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L-3,4-dihydroxyphenylalanine (L-DOPA), a naturally occurring tyrosine derivative, is prevalent in environments that include mosquito habitats, potentially serving as part of their diet. Given its role as a precursor for melanin synthesis we investigated the effect of dietary L-DOPA on mosquito physiology and immunity to Plasmodium falciparum and Cryptococcus neoformans infection. Dietary L-DOPA was incorporated into mosquito melanin via a non-canonical pathway and had profound transcriptional effects that were associated with enhanced immunity, increased pigmentation, and reduced lifespan. Increased melanization resulted in an enhanced capacity to absorb electromagnetic radiation that affected mosquito temperatures. Bacteria in the mosquito microbiome were sources of dopamine, which is a substrate for melanization. Our results illustrate how an environmentally abundant amino acid analogue can affect mosquito physiology and suggest its potential usefulness as an environmentally friendly vector control agent to reduce malaria transmission, warranting further research and field studies.

Cryptococcus neoformans is a fungal pathogen that can cause life-threatening brain infections in immunocompromised individuals. Unlike other fungal pathogens, it possesses a protective polysaccharide capsule that is crucial for its virulence. During infections, Cryptococcus cells release copious amounts of extracellular polysaccharides (exo-PS) that interfere with host immune responses. Both exo-PS and capsular-PS play pivotal roles in Cryptococcus infections and serve as essential targets for disease diagnosis and vaccine development strategies. However, understanding their structure is complicated by their polydispersity, complexity, sensitivity to sample isolation and processing, and scarcity of methods capable of isolating and analyzing them while preserving their native structure. In this study, we employ small-angle neutron scattering (SANS) and ultra-small-angle neutron scattering (USANS) for the first time to investigate both fungal cell suspensions and extracellular polysaccharides in solution. Our data suggests that exo-PS in solution exhibits collapsed chain-like behavior and demonstrates mass fractal properties that indicate a relatively condensed pore structure in aqueous environments. This observation is also supported by scanning electron microscopy (SEM). The local structure of the polysaccharide is characterized as a rigid rod, with a length scale corresponding to 3-4 repeating units. This research not only unveils insights into exo-PS and capsular-PS structures but also demonstrates the potential of USANS for studying changes in cell dimensions and the promise of contrast variation in future neutron scattering studies.

Understanding the mechanisms of antibody-mediated neutralization of SARS-CoV-2 is critical in combating the COVID-19 pandemic. Based on previous reports of antibody catalysis, we investigated the proteolysis of spike (S) by antibodies in COVID-19 convalescent plasma (CCP) and its contribution to viral neutralization. Quenched fluorescent peptides were designed based on S epitopes to sensitively detect antibody-mediated proteolysis. We observed epitope cleavage by CCP from different donors which persisted when plasma was heat-treated or when IgG was isolated from plasma. Further, purified CCP antibodies proteolyzed recombinant S domains, as well as authentic viral S. Cleavage of S variants suggests CCP antibody-mediated proteolysis is a durable phenomenon despite antigenic drift. We differentiated viral neutralization occurring via direct interference with receptor binding from that occurring by antibody-mediated proteolysis, demonstrating that antibody catalysis enhanced neutralization. These results suggest that antibody-catalyzed damage of S is an immunologically relevant function of neutralizing antibodies against SARS-CoV-2., Competing Interests: Declaration of interests None declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Practical experiments drive important scientific discoveries in biology, but theory-based research studies also contribute novel-sometimes paradigm-changing-findings. Here, we appraise the roles of theory-based approaches focusing on the experiment-dominated wet-biology research areas of microbial growth and survival, cell physiology, host-pathogen interactions, and competitive or symbiotic interactions. Additional examples relate to analyses of genome-sequence data, climate change and planetary health, habitability, and astrobiology. We assess the importance of thought at each step of the research process; the roles of natural philosophy, and inconsistencies in logic and language, as drivers of scientific progress; the value of thought experiments; the use and limitations of artificial intelligence technologies, including their potential for interdisciplinary and transdisciplinary research; and other instances when theory is the most-direct and most-scientifically robust route to scientific novelty including the development of techniques for practical experimentation or fieldwork. We highlight the intrinsic need for human engagement in scientific innovation, an issue pertinent to the ongoing controversy over papers authored using/authored by artificial intelligence (such as the large language model/chatbot ChatGPT). Other issues discussed are the way in which aspects of language can bias thinking towards the spatial rather than the temporal (and how this biased thinking can lead to skewed scientific terminology); receptivity to research that is non-mainstream; and the importance of theory-based science in education and epistemology. Whereas we briefly highlight classic works (those by Oakes Ames, Francis H.C. Crick and James D. Watson, Charles R. Darwin, Albert Einstein, James E. Lovelock, Lynn Margulis, Gilbert Ryle, Erwin R.J.A. Schrödinger, Alan M. Turing, and others), the focus is on microbiology studies that are more-recent, discussing these in the context of the scientific process and the types of scientific novelty that they represent. These include several studies carried out during the 2020 to 2022 lockdowns of the COVID-19 pandemic when access to research laboratories was disallowed (or limited). We interviewed the authors of some of the featured microbiology-related papers and-although we ourselves are involved in laboratory experiments and practical fieldwork-also drew from our own research experiences showing that such studies can not only produce new scientific findings but can also transcend barriers between disciplines, act counter to scientific reductionism, integrate biological data across different timescales and levels of complexity, and circumvent constraints imposed by practical techniques. In relation to urgent research needs, we believe that climate change and other global challenges may require approaches beyond the experiment., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Fungi play essential roles in global health, ecology, and economy, but their thermal biology is relatively unexplored. Mushrooms, the fruiting body of mycelium, were previously noticed to be colder than surrounding air through evaporative cooling. Here, we confirm those observations using infrared thermography and report that this hypothermic state is also observed in mold and yeast colonies. The relatively colder temperature of yeasts and molds is also mediated via evaporative cooling and associated with the accumulation of condensed water droplets on plate lids above colonies. The colonies appear coldest at their center and the surrounding agar appears warmest near the colony edges. The analysis of cultivated Pleurotus ostreatus mushrooms revealed that the hypothermic feature of mushrooms can be observed throughout the whole fruiting process and at the level of mycelium. The mushroom's hymenium was coldest, and different areas of the mushroom appear to dissipate heat differently. We also constructed a mushroom-based air-cooling prototype system capable of passively reducing the temperature of a semiclosed compartment by approximately 10 °C in 25 min. These findings suggest that the fungal kingdom is characteristically cold. Since fungi make up approximately 2% of Earth's biomass, their evapotranspiration may contribute to cooler temperatures in local environments.

Melanin is a complex pigment that is found in various fungal species and is associated with a multitude of protective functions against environmental stresses. In Cryptococcus neoformans, melanin is synthesized from exogenous substrate and deposited in the cell wall. Although melanin is often cited as a protector against mechanical stress, there is a paucity of direct experimental data supporting this claim. To probe whether melanin enhances cellular strength, we used ultrasonic cavitation and French cell press pressure to stress cryptococcal cells and then measured changes in cellular morphology and fragmentation for melanized and nonmelanized C. neoformans cells. Melanized yeast cells exhibited lower rates of fragmentation and greater cell areas than did nonmelanized yeast cells after sonication or French press passage. When subjected to French press passage, both melanized and nonmelanized cells exhibited responses that were dependent on their culture age. Our results indicate that melanization protects against some of the morphological changes, such as fragmentation and cellular shrinkage, that are initiated by mechanical energy derived from either sonic cavitation or French press passage, thus supporting the notion that this pigment provides mechanical strength for fungal cell walls. IMPORTANCE Melanin was shown in prior microbiological experiments to be associated with protection against environmental stressors, and it has often been cited as being associated with mechanical stress protection. However, there is a lack of direct experimentation to confirm this claim. We examined the responses of melanized and nonmelanized C. neoformans cells to sonication and French press passage, and we report differences in outcomes depending not only on melanization status but also on culture age. Such findings have important implications for the design and interpretation of laboratory experiments involving C. neoformans. In addition, the elucidation of some of the mechanical properties of melanin promotes further research into fungal melanin applications in health care and industry.

As human activity in space continues to increase, understanding how biological assets respond to spaceflight conditions is becoming more important. Spaceflight conditions include exposure to ionizing radiation, microgravity, spacecraft vibrations and hypervelocity; all of which can affect the viability of biological organisms. Previous studies have shown that melanin-producing fungi are capable of surviving the vacuum of space and Mars-simulated conditions in Low Earth Orbit. This survival has been associated in part with the protective effects of melanin, but a comparison of fungal viability in the presence or absence of melanin following spaceflight has never been tested. In this study, we evaluated the protective effects of melanin by comparing the viability of melanized and non-melanized clones of Cryptococcus neoformans yeasts following a roundtrip to the International Space Station. Yeast colonies were placed inside two MixStix silicone tubes; one stayed on Earth and the other was transported inside for 29 days before returning to Earth. Post-flight analysis based on colony-forming unit numbers shows that melanized yeast viability was 50% higher than non-melanized yeasts, while no difference was observed between the Earth-bound control samples. The results suggest that fungal melanin could increase the lifespan of biological assets in space., (© 2022 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded complete characterization. Cryptococcal polysaccharides are known to either remain attached to the cell as capsular polysaccharides (CPSs) or to be shed into the extracellular space as exopolysaccharides (EPSs). While many studies have examined the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and isolated capsular material. We show that sonication or Glucanex enzyme cocktail digestion yields soluble CPS preparations, while use of a French pressure cell press or Glucanex digestion followed by cell disruption removed the capsule and produced cell wall-associated polysaccharide aggregates that we call "capsule ghosts", implying an inherent organization that allows the CPS to exist independent of the cell wall surface. Since sonication and Glucanex digestion were noncytotoxic, it was also possible to observe the cryptococcal cells rebuilding their capsule, revealing the presence of reducing end glycans throughout the capsule. Finally, analysis of dimethyl sulfoxide-extracted and sonicated CPS preparations revealed the conservation of previously identified glucuronoxylomannan motifs only in the sonicated CPS. Together, these observations provide new insights into capsule architecture and synthesis, consistent with a model in which the capsule is assembled from the cell wall outward using smaller polymers, which are then compiled into larger ones., Competing Interests: Conflict of interest The authors have no conflicts of interest to report., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)