Cytopathic effects in Mimivirus infection: understanding the kinetics of virus-cell interaction.

BACKGROUND: Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. OBJECTIVES: In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. METHODS: Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. FINDINGS: The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. MAIN CONCLUSIONS: Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses.

Toll-like receptor 4 (TLR4) is the major pattern recognition receptor triggering the protective effect of a Candida albicans extracellular vesicle-based vaccine prototype in murine systemic candidiasis.

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.

Leishmania spp. amastigotes surrounding sensory nerve fibers in human painless skin ulcers: Evidence of pathogen-neuron proximity and absence of neuronal apoptosis.

In this present study, carried out between November 2020 and July 2023 at Londrina's University Hospital, patients with active lesions of cutaneous leishmaniasis (CL) were analyzed regarding pain perception and anatomopathological aspects of the ulcers. Pain was assessed using a numerical rating scale (NRS) to compare five patients diagnosed with CL with four control patients diagnosed with vascular skin ulcers. Histopathological evaluations were used to investigate the nociceptor neuron-Leishmania interface. Patients with CL ulcers reported less pain compared to patients with vascular ulcers (2.60 ± 2.30 and 7.25 ± 0.95, respectively, p = 0.0072). Histopathology evidenced Leishmania spp. amastigote forms nearby sensory nerve fibers in profound dermis. Schwann cells marker (S100 protein) was detected, and caspase-3 activation was not evidenced in the in the nerve fibers of CL patients' samples, suggesting absence of apoptotic activity in nerve endings. Additionally, samples taken from the active edge of the lesion were negative for bacilli acid-alcohol resistant (BAAR), which excludes concomitant leprosy, in which painless lesions are also observed. Thus, the present data unveil for the first time anatomopathological and microbiological details of painless ulcers in CL patients, which has important clinical implications for a better understanding on the intriguing painless clinical characteristic of CL.

ß-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

Cytopathic effects in Mimivirus infection: understanding the kinetics of virus-cell interaction

BACKGROUND Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. OBJECTIVES In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. METHODS Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. FINDINGS The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. MAIN CONCLUSIONS Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses.

Traversing the Cell Wall: The Chitinolytic Activity of Histoplasma capsulatum Extracellular Vesicles Facilitates Their Release.

Histoplasma capsulatum is the causative agent of histoplasmosis. Treating this fungal infection conventionally has significant limitations, prompting the search for alternative therapies. In this context, fungal extracellular vesicles (EVs) hold relevant potential as both therapeutic agents and targets for the treatment of fungal infections. To explore this further, we conducted a study using pharmacological inhibitors of chitinase (methylxanthines) to investigate their potential to reduce EV release and its subsequent impact on fungal virulence in an in vivo invertebrate model. Our findings revealed that a subinhibitory concentration of the methylxanthine, caffeine, effectively reduces EV release, leading to a modulation of H. capsulatum virulence. To the best of our knowledge, this is the first reported instance of a pharmacological inhibitor that reduces fungal EV release without any observed fungicidal effects.

SARS-CoV-2 uses CD4 to infect T helper lymphocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

Silver(I) 1,10-Phenanthroline Complexes Are Active against Fonsecaea pedrosoi Viability and Negatively Modulate Its Potential Virulence Attributes.

The genus Fonsecaea is one of the etiological agents of chromoblastomycosis (CBM), a chronic subcutaneous disease that is difficult to treat. This work aimed to evaluate the effects of copper(II), manganese(II) and silver(I) complexes coordinated with 1,10-phenanthroline (phen)/1,10-phenanthroline-5,6-dione (phendione) on Fonsecaea spp. Our results revealed that most of these complexes were able to inhibit F. pedrosoi, F. monophora and F. nubica conidial viability with minimum inhibitory concentration (MIC) values ranging from 0.6 to 100 µM. The most effective complexes against F. pedrosoi planktonic conidial cells, the main etiologic agent of CBM, were [Ag(phen)2]ClO4 and [Ag2(3,6,9-tdda)(phen)4].EtOH, (tdda: 3,6,9-trioxaundecanedioate), displaying MIC values equal to 1.2 and 0.6 µM, respectively. These complexes were effective in reducing the viability of F. pedrosoi biofilm formation and maturation. Silver(I)-tdda-phen, combined with itraconazole, reduced the viability and extracellular matrix during F. pedrosoi biofilm development. Moreover, both silver(I) complexes inhibited either metallo- or aspartic-type peptidase activities of F. pedrosoi as well as its conidia into mycelia transformation and melanin production. In addition, the complexes induced the production of intracellular reactive oxygen species in F. pedrosoi. Taken together, our data corroborate the antifungal action of metal-phen complexes, showing they represent a therapeutic option for fungal infections, including CBM.

Aspirin-Triggered Resolvin D1 (AT-RvD1) Protects Mouse Skin against UVB-Induced Inflammation and Oxidative Stress.

Intense exposure to UVB radiation incites excessive production of reactive oxygen species (ROS) and inflammation. The resolution of inflammation is an active process orchestrated by a family of lipid molecules that includes AT-RvD1, a specialized proresolving lipid mediator (SPM). AT-RvD1 is derived from omega-3, which presents anti-inflammatory activity and reduces oxidative stress markers. The present work aims to investigate the protective effect of AT-RvD1 on UVB-induced inflammation and oxidative stress in hairless mice. Animals were first treated with 30, 100, and 300 pg/animal AT-RvD1 (i.v.) and then exposed to UVB (4.14 J/cm2). The results showed that 300 pg/animal of AT-RvD1 could restrict skin edema, neutrophil and mast cell infiltration, COX-2 mRNA expression, cytokine release, and MMP-9 activity and restore skin antioxidant capacity as per FRAP and ABTS assays and control O2•- production, lipoperoxidation, epidermal thickening, and sunburn cells development. AT-RvD1 could reverse the UVB-induced downregulation of Nrf2 and its downstream targets GSH, catalase, and NOQ-1. Our results suggest that by upregulating the Nrf2 pathway, AT-RvD1 promotes the expression of ARE genes, restoring the skin's natural antioxidant defense against UVB exposition to avoid oxidative stress, inflammation, and tissue damage.

Upper respiratory tract viral load quantification in COVID-19 patients at hospital admission and its association with disease severity.

INTRODUCTION: There are aspects of COVID-19 pathogenesis that are still unknown. OBJECTIVE: To determine the relationship between severity, mortality and viral replication in patients with COVID-19. METHODS: Clinical characteristics, severity and mortality of 203 patients hospitalized for COVID-19 were analyzed and correlated with viral load (VL) and threshold cycle (TC) at admission; nasopharyngeal swab was obtained. RESULTS: Mean VLs in surviving patients with mild to moderate, moderate to severe and severe disease were the following: 6.8 x 106, 7.6 x 107 and 1.0 x 109, respectively; and in patients with critical disease who died, VL was 1.70 x 109. TCs were 26.06, 24.07, 22.66 and 21.78 for the same groups. In those who died, a higher mean VL was observed at admission in comparison with those who survived (1.7 x 109 vs. 9.84 x 106; p < 0.001). A significant correlation was observed between VL, severity and death (r = 0.254, p < 0.045 and r = 0.21, p < 0.015). High VL was associated with increased in-hospital mortality in comparison with low VL (OR = 2.926, p < 0.017). CONCLUSION: SARS-CoV-2 VL determined at hospital admission might classify risk simultaneously with other factors described in COVID-19.

INTRODUCCIÓN: Aún se desconocen aspectos de la patogenia de COVID-19. OBJETIVO: Determinar la relación entre gravedad, mortalidad y replicación viral en pacientes con COVID-19. MÉTODOS: Se analizaron características clínicas, gravedad de la enfermedad y mortalidad de 203 pacientes hospitalizados por COVID-19 y se correlacionaron con carga viral (CV) y ciclo umbral (Ct) al ingreso; se tomó hisopado nasofaríngeo. RESULTADOS: Las CV medias en los pacientes sobrevivientes fueron las siguientes ante enfermedad leve a moderada, moderada a grave y grave: 6.8 × 106, 7.6 × 107 y 1.0 × 109; y en los pacientes con enfermedad crítica que fallecieron, la CV fue de 1.70 × 109. Los Ct fueron 26.06, 24.07, 22.66 y 21.78 para esos mismos grupos. En quienes fallecieron se observó mayor CV media al ingreso en comparación con quienes sobrevivieron (1.7 × 109 versus 9.84 × 106), p < 0.001. Se evidenció correlación significativa entre CV, gravedad y muerte (r = 0.254, p < 0.045 y r = 0.21, p < 0.015). La CV alta se asoció a mayor mortalidad intrahospitalaria en comparación con la CV baja (RM = 2.926, p < 0.017). CONCLUSIÓN: La CV de SARS-CoV-2 determinada al ingreso hospitalario podría calificar el riesgo simultáneamente con otros factores descritos en COVID-19.

Screening of Pandemic Response Box Library Reveals the High Activity of Olorofim against Pathogenic Sporothrix Species.

The increase in the prevalence and severity of fungal infections and the resistance to available antifungals highlights the imperative need for novel therapeutics and the search for new targets. High-content screening of libraries containing hundreds of compounds is a powerful strategy for searching for new drug candidates. In this study, we screened the Pandemic Response Box library (Medicines for Malaria Venture) of 400 diverse molecules against the Sporothrix pathogenic species. The initial screen identified twenty-four candidates that inhibited the growth of Sporothrix brasiliensis by more than 80%. Some of these compounds are known to display antifungal activity, including olorofim (MMV1782354), a new antifungal drug. Olorofim inhibited and killed the yeasts of S. brasiliensis, S. schenckii, and S. globosa at concentrations lower than itraconazole, and it also showed antibiofilm activity. According to the results obtained by fluorimetry, electron microscopy, and particle characterization analyses, we observed that olorofim induced profound alterations on the cell surface and cell cycle arrest in S. brasiliensis yeasts. We also verified that these morphophysiological alterations impaired their ability to adhere to keratinocytes in vitro. Our results indicate that olorofim is a promising new antifungal against sporotrichosis agents.

Cuantificación de la carga viral en el tracto respiratorio superior en pacientes con COVID-19 al ingreso hospitalario y su asociación con la gravedad de la enfermedad / Upper respiratory tract viral load quantification in COVID-19 patients at hospital admission and its association with disease severity

Resumen Introducción: Aún se desconocen aspectos de la patogenia de COVID-19. Objetivo: Determinar la relación entre gravedad, mortalidad y replicación viral en pacientes con COVID-19. Métodos: Se analizaron características clínicas, gravedad de la enfermedad y mortalidad de 203 pacientes hospitalizados por COVID-19 y se correlacionaron con carga viral (CV) y ciclo umbral (Ct) al ingreso; se tomó hisopado nasofaríngeo. Resultados: Las CV medias en los pacientes sobrevivientes fueron las siguientes ante enfermedad leve a moderada, moderada a grave y grave: 6.8 × 106, 7.6 × 107 y 1.0 × 109; y en los pacientes con enfermedad crítica que fallecieron, la CV fue de 1.70 × 109. Los Ct fueron 26.06, 24.07, 22.66 y 21.78 para esos mismos grupos. En quienes fallecieron se observó mayor CV media al ingreso en comparación con quienes sobrevivieron (1.7 × 109 versus 9.84 × 106), p < 0.001. Se evidenció correlación significativa entre CV, gravedad y muerte (r = 0.254, p < 0.045 y r = 0.21, p < 0.015). La CV alta se asoció a mayor mortalidad intrahospitalaria en comparación con la CV baja (RM = 2.926, p < 0.017). Conclusión: La CV de SARS-CoV-2 determinada al ingreso hospitalario podría calificar el riesgo simultáneamente con otros factores descritos en COVID-19.

Abstract Introduction: There are aspects of COVID-19 pathogenesis that are still unknown. Objective: To determine the relationship between severity, mortality and viral replication in patients with COVID-19. Methods: Clinical characteristics, severity and mortality of 203 patients hospitalized for COVID-19 were analyzed and correlated with viral load (VL) and threshold cycle (TC) at admission; nasopharyngeal swab was obtained. Results: Mean VLs in surviving patients with mild to moderate, moderate to severe and severe disease were the following: 6.8 × 106, 7.6 × 107 and 1.0 × 109, respectively; and in patients with critical disease who died, VL was 1.70 × 109. TCs were 26.06, 24.07, 22.66 and 21.78 for the same groups. In those who died, a higher mean VL was observed at admission in comparison with those who survived (1.7 × 109 vs 9.84 × 106; p < 0.001). A significant correlation was observed between VL, severity and death (r = 0.254, p < 0.045 and r = 0.21, p < 0.015). High VL was associated with increased in-hospital mortality in comparison with low VL (OR = 2.926, p < 0.017). Conclusion: SARS-CoV-2 VL determined at hospital admission might classify risk simultaneously with other factors described in COVID-19.

The Flavonoid Hesperidin Methyl Chalcone Targets Cytokines and Oxidative Stress to Reduce Diclofenac-Induced Acute Renal Injury: Contribution of the Nrf2 Redox-Sensitive Pathway.

Hesperidin is derived from citrus fruits among other plants. Hesperidin was methylated to increase its solubility, generating hesperidin methyl chalcone (HMC), an emerging flavonoid that possess anti-inflammatory and antioxidant properties. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a powerful regulator of cellular resistance to oxidant products. Previous data evidenced HMC can activate Nrf2 signaling, providing antioxidant protection against diverse pathological conditions. However, its effects on kidney damage caused by non-steroidal anti-inflammatory drugs (NSAIDs) have not been evaluated so far. Mice received a nephrotoxic dose of diclofenac (200 mg/kg) orally followed by intra-peritoneal (i.p.) administration of HMC (0.03-3 mg/kg) or vehicle. Plasmatic levels of urea, creatinine, oxidative stress, and cytokines were assessed. Regarding the kidneys, oxidative parameters, cytokine production, kidney swelling, urine NGAL, histopathology, and Nrf2 mRNA expression and downstream targets were evaluated. HMC dose-dependently targeted diclofenac systemic alterations by decreasing urea and creatinine levels, and lipid peroxidation, as well as IL-6, IFN-γ, and IL-33 production, and restored antioxidant properties in plasma samples. In kidney samples, HMC re-established antioxidant defenses, inhibited lipid peroxidation and pro-inflammatory cytokines and upregulated IL-10, reduced kidney swelling, urine NGAL, and histopathological alterations. Additionally, HMC induced mRNA expression of Nrf2 and its downstream effectors HO-1 and Nqo1, as well as reduced the levels of Keap1 protein detected in renal tissue. The present data demonstrate HMC is a potential compound for the treatment of acute renal damage caused by diclofenac, a routinely prescribed non-steroidal anti-inflammatory drug.

Recognition of Cell Wall Mannosylated Components as a Conserved Feature for Fungal Entrance, Adaptation and Survival Within Trophozoites of Acanthamoeba castellanii and Murine Macrophages.

Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms.

Extracellular Vesicles Regulate Biofilm Formation and Yeast-to-Hypha Differentiation in Candida albicans.

In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation in vitro. By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence. IMPORTANCE The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.

Association between IL-10 systemic low level and highest pain score in patients during symptomatic SARS-CoV-2 infection.

BACKGROUND: Despite the wide variety of Covid-19 symptoms, pain and the related mechanisms underlying unsettled nociceptive status are still under-prioritized. Understanding the complex network of Covid-19-related pain may result in new lines of study. It is unknown whether patient's immunological background influences pain in the acute phase of Covid-19, including musculoskeletal pain. Thus, we evaluated the blood levels of selected molecules that are upregulated in SARS-CoV-2 infection and analyzed a possible correlation with pain during Covid-19. METHODS: A cohort of 20 hospitalized patients with confirmed diagnoses for Covid-19 were evaluated in the context of pain. Visual analogic scale (VAS) was applied to quantitate pain level. Blood tests were used to determine the systemic levels of cytokines (IL-10 and IL-1ß), substance P, and leptin. The data were correlated when appropriate to determine the association between pain-related markers and assessed pain intensity. RESULTS: Our findings show that systemic levels of IL-10 have strong negative correlation with pain intensity on Covid-19 patients. Additionally, we also show that leptin systemic levels were increased in Covid-19 patients with pain, however, with moderate positive correlation between these events. IL-1ß and SP levels did not differ between Covid-19 patients with or without pain. Men reported less pain compared to women. No differences were found between genders in the levels of the molecules evaluated in patients with pain. CONCLUSION: IL-10 has been described over the years as an anti-inflammatory and analgesic cytokine. The present data support that low IL-10 levels might contribute to Covid-19-associated pain.

Genomic Diversity Analysis Reveals a Strong Population Structure in Histoplasma capsulatum LAmA (Histoplasma suramericanum).

Histoplasmosis is a severe mycotic disease affecting thousands of immunocompetent and immunocompromised individuals with high incidence in Latin America, where the disease agents are Histoplasma capsulatum and Histoplasma suramericanum. In this work, we used whole-genome sequencing to infer the species diversity and the population structure of H. suramericanum in South America. We find evidence for strong population structure and little admixture within the species. Genome-level phylogenetic trees indicate the existence of at least three different discrete populations. We recovered the existence of a previously identified population, LAmB, and confirm that it is highly differentiated along the whole genome. We also find that H. suramericanum is composed of two populations, one in Northern South America, and another in the southern portion of the continent. Moreover, one of the lineages from the southern population is endemic to Rio de Janeiro and there was no association with clinical data and species isolated from patients with histoplasmosis. Our results point out the need to characterize the symptomatology of histoplasmosis caused by different species and lineages of Histoplasma spp.

Structural discrimination analysis for constraint selection in protein modeling.

MOTIVATION: Protein structure modeling can be improved by the use of distance constraints between amino acid residues, provided such data reflects-at least partially-the native tertiary structure of the target system. In fact, only a small subset of the native contact map is necessary to successfully drive the model conformational search, so one important goal is to obtain the set of constraints with the highest true-positive rate, lowest redundancy and greatest amount of information. In this work, we introduce a constraint evaluation and selection method based on the point-biserial correlation coefficient, which utilizes structural information from an ensemble of models to indirectly measure the power of each constraint in biasing the conformational search toward consensus structures. RESULTS: Residue contact maps obtained by direct coupling analysis are systematically improved by means of discriminant analysis, reaching in some cases accuracies often seen only in modern deep-learning-based approaches. When combined with an iterative modeling workflow, the proposed constraint classification optimizes the selection of the constraint set and maximizes the probability of obtaining successful models. The use of discriminant analysis for the valorization of the information of constraint datasets is a general concept with possible applications to other constraint types and modeling problems. AVAILABILITY AND IMPLEMENTATION: MSA for the targets in this work is available on https://github.com/m3g/2021_Bottino_Biserial. Modeling data supporting the findings of this study was generated at the Center for Computing in Engineering and Sciences, and is available from the corresponding author LM on request. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.