Background As the global coronavirus pandemic (COVID‐19) spreads across the world, new clinical challenges emerge in the hospital landscape. Among these challenges, the increased risk of coinfections is a major threat to the patients. Although still in a low number, due to the short time of the pandemic, studies that identified a significant number of hospitalised patients with COVID‐19 who developed secondary fungal infections that led to serious complications and even death have been published. Objectives In this scenario, we aim to determine the prevalence of invasive fungal infections (IFIs) and describe possible associated risk factors in patients admitted due to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. Patients/Methods We designed an open prospective observational study at the Rey Juan Carlos University Hospital (Mostoles, Spain), during the period from February 1 to April 30, 2020. Results In this article, we reported seven patients with COVID‐19‐associated pulmonary aspergillosis (CAPA) who had a poor prognosis. Severely ill patients represent a high‐risk group; therefore, we must actively investigate the possibility of aspergillosis in all of these patients. Larger cohort studies are needed to unravel the role of COVID‐19 immunosuppressive therapy as a risk factor for aspergillosis. Conclusions As the pandemic continues to spread across the world, further reports are needed to assess the frequency of emergent and highly resistant reemergent fungal infections during severe COVID‐19. These coinfections are leading a significant number of patients with COVID‐19 to death due to complications following the primary viral disease.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a C. neoformans mutant lacking the gene encoding sterylglucosidase (Δsgl1) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δsgl1 strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the SGL1 gene in the acapsular mutant Δcap59 to generate a double mutant (strain Δcap59/Δsgl1) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δcap59/Δsgl1 mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of Galleria mellonella against C. neoformans infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δsgl1 EVs as a vaccination strategy for cryptococcosis. IMPORTANCE The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipid-containing structures in a vaccination model to prevent cryptococcosis.

Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, L-dihydroxyphenylalanine (L-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence. Melanins are polymers with diverse molecular structures, typically black or dark brown, formed by the oxidative polymerization of phenolic and indolic compounds. They are produced by a broad range of organisms, from bacteria to humans. Several fungi can produce melanins, and the functions of these pigments are related to microbial survival under several unfavorable environmental and host conditions (10, 14). The major melanin type encountered among fungi is 1,8-dihydroxynaphthalene melanin (DHN-melanin), which is synthesized from acetyl coenzyme A via the polyketide pathway. This form of melanin is synthesized by several plant and human fungal pathogens. In addition to DHN-melanin, certain fungi can also produce melanin via dihydroxyphenylalanine (DOPA), in which tyrosinases or laccases hydroxylate tyrosine via DOPA to dopaquinone, which then auto-oxidizes and polymerizes, resulting in a polyphenolic heteropolymer that is black (9). Some fungi produce a soluble melanin from L-tyrosine through p-hydroxyphenylpyruvate and homogentisic acid. This soluble pigment is called pyomelanin, and it is similar to the alkaptomelanin produced by humans. Aspergillus fumigatus, Madurella mycetomatis, and Yarrowia lipolytica are examples of fungi that can produce this type of soluble pigment (4,18,20)...

We propose to use optical tweezers to probe the Casimir interaction between microspheres inside a liquid medium for geometric aspect ratios far beyond the validity of the widely employed proximity force approximation. This setup has the potential for revealing unprecedented features associated to the non-trivial role of the spherical curvatures. For a proof of concept, we measure femtonewton double layer forces between polystyrene microspheres at distances above 400 nm by employing very soft optical tweezers, with stiffness of the order of fractions of a fN/nm. As a future application, we propose to tune the Casimir interaction between a metallic and a polystyrene microsphere in saline solution from attraction to repulsion by varying the salt concentration. With those materials, the screened Casimir interaction may have a larger magnitude than the unscreened one. This line of investigation has the potential for bringing together different fields including classical and quantum optics, statistical physics and colloid science, while paving the way for novel quantitative applications of optical tweezers in cell and molecular biology.

Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence.

Background Over 100 million people worldwide suffer from birch pollen allergy. However, identification of molecular determinants driving Th2-biased allergic sensitization to Bet v 1, the major birch pollen allergen, remains elusive. Objective Here, we examined whether Bet v 1 or the pollen matrix is responsible for activation of antigen-presenting cells and the subsequent Th2 polarization, relevant in the process of allergic sensitization. Methods The allergenicity of Bet v 1 and of birch pollen extract (BPE) was addressed by stimulation of murine and human dendritic cells and by in vivo monitoring of Th2 polarization. Further, Bet v 1 was depleted from BPE by immunoprecipitation in order to analyze its involvement in the occurrence of a Th2 response. Results The allergen alone did neither stimulate dendritic cells in vitro nor induced Th2 polarization in vivo, even in the presence of the natural LPS concentration determined in the BPE. In contrast, BPE was shown to activate dendritic cells and strongly promoted a Th2 polarization. Even upon immunization with Bet v 1-depleted BPE the amount of induced Th2 cells remained unaltered. Conclusion This finding indicates that the Th2-polarizing potential of BPE is Bet v 1 independent; therefore, sensitization to Bet v 1 is induced by an as-yet-undetermined pollen compound or mechanism in the pollen environment. These data suggest that sensitization is not exclusively linked to the intrinsic properties of individual proteins. These findings are relevant in understanding allergic sensitization towards pollen allergens and might pave the way for future prophylactic approaches.

Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.

BackgroundThe viscoelastic properties of cells have been investigated by a variety of techniques. However, the experimental data reported in literature for viscoelastic moduli differ by up to three orders of magnitude. This has been attributed to differences in techniques and models for cell response as well as to the natural variability of cells.ResultsIn this work we develop and apply a new methodology based on optical tweezers to investigate the rheological behavior of fibroblasts, neurons and astrocytes in the frequency range from 1Hz to 35Hz, determining the storage and loss moduli of their membrane-cortex complex. To avoid distortions associated with cell probing techniques, we use a previously developed method that takes into account the influence of under bead cell thickness and bead immersion. These two parameters were carefully measured for the three cell types used. Employing the soft glass rheology model, we obtain the scaling exponent and the Young’s modulus for each cell type. The obtained viscoelastic moduli are in the order of Pa. Among the three cell types, astrocytes have the lowest elastic modulus, while neurons and fibroblasts exhibit a more solid-like behavior.ConclusionsAlthough some discrepancies with previous results remain and may be inevitable in view of natural variability, the methodology developed in this work allows us to explore the viscoelastic behavior of the membrane-cortex complex of different cell types as well as to compare their viscous and elastic moduli, obtained under identical and well-defined experimental conditions, relating them to the cell functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13628-016-0031-4) contains supplementary material, which is available to authorized users.