The buoyancy of cryptococcal cells and its implications for transport and persistence of Cryptococcus in aqueous environments.

Cryptococcus is a genus of saprophytic fungi with global distribution. Two species complexes, C. neoformans and C. gattii, pose health risks to humans and animals. Cryptococcal infections result from inhalation of aerosolized spores and/or desiccated yeasts from terrestrial reservoirs such as soil, trees, and avian guano. More recently, C. gattii has been implicated in infections in marine mammals, suggesting that inhalation of liquid droplets or aerosols from the air-water interface is also an important, yet understudied, mode of respiratory exposure. Water transport has also been suggested to play a role in the spread of C. gattii from tropical to temperate environments. However, the dynamics of fungal survival, persistence, and transport via water have not been fully studied. The size of the cryptococcal capsule was previously shown to reduce cell density and increase buoyancy. Here, we demonstrate that cell buoyancy is also impacted by the salinity of the media in which cells are suspended, with formation of a halocline interface significantly slowing the rate of settling of cryptococcal cells through water, resulting in persistence of C. neoformans within 1 cm of the air-water interface for over 60 min and C. gattii for 4-6 h. Our data also showed that during culture in yeast peptone dextrose media (YPD), polysaccharide accumulating in the supernatant formed a raft that augmented buoyancy and further slowed settling of cryptococcal cells. These findings illustrate new mechanisms by which cryptococcal cells may persist in aquatic environments, with important implications for aqueous transport and pathogen exposure.

Host and fungal factors both contribute to cryptococcosis-associated hyperammonemia (cryptammonia).

Cryptococcus neoformans and Cryptococcus gattii are both known urease producers and have the potential to cause hyperammonemia. We hypothesized that the risk of hyperammonemia is increased by renal failure, burden of cryptococcal infection, and fungal strain characteristics. We performed a retrospective review of plasma ammonia levels in patients with cryptococcal infections. Risk factors for hyperammonemia were statistically compared between patients with and without hyperammonemia (>53 µmol/L). Cryptococcal cells from three patients included in the study were recovered from our biorepository. Strain characteristics including urease activity, ammonia production, growth curves, microscopy, melanin production, and M13 molecular typing were analyzed and compared with a wild-type (WT) C. neoformans strain. We included 29 patients, of whom 37.9% had hyperammonemia, 59% had disseminated cryptococcal infection (DCI), and 41% had isolated central nervous system infection. Thirty-eight percent of patients had renal failure and 28% had liver disease. Renal failure was associated with 4.4 times (95% confidence interval [CI] 1.5, 13.0) higher risk of hyperammonemia. This risk was higher in DCIs (RR 6.2, 95% CI 1.0, 40.2) versus isolated cryptococcal meningitis (RR 2.5, 95% CI, 0.40, 16.0). Liver disease and cryptococcal titers were not associated with hyperammonemia. C. neoformans from one patient with extreme hyperammonemia demonstrated a 4- to 5-fold increase in extracellular urease activity, slow growth, enlarged cell size phenotypes, and diminished virulence factors. Hyperammonemia was strongly associated with renal failure in individuals with DCI, surpassing associations with liver failure or cryptococcal titers. However, profound hyperammonemia in one patient was attributable to high levels of urease secretion unique to that cryptococcal strain. Prospective studies are crucial to exploring the significance of this association.IMPORTANCECryptococcus produces and secretes the urease enzyme to facilitate its colonization of the host. Urease breaks down urea into ammonia, overwhelming the liver's detoxification process and leading to hyperammonemia in some hosts. This underrecognized complication exacerbates organ dysfunction alongside the infection. Our study investigated this intricate relationship, uncovering a strong association between the development of hyperammonemia and renal failure in patients with cryptococcal infections, particularly those with disseminated infections. We also explore mechanisms underlying increased urease activity, specifically in strains associated with extreme hyperammonemia. Our discoveries provide a foundation for advancing research into cryptococcal metabolism and identifying therapeutic targets to enhance patient outcomes.

Comprehensive characterization of extracellular vesicles produced by environmental (Neff) and clinical (T4) strains of Acanthamoeba castellanii.

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.

Immunoglobulin constant regions provide stabilization to the paratope and enforce epitope specificity.

Constant domains in antibody molecules at the level of the Fab (CH1 and CL) have long been considered to be simple scaffolding elements that physically separate the paratope-defining variable (V) region from the effector function-mediating constant (C) regions. However, due to recent findings that C domains of different isotypes can modulate the fine specificity encoded in the V region, elucidating the role of C domains in shaping the paratope and influencing specificity is a critical area of interest. To dissect the relative contributions of each C domain to this phenomenon, we generated antibody fragments with different C regions omitted, using a set of antibodies targeting capsular polysaccharide from the fungal pathogen, Cryptococcus neoformans. Antigen specificity mapping and functional activity measurements revealed that V region-only antibody fragments exhibited poly-specificity to antigenic variants and extending to recognition of self-antigens, while measurable hydrolytic activity of the capsule was greatly attenuated. To better understand the mechanistic origins of the remarkable loss of specificity that accompanies the removal of C domains from identical paratopes, we performed molecular dynamics simulations which revealed increased paratope plasticity in the scFv relative to the corresponding Fab. Together, our results and provide insight into how the remarkable specificity of immunoglobulins is governed and maintained at the level of the Fab through the enforcement of structural restrictions on the paratope by CH1 domains.

The Logic and History of Passive Immunity and Antibody Therapies.

This volume takes a broad overview of antibody-based therapies prior to and during the COVID pandemic and examines their potential use in future pandemics. Passive antibody therapy was the first effective antimicrobial treatment and its development in the early twentieth century helped catalyze immunological and microbiological research. During the era of serum therapy (1890-1940) antibody-based therapies were developed against both viral and bacterial diseases. Effective treatment required an understanding of how to quantify antibodies, how to develop serotype-specific sera and recognition of the need to treat early in disease. Thus, although the era of serum therapy essentially ended with the development of small molecule antimicrobial therapy in the 1940s, antibody-based therapies led to important new scientific understanding, while remaining in use for some toxin and venom-caused diseases and in the prevention of outbreaks of viral hepatitis. A renewed interest in antibody-based therapies was seen in the widespread deployment of convalescent plasma and monoclonal antibodies during the COVID-19 pandemic. Convalescent plasma will likely be the first specific therapy during outbreaks with new pathogens for which there is no other therapy. For all forms of antibody-based therapies, effectiveness relies on the key principles of antibody therapy, namely, treatment early in disease with preparations containing sufficient antibody specific to the microbe in question.

Applying lessons of COVID-19 and other emerging infectious diseases to future outbreaks.

Infectious diseases are emerging and re-emerging far more frequently than many appreciate. In the past two decades alone, there have been numerous outbreaks (e.g., Ebola, chikungunya, Zika, and Mpox) and pandemics (i.e., swine flu and coronavirus disease 2019) with profound effects to public health, the economy, and society at large. Rather than view these in isolation, there are important lessons pertaining to how best to contend with future outbreaks of emerging infectious diseases. Those lessons span definition (i.e., what constitutes a pandemic), through deficiencies in surveillance, data collection and reporting, the execution of research in a rapidly changing environment, the nuances of study design and hierarchy of clinical evidence, triage according to clinical need as supply chains become overwhelmed, and the challenges surrounding forecasting of outbreaks. Understanding those lessons and drawing on both the successes and failures of the past are imperative if we are to overcome the challenges of outbreak/pandemic responsiveness.

Semisynthetic Glycoconjugate Vaccine Candidates against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.

Methods of Cryptococcal Polysaccharide Analysis Using ELISA.

One of the standard assays for the fungal pathogen Cryptococcus neoformans is the glucuronoxylomannan (GXM) ELISA. This assay utilizes monoclonal antibodies targeted against the critical virulence factor, the polysaccharide (PS) capsule. GXM ELISA is one of the most used assays in the field used for diagnosis of cryptococcal infection, quantification of PS content, and determination of binding specificity for antibodies. Here we present three variations of the GXM ELISA used by our group-indirect, capture, and competition ELISAs. We have also provided some history, perspective, and notes on these methods, which we hope will help the reader choose, and implement, the best assay for their research.While it has long been referred to as the GXM ELISA, we also suggest a name update to better reflect our updated understanding of the polysaccharide antigens targeted by this assay. The Cryptococcal PS ELISA is a more accurate description of this set of methodologies and the antigens they measure. Finally, we discuss the limitations of this assay and put forth future plans for expanding the antigens assayed by ELISA.

A concept for international societally relevant microbiology education and microbiology knowledge promulgation in society.

EXECUTIVE SUMMARY: Microbes are all pervasive in their distribution and influence on the functioning and well-being of humans, life in general and the planet. Microbially-based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision-makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative-the IMiLI-is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence-based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner-centric, not academic microbiology-centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators-learners. As such, they will collect and analyse feedback from the end-users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators-learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships-a global societally relevant microbiology education ecosystem-in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. ABSTRACT: The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well-being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us-individuals/communities/nations/the human world-and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. HERE, WE PRESENT: our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision-making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner-centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091-1111). Importantly, although the primary target of the freely available (CC BY-NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity-driven, web-based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

Passive immunotherapies for the next influenza pandemic.

Influenzavirus is among the most relevant candidates for a next pandemic. We review here the phylogeny of former influenza pandemics, and discuss candidate lineages. After briefly reviewing the other existing antiviral options, we discuss in detail the evidences supporting the efficacy of passive immunotherapies against influenzavirus, with a focus on convalescent plasma.

An update on the anti-spike monoclonal antibody pipeline for SARS-CoV-2.

BACKGROUND: Anti-spike monoclonal antibodies represent one of the most tolerable prophylaxis and therapies for COVID-19 in frail and immunocompromised patients. Unfortunately, viral evolution in Omicron has led all of them to failure. OBJECTIVES: We review here the current pipeline of anti-spike mAb's, discussing in detail the most promising candidates. SOURCES: We scanned PubMed, ClinicalTrials.gov and manufacturers' press releases for clinical studies on anti-spike monoclonal antibodies. CONTENT: We present state-of-art data clinical progress for AstraZeneca's AZD3152, Invivyd's VYD222, Regeneron's REGN-17092 and Aerium Therapeutics' AER-800. IMPLICATIONS: The anti-spike monoclonal antibody clinical pipeline is currently limited to few agents (most being single antibodies) with unknown efficacy against the dominant JN.1 sublineage. The field of antibody-based therapies requires boosting by both manufacturers and institutions.

Fragile science.

Science currently faces major external and internal threats. External threats include persistent anti-science attacks, the post-pandemic politicization of public health, and chronic underfunding. Internal threats include a proliferation of low-quality studies, an epidemic of retractions, and questions regarding the reproducibility of important research findings. These threats occur just as humanity faces an unprecedented onslaught of existential challenges including climate change, a failing green revolution, pandemics, and severe environmental degradation of the planet, each of which will require scientific solutions. History shows that science is fragile and vulnerable to theocratic, ideological, and authoritarian forces. In this moment of crisis, it is important for all scientists to become foot soldiers in the defense of science.

Safety and Efficacy of Convalescent Plasma Combined with Other Pharmaceutical Agents for Treatment of COVID-19 in Hospitalized Patients: A Systematic Review and Meta-Analysis.

Plasma collected from people recovered from COVID-19 (COVID-19 convalescent plasma, CCP) was the first antibody-based therapy employed to fight the pandemic. CCP was, however, often employed in combination with other drugs, such as the antiviral remdesivir and glucocorticoids. The possible effect of such interaction has never been investigated systematically. To assess the safety and efficacy of CCP combined with other agents for treatment of patients hospitalized for COVID-19, a systematic literature search using appropriate Medical Subject Heading (MeSH) terms was performed through PubMed, EMBASE, Cochrane central, medRxiv and bioRxiv. The main outcomes considered were mortality and safety of CCP combined with other treatments versus CCP alone. This review was carried out in accordance with Cochrane methodology including risk of bias assessment and grading of the quality of evidence. Measure of treatment effect was the risk ratio (RR) together with 95% confidence intervals (CIs). A total of 11 studies (8 randomized controlled trials [RCTs] and 3 observational) were included in the systematic review, 4 studies with CCP combined with remdesivir and 6 studies with CCP combined with corticosteroids, all involving hospitalized patients. One RCT reported information on both remdesivir and steroids use with CCP. The use of CCP combined with remdesivir was associated with a significantly reduced risk of death (RR 0.74; 95% CI 0.56-0.97; p = 0.03; moderate certainty of evidence), while the use of steroids with CCP did not modify the mortality risk (RR 0.72; 95% CI 0.34-1.51; p = 0.38; very low certainty of evidence). Not enough safety data were retrieved form the systematic literature analysis. The current evidence from the literature suggests a potential beneficial effect on mortality of combined CCP plus remdesivir compared to CCP alone in hospitalized COVID-19 patients. No significant clinical interaction was found between CCP and steroids.

Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia.

Cryptococcus neoformans causes lethal meningitis and accounts for approximately 10%-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this fungus invades the mammalian brain. To investigate the dynamics of C. neoformans tissue invasion, we mapped fungal localization and host cell interactions in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an in situ imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. We confirm high fungal burden in mouse upper airway after nasal inoculation. Yeast in turbinates were frequently titan cells, with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of the upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of C. neoformans, by finding viable fungi in the bloodstream of mice a few days after intranasal infection. As early as 24 h post systemic infection, the majority of C. neoformans cells traversed the blood-brain barrier, and were engulfed or in close proximity to microglia. Our work presents a new method for investigating microbial invasion, establishes that C. neoformans can breach multiple tissue barriers within the first days of infection, and demonstrates microglia as the first cells responding to C. neoformans invasion of the brain.IMPORTANCECryptococcal meningitis causes 10%-15% of AIDS-associated deaths globally. Still, brain-specific immunity to cryptococci is a conundrum. By employing innovative imaging, this study reveals what occurs during the first days of infection in brain and in airways. We found that titan cells predominate in upper airways and that cryptococci breach the upper airway mucosa, which implies that, at least in mice, the upper airways are a site for fungal dissemination. This would signify that mucosal immunity of the upper airway needs to be better understood. Importantly, we also show that microglia, the brain-resident macrophages, are the first responders to infection, and microglia clusters are formed surrounding cryptococci. This study opens the field to detailed molecular investigations on airway immune response, how fungus traverses the blood-brain barrier, how microglia respond to infection, and ultimately how microglia monitor the blood-brain barrier to preserve brain function.

Analysis of justification for and gender bias in author order among those contributing equally.

The practice of designating two or more authors as equal contributors (EC) on a scientific publication is increasingly common as a form of sharing credit. However, EC authors are often unclearly attributed on CVs or citation engines, and it is unclear how research teams determine author order within an EC listing. In response to studies showing that male authors were more likely to be placed first in an EC listing, the American Society of Microbiology (ASM) required that authors explain the reasons for author order beginning in 2020. In this study we analyze data from over 2500 ASM publications to see how this policy affected gender bias and how research teams are making decisions on author order. Data on publications from 2018-2021 show that gender bias was largely nonsignificant both before and after authors were asked by ASM to provide an EC statement. The most likely reasons for EC order included alphabetical order, seniority, and chance, although there were differences for publications from different geographic regions. However, many research teams used unique methods in order selection, highlighting the importance of EC statements to provide clarity for readers, funding agencies, and tenure committees.

Analysis of justification for author order and gender bias in author order among those contributing equally.

The practice of designating two or more authors as equal contributors (ECs) on a scientific publication is increasingly common as a form of sharing credit. However, EC authors are often unclearly attributed on curriculum vitae (CVs) or citation engines, and it is unclear how research teams determine author order within an EC listing. In response to studies showing that male authors were more likely to be placed first in an EC listing, the American Society for Microbiology (ASM) required that authors explain the reasons for author order beginning in 2020. In this study, we analyze data from over 2,500 ASM publications to see how this policy affected gender bias and how research teams are making decisions on author order. Data on publications from 2018 to 2021 show that gender bias was largely nonsignificant both before and after authors were asked by ASM to provide an EC statement. The most likely reasons for EC order included alphabetical order, seniority, and chance, although there were differences for publications from different geographic regions. However, many research teams used unique methods in order selection, highlighting the importance of EC statements to provide clarity for readers, funding agencies, and tenure committees. IMPORTANCE: First-author publications are important for early career scientists to secure funding and educational opportunities. However, an analysis published in eLife in 2019 noted that female authors are more likely to be placed second even when both authors report they have contributed equally. American Society for Microbiology announced in response that they would require submissions to include a written justification of author order. In this paper, we analyze the resultant data and show that laboratories are most likely to use some combination of alphabetical order, seniority, and chance to determine author order. However, the prevalence of these methods varies based on the research team's geographic location. These findings highlight the importance of equal contributor statements to provide clarity for readers, funding agencies, and tenure committees. Furthermore, this work is critically important for understanding how these decisions are made and provides a glimpse of the sociology of science.

The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans' capsular polysaccharide and its shed exopolysaccharide function both as key virulence factors and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this study, NOE and J-coupling values from NMR experiments were consistent with a converged structure of the synthetic decasaccharide, GXM10-Ac3, calculated from MD simulations. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is a hexasaccharide with a three-residue α-mannan backbone, modified by ß-(1→2)-xyloses (Xyl) on the first two mannoses (Man) and a ß-(1→2)-glucuronic acid (GlcA) on the third Man. Combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with Xyl/GlcA branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This derived GXM model displays high flexibility while maintaining a structural identity, yielding insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.