Geospatial pattern of topsoil pollution and multi-endpoint toxicity in the petrochemical area of Augusta-Priolo (eastern Sicily, Italy).

The present study was aimed at identifying geospatial patterns of pollutants including concentrations and toxicity as complex environmental mixtures, in topsoil samples close to petrochemical facilities in the heavily industrialized area of Augusta and Priolo in south-eastern Sicily (Italy). Elemental analysis of soil was conducted by ICP-MS for 23 metals and 16 rare earth elements (REEs). Organic analyses were primarily focused on polycyclic aromatic hydrocarbons (PAHs) (16 parent homologs) and total aliphatic hydrocarbons (C10 - C40). Topsoil samples were tested for toxicity in multiple bioassay models including: 1) developmental defects and cytogenetic anomalies in sea urchin Sphaerechinus granularis early life stages; 2) growth inhibition of diatom Phaeodactylum tricornutum; 3) mortality in nematode Caenorhabditis elegans; and 4) induction of mitotic abnormalities in onion Allium cepa. Samples collected at sites closest to defined petrochemical facilities were highest in select pollutants and correlated with biological effects in different toxicity endpoints. A noteworthy finding was the increased level of total REEs in sites closest to petrochemical facilities, suggesting their contributions to identifying petrochemical sources of pollutants to the environment. The combined data obtained in the different bioassays allowed exploration of geospatial patterns of effect in biota as a function of contaminant levels. In conclusion, this study provides consistent data of soil toxicity, metal and REE contamination at Augusta-Priolo sampling sites, and may provide an appropriate baseline for epidemiological studies on high incidences of congenital birth defects in the area and identification of at-risk localities.

Polymer Chemical Identity as a Key Factor in Microplastic-Insecticide Antagonistic Effects during Embryogenesis of Sea Urchin Arbacia lixula.

As a proxy for pollutants that may be simultaneously present in urban wastewater streams, the effects of two microplastics-polystyrene (PS; 10, 80 and 230 µm diameter) and polymethylmethacrylate (PMMA; 10 and 50 µm diameter)-on fertilisation and embryogenesis in the sea urchin Arbacia lixula with co-exposure to the pyrethroid insecticide cypermethrin were investigated. Synergistic or additive effects were not seen for plastic microparticles (50 mg L-1) in combination with cypermethrin (10 and 1000 µg L-1) based on evaluation of skeletal abnormalities or arrested development and death of significant numbers of larvae during the embryotoxicity assay. This behaviour was also apparent for male gametes pretreated with PS and PMMA microplastics and cypermethrin, where a reduction in sperm fertilisation ability was not evidenced. However, a modest reduction in the quality of the offspring was noted, suggesting that there may be some transmissible damage to the zygotes. PMMA microparticles were more readily taken up than PS microparticles, which could suggest surface chemical identity as potentially modulating the affinity of larvae for specific plastics. In contrast, significantly reduced toxicity was noted for the combination of PMMA microparticles and cypermethrin (100 µg L-1), and may be related to less ready desorption of the pyrethroid than PS, as well as cypermethrin activating mechanisms that result in reduced feeding and hence decreased ingestion of microparticles.

Evaluation of Rare Earth Element-Associated Hormetic Effects in Candidate Fertilizers and Livestock Feed Additives.

Rare earth elements (REEs) are recognized as emerging contaminants with implications in human and environmental health. Apart from their adverse effects, REEs have been reported as having positive effects when amended to fertilizers and livestock feed additives, thus suggesting a hormetic trend, implying a concentration-related shift from stimulation to inhibition and toxicity, with analogous trends that have been assessed for a number of xenobiotics. In view of optimizing the success of REE mixtures in stimulating crop yield and/or livestock growth or egg production, one should foresee the comparative concentration-related effects of individual REEs (e.g., Ce and La) vs. their mixtures, which may display distinct trends. The results might prompt further explorations on the use of REE mixtures vs. single REEs aimed at optimizing the preparation of fertilizers and feed additives, in view of the potential recognition of their use in agronomy and zootechny.

Resolving the effects of environmental micro- and nanoplastics exposure in biota: A knowledge gap analysis.

The pervasive spread of microplastics (MPs) and nanoplastics (NPs) has raised significant concerns on their toxicity in both aquatic and terrestrial environments. These polymer-based materials have implications for plants, wildlife and human health, threatening food chain integrity and ultimate ecosystem resilience. An extensive - and growing - body of literature is available on MP- and NP-associated effects, including in a number of aquatic biota, with as yet limited reports in terrestrial environments. Effects range from no detectable, or very low level, biological effects to more severe outcomes such as (but not limited to) increased mortality rates, altered immune and inflammatory responses, oxidative stress, genetic damage and dysmetabolic changes. A well-established exposure route to MPs and NPs involves ingestion with subsequent incorporation into tissues. MP and NP exposures have also been found to lead to genetic damage, including effects related to mitotic anomalies, or to transmissible damage from sperm cells to their offspring, especially in echinoderms. Effects on the proteome, transcriptome and metabolome warrant ad hoc investigations as these integrated "omics" workflows could provide greater insight into molecular pathways of effect. Given their different physical structures, chemical identity and presumably different modes of action, exposure to different types of MPs and NPs may result in different biological effects in biota, thus comparative investigations of different MPs and NPs are required to ascertain the respective effects. Furthermore, research on MP and NP should also consider their ability to act as vectors for other toxicants, and possible outcomes of exposure may even include effects at the community level, thus requiring investigations in mesocosm models.

Review of Rare Earth Elements as Fertilizers and Feed Additives: A Knowledge Gap Analysis.

Rare earth elements (REEs) are key constituents of modern technology and play important roles in various chemical and industrial applications. They also are increasingly used in agricultural and zootechnical applications, such as fertilizers and feed additives. Early applications of REEs in agriculture have originated in China over the past several decades with the objective of increasing crop productivity and improving livestock yield (e.g., egg production or piglet growth). Outside China, REE agricultural or zootechnical uses are not currently practiced. A number of peer-reviewed manuscripts have evaluated the adverse and the positive effects of some light REEs (lanthanum and cerium salts) or REE mixtures both in plant growth and in livestock yield. This information was never systematically evaluated from the growing body of scientific literature. The present review was designed to evaluate the available evidence for adverse and/or positive effects of REE exposures in plant and animal biota and the cellular/molecular evidence for the REE-associated effects. The overall information points to shifts from toxic to favorable effects in plant systems at lower REE concentrations (possibly suggesting hormesis). The available evidence for REE use as feed additives may suggest positive outcomes at certain doses but requires further investigations before extending this use for zootechnical purposes.

The Influence of Different Classes of Amino Acids on Calcium Phosphates Seeded Growth.

Amino acids (AAs) attract attention for elucidating the role of proteins in biomineralization and the preparation of functionalized biomaterials. The influence that AAs exert on calcium phosphate (CaP) mineralization is still not completely understood, as contradictory results have been reported. In this paper, the influence of the addition of different classes of AAs, charged (L-aspartic acid, Asp; L-lysine, Lys), polar (L-asparagine, Asn; L-serine, Ser; L-tyrosine, Tyr), and non-polar (L-phenylalanine, Phe), on CaP growth in the presence of octacalcium phosphate (OCP) and calcium hydrogenphosphate dihydrate (DCPD) seeds was investigated. In control systems (without AAs), a calcium-deficient apatite (CaDHA) layer was formed on the surface of OCP, while a mixture of CaDHA and OCP in the form of spherical aggregates was formed on the surface of DCPD crystals. Charged and non-polar promoted, while polar AAs inhibited CaDHA formation on the OCP seeds. In the case of DCPD, Lys, Asp, and Phe promoted CaP formation, while the influence of other AAs was negligible. The most efficient promotor of precipitation in both cases was non-polar Phe. No significant influence of AAs on the composition and morphology of precipitates was observed. The obtained results are of interest for understanding biomineralization processes and additive controlled material synthesis.

Mild toxicity of polystyrene and polymethylmethacrylate microplastics in Paracentrotus lividus early life stages.

The vast category of microplastics in the marine environment, encompassing among other aspects their persistence, degradation and impact on biota, has become an important topic of research. In spite of environmental health concerns, much work has yet to be done on understanding the potential roles of polymer sources, composition and particle sizes in causing adverse effects which have already been observed in a number of biota. The present study was aimed at adding to current knowledge by verifying if, and to what extent, embryogenesis in the sea urchin species Paracentrotus lividus is adversely affected by polystyrene and polymethylmethacrylate virgin microparticles over a size range 1-230 µm and at concentrations of 0.1-10 mg L-1. Developing embryos which came in contact with the microplastics only after fertilisation did not display a significant increase of developmental defects. Unlike embryo exposures, when P. lividus sperm were exposed to the microplastics or their leachates, modest, yet significant effects were observed, both in terms of decreased fertilisation rate and increase of transmissible damage to offspring. Further, it was noted that larvae more readily ingested polymethylmethacrylate than polystyrene microparticles after 3 days which may represent a route for enhancing the toxicity of the former compared to the latter. Overall, these findings provide evidence for lesser sensitivity of P. lividus early life stages to microplastics compared to other urchins such as Sphaerechinus granularis. In turn, the more robust response of P. lividus highlights the importance of choosing an appropriate test species with the highest sensitivity when investigating mildly harmful materials.

Idiosyncratic epistasis creates universals in mutational effects and evolutionary trajectories.

Patterns of epistasis and shapes of fitness landscapes are of wide interest because of their bearings on a number of evolutionary theories. The common phenomena of slowing fitness increases during adaptations and diminishing returns from beneficial mutations are believed to reflect a concave fitness landscape and a preponderance of negative epistasis. Paradoxically, fitness decreases tend to decelerate and harm from deleterious mutations shrinks during the accumulation of random mutations-patterns thought to indicate a convex fitness landscape and a predominance of positive epistasis. Current theories cannot resolve this apparent contradiction. Here, we show that the phenotypic effect of a mutation varies substantially depending on the specific genetic background and that this idiosyncrasy in epistasis creates all of the above trends without requiring a biased distribution of epistasis. The idiosyncratic epistasis theory explains the universalities in mutational effects and evolutionary trajectories as emerging from randomness due to biological complexity.

Social Network Predicts Exposure to Respiratory Infection in a Wild Chimpanzee Group.

Respiratory pathogens are expected to spread through social contacts, but outbreaks often occur quickly and unpredictably, making it challenging to simultaneously record social contact and disease incidence data, especially in wildlife. Thus, the role of social contacts in the spread of infectious disease is often treated as an assumption in disease simulation studies, and few studies have empirically demonstrated how pathogens spread through social networks. In July-August 2015, an outbreak of respiratory disease was observed in a wild chimpanzee community in Kibale National Park, Uganda, during an ongoing behavioral study of male chimpanzees, offering a rare opportunity to evaluate how social behavior affects individual exposure to socially transmissible diseases. From May to August 2015, we identified adult and adolescent male chimpanzees displaying coughs and rhinorrhea and recorded 5-m proximity data on males (N = 40). Using the network k-test, we found significant relationships between male network connectivity and the distribution of cases within the network, supporting the importance of short-distance contacts for the spread of the respiratory outbreak. Additionally, chimpanzees central to the network were more likely to display clinical signs than those with fewer connections. Although our analyses were limited to male chimpanzees, these findings underscore the value of social connectivity data in predicting disease outcomes and elucidate a potential evolutionary cost of being social.

Microplastic-induced damage in early embryonal development of sea urchin Sphaerechinus granularis.

Two microplastic sets, polystyrene (PS) and polymethyl methacrylate (PMMA), were tested for adverse effects on early life stages of Sphaerechinus granularis sea urchins. Microparticulate PS (10, 80 and 230 µm diameter) and PMMA (10 and 50 µm diameter) were tested on developing S. granularis embryos from 10 min post-fertilisation (p-f) to the pluteus larval stage (72 h p-f), at concentrations ranging from 0.1 to 5 mg L-1. Both PS and PMMA exposures resulted in significant concentration-related increase of developmental defects and of microplastic uptake in plutei. Moreover, embryo exposures to PS and PMMA (5 and 50 mg L-1) from 10 min to 5 h p-f resulted in a significant increase of cytogenetic abnormalities, expressed as significantly increased mitotic aberrations, while mitotoxicity (as % embryos lacking active mitoses) was observed in embryos exposed to PS, though not to PMMA. When S. granularis sperm suspensions were exposed for 10 min to PS or to PMMA (0.1-5 mg L-1), a significant decrease of fertilisation success was observed following sperm exposure to 0.1 mg L-1 PS, though not to higher PS concentrations nor to PMMA. Sperm pretreatment, however, resulted in significant offspring damage, as excess developmental defects in plutei, both following sperm exposure to PS and PMMA, thus suggesting transmissible damage from sperm pronuclei to the offspring. The overall results point to relevant developmental, cytogenetic and genotoxic effects of PS and PMMA microplastics to S. granularis early life stages, warranting further investigations of other microplastics and other target biota.

Soil pollution and toxicity in an area affected by emissions from a bauxite processing plant and a power plant in Gardanne (southern France).

Soil pollution and toxicity have been investigated in the Gardanne area (southern France) at a range of sites around a recognized pollution source, a bauxite processing plant (BPP), and a power plant (PP). Soil samples were submitted to inorganic and organic analyses and tested for toxicity in two invertebrate models. Inorganic analysis was based on determining elemental concentrations by ICP-MS, encompassing a total of 26 elements including 13 rare earth elements (REEs), of the soil samples and their leachates after 24 or 48 h in seawater. Organic analyses were performed by measuring the sums of 16 polycyclic aromatic hydrocarbons (PAHs) and of total hydrocarbons (C-10 to C-40). Bioassays were carried out on the early life stages of three sea urchin species (Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis), and on a nematode (Caenorhabditis elegans). Sea urchin bioassays were evaluated by the effects of soil samples (0.1-0.5% dry wt/vol) on developing embryos and on sperm, and scored as: a) % developmental defects, b) inhibition of sperm fertilization success and offspring damage, and c) frequencies of mitotic aberrations. C. elegans 24 h-mortality assay showed significant toxicity associated with soil samples. The effects of soil samples showed heightened toxicity at two groups of sites, close to the BPP main entrance and around the PP, which was consistent with the highest concentrations found for metals and PAHs, respectively. Total hydrocarbon concentrations displayed high concentrations both close to BPP main entrance and to the PP. Further studies of the health effects of such materials in Gardanne are warranted.

Topsoil and urban dust pollution and toxicity in Taranto (southern Italy) industrial area and in a residential district.

Adverse environmental conditions in the Taranto area (southern Italy) were investigated in studies of air, marine sediment, and human health. The present study aimed at providing unprecedented information on soil pollution and toxicity in a set of sites around recognized pollution sources in the Taranto area, since previous studies were focused on marine or air pollution, or on human health effects. The investigated area included a steel foundry and a power plant, as well as some sites located in an adjacent neighborhood. Surface soil samples and urban dust were collected and submitted to inorganic and organic analyses and tested for toxicity in two invertebrate bioassay models; a sea urchin (Sphaerechinus granularis) and an annelid (Caenorhabditis elegans). Inorganic analysis was carried out using ICP-MS for elemental composition for a total of 34 elements, whose levels were evaluated as a function of bioassay data analyzed through principal component analysis (PCA). Other analyses included asbestos search by powder X-ray diffraction (PXRD) and organic analysis for polycyclic aromatic hydrocarbons (PAHs) and aliphatic compounds (C10-C40). Toxicity bioassays were carried out on a sea urchin (Sphaerechinus granularis), and an annelid (Caenorhabditis elegans). Sea urchin bioassays evaluated effects of topsoil or street dust sample exposures (0.1 to 0.5% dry wt/vol) on developing embryos and on sperm, and scored as (a) % developmental defects, (b) inhibition of fertilization success and offspring damage, and (c) frequencies of mitotic aberrations. C. elegans mortality assay displayed significant toxicity associated with soil samples. The overall effects of samples showed very high toxicity at four out of nine sites. These effects were consistent with the highest levels measured for metals and PAHs. Further studies of health effects related to dust exposures in residential areas are warranted. Graphical abstract ᅟ.

Mutation and Epistasis in Influenza Virus Evolution.

Influenza remains a persistent public health challenge, because the rapid evolution of influenza viruses has led to marginal vaccine efficacy, antiviral resistance, and the annual emergence of novel strains. This evolvability is driven, in part, by the virus's capacity to generate diversity through mutation and reassortment. Because many new traits require multiple mutations and mutations are frequently combined by reassortment, epistatic interactions between mutations play an important role in influenza virus evolution. While mutation and epistasis are fundamental to the adaptability of influenza viruses, they also constrain the evolutionary process in important ways. Here, we review recent work on mutational effects and epistasis in influenza viruses.

Heavy Rare Earth Elements Affect Sphaerechinus granularis Sea Urchin Early Life Stages by Multiple Toxicity Endpoints.

Heavy rare earth elements (HREEs) were tested for adverse effects to early life stages of the sea urchin Sphaerechinus granularis. Embryos were exposed to analytically measured HREE concentrations ranging from 10-7 to 10-5 M. No significant developmental defect (DD) increases were observed in embryos exposed to 10-7 M HREEs, whereas 10-5 M HREEs resulted in significant DD increase up to 96% for HoCl3 versus 14% in controls. Embryos exposed to 10-6 M HREEs showed the highest DD frequency in embryos exposed to 10-6 M DyCl3 and HoCl3. Cytogenetic analysis of HREE-exposed embryos revealed a significant decrease in mitotic activity, with increased mitotic aberrations. When S. granularis sperm were exposed to HREEs, the offspring of sperm exposed to 10-5 M GdCl3 and LuCl3 showed significant DD increases. The results warrant investigations on HREEs in other test systems, and on REE-containing complex mixtures.

Evidence for the Selective Basis of Transition-to-Transversion Substitution Bias in Two RNA Viruses.

The substitution rates of transitions are higher than expected by chance relative to those of transversions. Many have argued that selection disfavors transversions, as nonsynonymous transversions are less likely to conserve biochemical properties of the original amino acid. Only recently has it become feasible to directly test this selective hypothesis by comparing the fitness effects of a large number of transition and transversion mutations. For example, a recent study of six viruses and one beta-lactamase gene did not find evidence supporting the selective hypothesis. Here, we analyze the relative fitness effects of transition and transversion mutations from our recently published genome-wide study of mutational fitness effects in influenza virus. In contrast to prior work, we find that transversions are significantly more detrimental than transitions. Using what we believe to be an improved statistical framework, we also identify a similar trend in two HIV data sets. We further demonstrate a fitness difference in transition and transversion mutations using four deep mutational scanning data sets of influenza virus and HIV, which provided adequate statistical power. We find that three of the most commonly cited radical/conservative amino acid categories are predictive of fitness, supporting their utility in studies of positive selection and codon usage bias. We conclude that selection is a major contributor to the transition:transversion substitution bias in viruses and that this effect is only partially explained by the greater likelihood of transversion mutations to cause radical as opposed to conservative amino acid changes.

Epistatic Interactions within the Influenza A Virus Polymerase Complex Mediate Mutagen Resistance and Replication Fidelity.

Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza A virus using three nucleoside analogs as well as the virus's high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C-to-U and G-to-A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB1 T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide} resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza A virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates. IMPORTANCE RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis.

Comparative toxicity of seven rare earth elements in sea urchin early life stages.

The widespread use of rare earth elements (REEs) in a number of technological applications raises unanswered questions related to REE-associated adverse effects. We have previously reported on the multiple impact of some REEs on the early life stages of the sea urchin Paracentrotus lividus. The present investigation was to evaluate REE toxicity to early life stages in two unrelated sea urchin species, Sphaerechinus granularis and Arbacia lixula. The comparative toxicities were tested of seven REEs, namely yttrium, lanthanum, cerium, neodymium, samarium, europium and gadolinium as chloride salts at concentrations ranging from 10-7 to 10-4 M. The evaluated endpoints included developmental defects and cytogenetic anomalies in REE-exposed embryos/larvae, and decreased fertilization success and offspring damage following sperm exposure. The results showed different toxicity patterns for individual REEs that varied according to test species and to treatment protocol, thus showing toxicity scaling for the different REEs. Further, the observed effects were compared with those reported for P. lividus either following embryo or sperm exposures. S. granularis showed a significantly higher sensitivity both compared to A. lixula and to P. lividus. This study provides clear-cut evidence for distinct toxicity patterns among a series of REEs. The differences in species sensitivity at micromolar REE levels may warrant investigations on species susceptibility to impacts along polluted coasts.

Effect of Protein Corona on Silver Nanoparticle Stabilization and Ion Release Kinetics in Artificial Seawater.

In parallel with the growing use of nanoparticle-containing products, their release into the environment over the coming years is expected to increase significantly. With many large population centers located in near-coastal areas, and increasing evidence that various nanoparticles may be toxic to a range of organisms, biota in estuarine and coastal waters may be particularly vulnerable. While size effects may be important in cases, silver nanoparticles have been found to be toxic in large part due to their release of silver ions. However, there is relatively little data available on how nanoparticle coatings can affect silver ion release in estuarine or marine waters. We have found that albumin, as a model for biocorona-forming macromolecules which nanoparticles may encounter in wastewater streams, stabilizes silver colloids from agglomeration in high salinity marine waters by electrosteric repulsion for long time periods. A minimum mass ratio of about 130 for albumin:silver nanoparticles (40 nm) was required for stable dispersion in seawater. Increasing albumin concentration was also found to reduce dissolution of nanoparticles in seawater with up to 3.3 times lower concentrations of silver ions noted. Persistent colloids and slow sustained ion release may have important consequences for biota in these environmental compartments.

Antibody-based donor-acceptor spatial reconfiguration in decorated lanthanide-doped nanoparticle colloids for the quantification of okadaic acid biotoxin.

With the increasing movement away from the mouse bioassay for the detection of toxins in commercially harvested shellfish, there is a growing demand for the development of new and potentially field-deployable tests in its place. In this direction we report the development of a simple and sensitive nanoparticle-based luminescence technique for the detection of the marine biotoxin okadaic acid. Photoluminescent lanthanide nanoparticles were conjugated with fluorophore-labelled anti-okadaic acid antibodies which, upon binding to okadaic acid, gave rise to luminescence resonance energy transfer from the nanoparticle to the organic fluorophore dye deriving from a reduction in distance between the two. The intensity ratio of the fluorophore: nanoparticle emission peaks was found to correlate with okadaic acid concentration, and the sensor showed a linear response in the 0.37-3.97 µM okadaic acid range with a limit of detection of 0.25 µM. This work may have important implications for the development of new, cheap, and versatile biosensors for a range of biomolecules and that are sufficiently simple to be applied in the field or at point-of-care.

Cryptococcal heat shock protein 70 homolog Ssa1 contributes to pulmonary expansion of Cryptococcus neoformans during the afferent phase of the immune response by promoting macrophage M2 polarization.

Numerous virulence factors expressed by Cryptococcus neoformans modulate host defenses by promoting nonprotective Th2-biased adaptive immune responses. Prior studies demonstrate that the heat shock protein 70 homolog, Ssa1, significantly contributes to serotype D C. neoformans virulence through the induction of laccase, a Th2-skewing and CNS tropic factor. In the present study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A strain of C. neoformans (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response, whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 d. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate phase, but not the adaptive phase, of the immune response. We conclude that Ssa1's virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neoformans, but ultimately has no effect on cryptococcal control by adaptive immunity.