out, followed by in vitro susceptibility tests. Exophiala dermatitidis quickly developed resistance, whereas F. pedrosoi took seven passages in carmofur-supplemented medium to develop resistance. Moreover, resistance was permanent in E. dermatitidis but transient in F. pedrosoi. Hence, carmofur has exhibited certain advantages, albeit accompanied by limitations such as the development of resistance, which was expected as with 5-FC. This underscores its therapeutic potential in combination with other drugs, emphasizing the need for a meticulous evaluation of its application in the fight against dematiaceous fungi.

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