Glutathione-mediated redox regulation in Cryptococcus neoformans impacts virulence.

The fungal pathogen Cryptococcus neoformans is well adapted to its host environment. It has several defence mechanisms to evade oxidative and nitrosative agents released by phagocytic host cells during infection. Among them, melanin production is linked to both fungal virulence and defence against harmful free radicals that facilitate host innate immunity. How C. neoformans manipulates its redox environment to facilitate melanin formation and virulence is unclear. Here we show that the antioxidant glutathione is inextricably linked to redox-active processes that facilitate melanin and titan cell production, as well as survival in macrophages and virulence in a murine model of cryptococcosis. Comparative metabolomics revealed that disruption of glutathione biosynthesis leads to accumulation of reducing and acidic compounds in the extracellular environment of mutant cells. Overall, these findings highlight the importance of redox homeostasis and metabolic compensation in pathogen adaptation to the host environment and suggest new avenues for antifungal drug development.

Artificial intelligence for basal cell carcinoma: diagnosis and distinction from histological mimics.

We trained an artificial intelligence (AI) algorithm to identify basal cell carcinoma (BCC), and to distinguish BCC from histological mimics. A total of 1061 glass slides were collected: 616 containing BCC and 445 without BCC. BCC slides were collected prospectively, reflecting the range of specimen types and morphological variety encountered in routine pathology practice. Benign and malignant histological mimics of BCC were selected prospectively and retrospectively, including cases considered diagnostically challenging for pathologists. Glass slides were digitally scanned to create a whole slide image (WSI), which was divided into patches representing a tissue area of 65,535 µm2. Pathologists annotated the data, yielding 87,205 patches labelled BCC present and 1,688,697 patches labelled BCC absent. The COMPASS model (COntext-aware Multi-scale tool for Pathologists Assessing SlideS) based on Convolutional Neural Networks, was trained to provide a probability of BCC being present at the patch level and the slide level. The test set comprised 246 slides, 147 of which contained BCC. The COMPASS AI model demonstrated high accuracy, classifying WSIs as containing BCC with a sensitivity of 98.0% and a specificity of 97.0%, representing 240 WSIs classified correctly, three false positives, and three false negatives. Using BCC as a proof of concept, we demonstrate how AI can account for morphological variation within an entity, and accurately distinguish from histologically similar entities. Our study highlights the potential for AI in routine pathology practice.

Disaster mycology / Micología de desastres

Natural and human-made disasters have long played a role in shaping the environment and microbial communities, also affecting non-microbial life on Earth. Disaster microbiology is a new concept based on the notion that a disaster changes the environment causing adaptation or alteration of microbial populations-growth, death, transportation to a new area, development traits, or resistance-that can have downstream effects on the affected ecosystem. Such downstream effects include blooms of microbial populations and the ability to colonize a new niche or host, cause disease, or survive in former extreme conditions. Throughout history, fungal populations have been affected by disasters. There are prehistoric archeological records of fungal blooms after asteroid impacts and fungi implicated in the fall of the dinosaurs. In recent times, drought and dust storms have caused disturbance of soil fungi, and hurricanes have induced the growth of molds on wet surfaces, resulting in an increased incidence of fungal disease. Probably, the anticipated increase in extreme heat would force fungi adaptation to survive at high temperatures, like those in the human body, and thus be able to infect mammals. This may lead to a drastic rise of new fungal diseases in humans.

Los desastres naturales o los causados por el hombre impactan la formación de ecosistemas y comunidades microbianas, y también afectan las formas de vida no microbianas. Este concepto es conocido como "microbiología de desastres", una subespecialización de la microbiología, basada en los cambios ambientales generados por un desastre y las posibles adaptaciones o alteraciones de las poblaciones microbianas -crecimiento, muerte, trasporte a una nueva región, o adquisición de resistencia o de nuevas características- que influirán en el moldeamiento del ecosistema transformado. Algunos de los efectos de estas adaptaciones pueden ser: el surgimiento de poblaciones microbianas, la habilidad de colonizar nuevos nichos u huéspedes, la generación de nuevas enfermedades, o el crecimiento de microorganismos en condiciones que antes eran "extremas" para ellos. A lo largo de la historia, varias poblaciones de hongos han sido afectadas por desastres. Existen registros arqueológicos prehistóricos que evidencian la presencia y el crecimiento de hongos luego del impacto de asteroides, y otros de hongos relacionados con la extinción de los dinosaurios. Actualmente, las sequías y las tormentas de polvo causan perturbaciones en las comunidades de hongos del suelo, y los huracanes inducen el crecimiento de hongos filamentosos en superficies húmedas, lo que aumenta la cantidad de enfermedades por hongos. Además, con el aumento de las temperaturas extremas es posible que los hongos puedan adaptarse para sobrevivir a temperaturas más altas, equivalentes a las temperaturas corporales, y nuevas especies puedan infectar mamíferos. Esto puede llevar a un aumento drástico de las infecciones fúngicas en humanos.

A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule.

Classic antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.

Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles.

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Childhood cancer incidence rates and hazardous air pollutants in California: an exploratory analysis.

Hazardous air pollutants (HAPs) are compounds shown to cause cancer or other adverse health effects. We analyzed population-based childhood cancer incidence rates in California (USA) from 1988 to 1994, by HAP exposure scores, for all California census tracts. For each census tract, we calculated exposure scores by combining cancer potency factors with outdoor HAP concentrations modeled by the U.S. Environmental Protection Agency. We evaluated the relationship between childhood cancer rates and exposure scores for 25 potentially carcinogenic HAPs emitted from mobile, area, and point sources and from all sources combined. Our study period saw 7,143 newly diagnosed cancer cases in California; of these, 6,989 (97.8%) could be assigned to census tracts and included in our analysis. Using Poisson regression, we estimated rate ratios (RRs) adjusted for age, race/ethnicity, and sex. We found little evidence for elevated cancer RRs for all sites or for gliomas among children living in high-ranking combined-source exposure areas. We found elevated RRs and a significant trend with increasing exposure level for childhood leukemia in tracts ranked highest for exposure to the combined group of 25 HAPs (RR = 1.21; 95% confidence interval, 1.03, 1.42) and in tracts ranked highest for point-source HAP exposure (RR = 1.32; 95% confidence interval, 1.11, 1.57). Our findings suggest an association between increased childhood leukemia rates and high HAP exposure, but studies involving more comprehensive exposure assessment and individual-level exposure data will be important for elucidating this relationship.