TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6.

Human tripartite motif protein 5α (TRIM5α) is a well-characterized restriction factor for some RNA viruses, including HIV1-5; however, reports are limited for DNA viruses6,7. Here we demonstrate that TRIM5α also restricts orthopoxviruses and, via its SPRY domain, binds to the orthopoxvirus capsid protein L3 to diminish virus replication and activate innate immunity. In response, several orthopoxviruses, including vaccinia, rabbitpox, cowpox, monkeypox, camelpox and variola viruses, deploy countermeasures. First, the protein C6 binds to TRIM5 via the RING domain to induce its proteasome-dependent degradation. Second, cyclophilin A (CypA) is recruited via interaction with the capsid protein L3 to virus factories and virions to antagonize TRIM5α; this interaction is prevented by cyclosporine A (CsA) and the non-immunosuppressive derivatives alisporivir and NIM811. Both the proviral effect of CypA and the antiviral effect of CsA are dependent on TRIM5α. CsA, alisporivir and NIM811 have antiviral activity against orthopoxviruses, and because these drugs target a cellular protein, CypA, the emergence of viral drug resistance is difficult. These results warrant testing of CsA derivatives against orthopoxviruses, including monkeypox and variola.

Genome sequencing and analysis of the Tasmanian devil and its transmissible cancer.

The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.

Filamin B restricts vaccinia virus spread and is targeted by vaccinia virus protein C4.

Vaccinia virus (VACV) is a large DNA virus that encodes scores of proteins that modulate the host immune response. VACV protein C4 is one such immunomodulator known to inhibit the activation of both the NF-κB signaling cascade and the DNA-PK-mediated DNA sensing pathway. Here, we show that the N-terminal region of C4, which neither inhibits NF-κB nor mediates interaction with DNA-PK, still contributes to virus virulence. Furthermore, this domain interacts directly and with high affinity to the C-terminal domain of filamin B (FLNB). FLNB is a large actin-binding protein that stabilizes the F-actin network and is implicated in other cellular processes. Deletion of FLNB from cells results in larger VACV plaques and increased infectious viral yield, indicating that FLNB restricts VACV spread. These data demonstrate that C4 has a new function that contributes to virulence and engages the cytoskeleton. Furthermore, we show that the cytoskeleton performs further previously uncharacterized functions during VACV infection. IMPORTANCE: Vaccinia virus (VACV), the vaccine against smallpox and monkeypox, encodes many proteins to counteract the host immune response. Investigating these proteins provides insights into viral immune evasion mechanisms and thereby indicates how to engineer safer and more immunogenic VACV-based vaccines. Here, we report that the N-terminal domain of VACV protein C4 interacts directly with the cytoskeletal protein filamin B (FLNB), and this domain of C4 contributes to virus virulence. Furthermore, VACV replicates and spreads better in cells lacking FLNB, thus demonstrating that FLNB has antiviral activity. VACV utilizes the cytoskeleton for movement within and between cells; however, previous studies show no involvement of C4 in VACV replication or spread. Thus, C4 associates with FLNB for a different reason, suggesting that the cytoskeleton has further uncharacterized roles during virus infection.

PNPASE regulates RNA import into mitochondria.

RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, we localized polynucleotide phosphorylase (PNPASE), a 3' --> 5' exoribonuclease and poly-A polymerase, in the mitochondrial intermembrane space, a location lacking resident RNAs. Here, we show a new role for PNPASE in regulating the import of nuclear-encoded RNAs into the mitochondrial matrix. PNPASE reduction impaired mitochondrial RNA processing and polycistronic transcripts accumulated. Augmented import of RNase P, 5S rRNA, and MRP RNAs depended on PNPASE expression and PNPASE-imported RNA interactions were identified. PNPASE RNA processing and import activities were separable and a mitochondrial RNA targeting signal was isolated that enabled RNA import in a PNPASE-dependent manner. Combined, these data strongly support an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria.

Correlation of HER2 Protein Level With mRNA Level Quantified by RNAscope in Breast Cancer.

Trastuzumab deruxtecan (T-DXd) has been approved by the US Food and Drug Administration (FDA) to treat patients with metastatic HER2-positive and HER2-low breast cancer, and clinical trials are examining its efficacy against early-stage breast cancer. Current HER2 immunohistochemical (IHC) assays are suboptimal in evaluating HER2-low breast cancers and identifying which patients would benefit from T-DXd. HER2 expression in 526 breast cancer tissue microarray (TMA) cores was measured using the FDA-approved PATHWAY and HercepTest IHC assays, and the corresponding RNA levels were evaluated by RNAscope. HER2 protein levels by regression analysis using a quantitative immunofluorescence score against cell line arrays with known HER2 protein levels determined by mass spectrometry were available in 48 of the cores. RNAscope was also performed in 32 metastatic biopsies from 23 patients who were subsequently treated with T-DXd, and the results were correlated with response rate. HER2 RNA levels by RNAscope strongly correlated with HER2 protein levels (P < .0001) and with HER2 IHC H-scores from the PATHWAY and HercepTest assays (P < .0001). However, neither protein levels nor RNA levels significantly differed between cases scored 0, ultralow, and 1+ by PATHWAY and HercepTest. The RNA levels were significantly higher (P = .030) in responders (6.4 ± 8.2 dots/cell, n = 12) than those in nonresponders (2.6 ± 2.2, n = 20) to T-DXd. RNAscope is a simple assay that can be objectively quantified and is a promising alternative to current IHC assays in evaluating HER2 expression in breast cancers, especially HER2-low cases, and may identify patients who would benefit from T-DXd.

Smallpox vaccination induces a substantial increase in commensal skin bacteria that promote pathology and influence the host response.

Interactions between pathogens, host microbiota and the immune system influence many physiological and pathological processes. In the 20th century, widespread dermal vaccination with vaccinia virus (VACV) led to the eradication of smallpox but how VACV interacts with the microbiota and whether this influences the efficacy of vaccination are largely unknown. Here we report that intradermal vaccination with VACV induces a large increase in the number of commensal bacteria in infected tissue, which enhance recruitment of inflammatory cells, promote tissue damage and influence the host response. Treatment of vaccinated specific-pathogen-free (SPF) mice with antibiotic, or infection of genetically-matched germ-free (GF) animals caused smaller lesions without alteration in virus titre. Tissue damage correlated with enhanced neutrophil and T cell infiltration and levels of pro-inflammatory tissue cytokines and chemokines. One month after vaccination, GF and both groups of SPF mice had equal numbers of VACV-specific CD8+ T cells and were protected from disease induced by VACV challenge, despite lower levels of VACV-neutralising antibodies observed in GF animals. Thus, skin microbiota may provide an adjuvant-like stimulus during vaccination with VACV and influence the host response to vaccination.

The actin nucleator Spir-1 is a virus restriction factor that promotes innate immune signalling.

Cellular proteins often have multiple and diverse functions. This is illustrated with protein Spir-1 that is an actin nucleator, but, as shown here, also functions to enhance innate immune signalling downstream of RNA sensing by RIG-I/MDA-5. In human and mouse cells lacking Spir-1, IRF3 and NF-κB-dependent gene activation is impaired, whereas Spir-1 overexpression enhanced IRF3 activation. Furthermore, the infectious virus titres and sizes of plaques formed by two viruses that are sensed by RIG-I, vaccinia virus (VACV) and Zika virus, are increased in Spir-1 KO cells. These observations demonstrate the biological importance of Spir-1 in the response to virus infection. Like cellular proteins, viral proteins also have multiple and diverse functions. Here, we also show that VACV virulence factor K7 binds directly to Spir-1 and that a diphenylalanine motif of Spir-1 is needed for this interaction and for Spir-1-mediated enhancement of IRF3 activation. Thus, Spir-1 is a new virus restriction factor and is targeted directly by an immunomodulatory viral protein that enhances virus virulence and diminishes the host antiviral responses.

Selective modulation of cell surface proteins during vaccinia infection: A resource for identifying viral immune evasion strategies.

The interaction between immune cells and virus-infected targets involves multiple plasma membrane (PM) proteins. A systematic study of PM protein modulation by vaccinia virus (VACV), the paradigm of host regulation, has the potential to reveal not only novel viral immune evasion mechanisms, but also novel factors critical in host immunity. Here, >1000 PM proteins were quantified throughout VACV infection, revealing selective downregulation of known T and NK cell ligands including HLA-C, downregulation of cytokine receptors including IFNAR2, IL-6ST and IL-10RB, and rapid inhibition of expression of certain protocadherins and ephrins, candidate activating immune ligands. Downregulation of most PM proteins occurred via a proteasome-independent mechanism. Upregulated proteins included a decoy receptor for TRAIL. Twenty VACV-encoded PM proteins were identified, of which five were not recognised previously as such. Collectively, this dataset constitutes a valuable resource for future studies on antiviral immunity, host-pathogen interaction, poxvirus biology, vector-based vaccine design and oncolytic therapy.

Population genetic structure and demographic history reconstruction of introduced flathead catfish (Pylodictis olivaris) in two US Mid-Atlantic rivers.

Population genetic analysis of invasive populations can provide valuable insights into the source of introductions, pathways for expansion, and their demographic histories. Flathead catfish (Pylodictis olivaris) are a prolific invasive species with high fecundity, long-distance dispersal, and piscivorous feeding habits that can lead to declines in native fish populations. In this study, we analyse the genetics of invasive P. olivaris in the Mid-Atlantic region to assess their connectivity and attempt to reconstruct the history of introduced populations. Based on an assessment across 13 microsatellite loci, P. olivaris from the Susquehanna River system (N = 537), Schuylkill River (N = 33), and Delaware River (N = 1) have low genetic diversity (global Hobs = 0.504), although we detected no evidence of substantial inbreeding (FIS = -0.083 to 0.022). P. olivaris from these different river systems were genetically distinct, suggesting separate introductions. However, population structure was much weaker within each river system and exhibited a pattern of high connectivity, with some evidence of isolation by distance. P. olivaris from the Susquehanna and Schuylkill rivers showed evidence for recent genetic bottlenecks, and demographic models were consistent with historical records, which suggest that populations were established by recent founder events consisting of a small number of individuals. Our results show the risk posed by small introductions of P. olivaris, which can spread widely once a population is established, and highlight the importance of prevention and sensitive early detection methods to prevent the spread of P. olivaris in the future.

Temporal analysis of water chemistry and smallmouth bass (Micropterus dolomieu) health at two sites with divergent land use in the Susquehanna River watershed, Pennsylvania, USA.

Monitoring wild fish health and exposure effects in impacted rivers and streams with differing land use has become a valuable research tool. Smallmouth bass (Micropterus dolomieu) are a sensitive, indicator species that exhibit signs of immunosuppression and endocrine disruption in response to water quality changes and contaminant exposure. To determine the impact of agriculture and development on smallmouth bass health, two sites (a developed/agriculture site and a forested site) in the Susquehanna River watershed, Pennsylvania were selected where bass and water chemistry were sampled from 2015 to 2019. Smallmouth bass were sampled for histopathology to assess parasite and macrophage aggregate density in the liver and spleen, condition factor (Ktl), hepatic gene transcript abundance, hepatosomatic index (HSI), and a health assessment index (HAI). Land use at the developed/agriculture site included greater pesticide application rates and phytoestrogen crop cover and more detections and higher concentrations of pesticides, wastewater-associated contaminants, hormones, phytoestrogens, and mycotoxins than at the forested site. Additionally, at the developed/agriculture site, indicators of molecular changes, including oxidative stress, immune/inflammation, and lipid metabolism-related hepatic gene transcripts, were associated with more contaminants and land use variables. At both sites, there were multiple associations of contaminants with liver and/or spleen macrophage aggregate density, indicating that changes at the molecular level seemed to be a better indicator of exposures unique to each site. The findings illustrate the importance of timing for land management practices, the complex mixtures aquatic animals are exposed to, and the temporal changes in contaminant concentration. Agricultural practices that affect hepatic gene transcripts associated with immune function and disease resistance were demonstrated which could negatively affect smallmouth bass populations.

Transcriptome software results show significant variation among different commercial pipelines.

BACKGROUND: We have been documenting the biological responses to low levels of radiation (natural background) and very low level radiation (below background), and thus these studies are testing mild external stimuli to which we would expect relatively mild biological responses. We recently published a transcriptome software comparison study based on RNA-Seqs from a below background radiation treatment of two model organisms, E. coli and C. elegans (Thawng and Smith, BMC Genomics 23:452, 2022). We reported DNAstar-D (Deseq2 in the DNAstar software pipeline) to be the more conservative, realistic tool for differential gene expression compared to other transcriptome software packages (CLC, Partek and DNAstar-E (using edgeR). Here we report two follow-up studies (one with a new model organism, Aedes aegypti and another software package (Azenta) on transcriptome responses from varying dose rates using three different sources of natural radiation. RESULTS: When E. coli was exposed to varying levels of K40, we again found that the DNAstar-D pipeline yielded a more conservative number of DEGs and a lower fold-difference than the CLC pipeline and DNAstar-E run in parallel. After a 30 read minimum cutoff criterion was applied to the data, the number of significant DEGs ranged from 0 to 81 with DNAstar-D, while the number of significant DEGs ranged from 4 to 117 and 14 to 139 using DNAstar-E and the CLC pipelines, respectively. In terms of the extent of expression, the highest foldchange DEG was observed in DNAstar-E with 19.7-fold followed by 12.5-fold in CLC and 4.3-fold in DNAstar-D. In a recently completed study with Ae. Aegypti and using another software package (Azenta), we analyzed the RNA-Seq response to similar sources of low-level radiation and again found the DNAstar-D pipeline to give the more conservative number and fold-expression of DEGs compared to other softwares. The number of significant DEGs ranged 31-221 in Azenta and 31 to 237 in CLC, 19-252 in DNAstar-E and 0-67 in DNAStar-D. The highest fold-change of DEGs were found in CLC (1,350.9-fold), with DNAstar-E (5.9 -fold) and Azenta (5.5-fold) intermediate, and the lowest levels of expression (4-fold) found in DNAstar-D. CONCLUSIONS: This study once again highlights the importance of choosing appropriate software for transcriptome analysis. Using three different biological models (bacteria, nematode and mosquito) in four different studies testing very low levels of radiation (Van Voorhies et al., Front Public Health 8:581796, 2020; Thawng and Smith, BMC Genomics 23:452, 2022; current study), the CLC software package resulted in what appears to be an exaggerated gene expression response in terms of numbers of DEGs and extent of expression. Setting a 30-read cutoff diminishes this exaggerated response in most of the software tested. We have further affirmed that DNAstar-Deseq2 gives a more conservative transcriptome expression pattern which appears more suitable for studies expecting subtle gene expression patterns.

ICTV Virus Taxonomy Profile: Poxviridae 2023.

Poxviridae is a family of enveloped, brick-shaped or ovoid viruses. The genome is a linear molecule of dsDNA (128-375 kbp) with covalently closed ends. The family includes the sub-families Entomopoxvirinae, whose members have been found in four orders of insects, and Chordopoxvirinae, whose members are found in mammals, birds, reptiles and fish. Poxviruses are important pathogens in various animals, including humans, and typically result in the formation of lesions, skin nodules, or disseminated rash. Infections can be fatal. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Poxviridae, which is available at ictv.global/report/poxviridae.

Dysregulation of Cellular VRK1, BAF, and Innate Immune Signaling by the Vaccinia Virus B12 Pseudokinase.

Poxvirus proteins remodel signaling throughout the cell by targeting host enzymes for inhibition and redirection. Recently, it was discovered that early in infection the vaccinia virus (VACV) B12 pseudokinase copurifies with the cellular kinase VRK1, a proviral factor, in the nucleus. Although the formation of this complex correlates with inhibition of cytoplasmic VACV DNA replication and likely has other downstream signaling consequences, the molecular mechanisms involved are poorly understood. Here, we further characterize how B12 and VRK1 regulate one another during poxvirus infection. First, we demonstrate that B12 is stabilized in the presence of VRK1 and that VRK1 and B12 coinfluence their respective solubility and subcellular localization. In this regard, we find that B12 promotes VRK1 colocalization with cellular DNA during mitosis and that B12 and VRK1 may be tethered cooperatively to chromatin. Next, we observe that the C-terminal tail of VRK1 is unnecessary for B12-VRK1 complex formation or its proviral activity. Interestingly, we identify a point mutation of B12 capable of abrogating interaction with VRK1 and which renders B12 nonrepressive during infection. Lastly, we investigated the influence of B12 on the host factor BAF and antiviral signaling pathways and find that B12 triggers redistribution of BAF from the cytoplasm to the nucleus. In addition, B12 increases DNA-induced innate immune signaling, revealing a new functional consequence of the B12 pseudokinase. Together, this study characterizes the multifaceted roles B12 plays during poxvirus infection that impact VRK1, BAF, and innate immune signaling. IMPORTANCE Protein pseudokinases comprise a considerable fraction of the human kinome, as well as other forms of life. Recent studies have demonstrated that their lack of key catalytic residues compared to their kinase counterparts does not negate their ability to intersect with molecular signal transduction. While the multifaceted roles pseudokinases can play are known, their contribution to virus infection remains understudied. Here, we further characterize the mechanism of how the VACV B12 pseudokinase and human VRK1 kinase regulate one another in the nucleus during poxvirus infection and inhibit VACV DNA replication. We find that B12 disrupts regulation of VRK1 and its downstream target BAF, while also enhancing DNA-dependent innate immune signaling. Combined with previous data, these studies contribute to the growing field of nuclear pathways targeted by poxviruses and provide evidence of unexplored roles of B12 in the activation of antiviral immunity.

An Automated Pipeline for Differential Cell Counts on Whole-Slide Bone Marrow Aspirate Smears.

The pathologic diagnosis of bone marrow disorders relies in part on the microscopic analysis of bone marrow aspirate (BMA) smears and the manual counting of marrow nucleated cells to obtain a differential cell count (DCC). This manual process has significant limitations, including the analysis of only a small subset of optimal slide areas and nucleated cells, as well as interobserver variability due to differences in cell selection and classification. To address these shortcomings, we developed an automated machine learning-based pipeline for obtaining 11-component DCCs on whole-slide BMAs. This pipeline uses a sequential process of identifying optimal BMA regions with high proportions of marrow nucleated cells, detecting individual cells within these optimal areas, and classifying these cells into 1 of 11 DCC components. Convolutional neural network models were trained on 396,048 BMA region, 28,914 cell boundary, and 1,510,976 cell class images from manual annotations. The resulting automated pipeline produced 11-component DCCs that demonstrated a high statistical and diagnostic concordance with manual DCCs among a heterogeneous group of testing BMA slides with varying pathologies and cellularities. Additionally, we demonstrated that an automated analysis can reduce the intraslide variance in DCCs by analyzing the whole slide and marrow nucleated cells within all optimal regions. Finally, the pipeline outputs of region classification, cell detection, and cell classification can be visualized using whole-slide image analysis software. This study demonstrates the feasibility of a fully automated pipeline for generating DCCs on scanned whole-slide BMA images, with the potential for improving the current standard of practice for utilizing BMA smears in the laboratory analysis of hematologic disorders.

Elevated homocysteine levels: What inborn errors of metabolism might we be missing?

Elevated total plasma homocysteine (hyperhomocysteinemia) is a marker of cardiovascular, thrombotic, and neuropsychological disease. It has multiple causes, including the common nutritional vitamin B12 or folate deficiency. However, some rare but treatable, inborn errors of metabolism (IEM) characterized by hyperhomocysteinemia can be missed due to variable presentations and the lack of awareness. The aim of this study is to identify undiagnosed IEM in adults with significantly elevated homocysteine using key existing clinical data points, then IEM specific treatment can be offered to improve outcome. We conducted a retrospective study with data mining and chart review of patients with plasma total homocysteine >30 µmol/L over a two-year period. We offer biochemical and genetic testing to patients with significant hyperhomocysteinemia without a clear explanation to diagnose IEM. We identified 22 subjects with significant hyperhomocysteinemia but no clear explanation. Subsequently, we offered genetic testing to seven patients and diagnosed one patient with classic homocystinuria due to cystathionine beta-synthase deficiency. With treatment, she lowered her plasma homocysteine and improved her health. This study stresses the importance of a thorough investigation of hyperhomocysteinemia in adults to identify rare but treatable IEM. We propose a metabolic evaluation algorithm for elevated homocysteine levels.

Corticosterone and immune responses to dehydration in squamate reptiles.

Many environments present some degree of seasonal water limitations; organisms that live in such environments must be adapted to survive periods without permanent water access. Often this involves the ability to tolerate dehydration, which can have adverse physiological effects and is typically considered a physiological stressor. While having many functions, the hormone corticosterone (CORT) is often released in response to stressors, yet increasing plasma CORT while dehydrated could be considered maladaptive, especially for species that experience predictable bouts of dehydration and have related coping mechanisms. Elevating CORT could reduce immunocompetence and have other negative physiological effects. Thus, such species likely have CORT and immune responses adapted to experiencing seasonal droughts. We evaluated how dehydration affects CORT and immune function in eight squamate species that naturally experience varied water limitation. We tested whether hydric state affected plasma CORT concentrations and aspects of immunocompetence (lysis, agglutination, bacterial killing ability and white blood cell counts) differently among species based on how seasonally water limited they are and whether this is constrained by phylogeny. The species represented four familial pairs, with one species of each pair inhabiting environments with frequent access to water and one naturally experiencing extended periods (>30 days) with no access to standing water. The effects of dehydration on CORT and immunity varied among species. Increases in CORT were generally not associated with reduced immunocompetence, indicating CORT and immunity might be decoupled in some species. Interspecies variations in responses to dehydration were more clearly grouped by phylogeny than by habitat type.

UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.

Egg viability and egg mass underlie immune tradeoffs and differences between urban and rural lizard egg yolk physiology.

Urbanization can cause innumerable abiotic and biotic changes that have the potential to influence the ecology, behavior, and physiology of native resident organisms. Relative to their rural conspecifics, urban Side-blotched Lizard (Uta stansburiana) populations in southern Utah have lower survival prospects and maximize reproductive investment via producing larger eggs and larger clutch sizes. While egg size is an important predictor of offspring quality, physiological factors within the egg yolk are reflective of the maternal environment and can alter offspring traits, especially during energetically costly processes, such as reproduction or immunity. Therefore, maternal effects may represent an adaptive mechanism by which urban-dwelling species can persist within a variable landscape. In this study, we assess urban and rural differences in egg yolk bacterial killing ability (BKA), corticosterone (CORT), oxidative status (d-ROMs), and energy metabolites (free glycerol and triglycerides), and their association with female immune status and egg quality. Within a laboratory setting, we immune challenged urban lizards via lipopolysaccharide injection (LPS) to test whether physiological changes associated with immune system activity impacted egg yolk investment. We found urban females had higher mite loads than rural females, however mite burden was related to yolk BKA in rural eggs, but not urban eggs. While yolk BKA differed between urban and rural sites, egg mass and egg viability (fertilized vs. unfertilized) were strong predictors of yolk physiology and may imply tradeoffs exist between maintenance and reproduction. LPS treatment caused a decrease in egg yolk d-ROMs relative to the control treatments, supporting results from previous research. Finally, urban lizards laid a higher proportion of unfertilized eggs, which differed in egg yolk BKA, CORT, and triglycerides in comparison to fertilized eggs. Because rural lizards laid only viable eggs during this study, these results suggest that reduced egg viability is a potential cost of living in an urban environment. Furthermore, these results help us better understand potential downstream impacts of urbanization on offspring survival, fitness, and overall population health.

Radiative absorption enhancements by black carbon controlled by particle-to-particle heterogeneity in composition.

Black carbon (BC) absorbs solar radiation, leading to a strong but uncertain warming effect on climate. A key challenge in modeling and quantifying BC's radiative effect on climate is predicting enhancements in light absorption that result from internal mixing between BC and other aerosol components. Modeling and laboratory studies show that BC, when mixed with other aerosol components, absorbs more strongly than pure, uncoated BC; however, some ambient observations suggest more variable and weaker absorption enhancement. We show that the lower-than-expected enhancements in ambient measurements result from a combination of two factors. First, the often used spherical, concentric core-shell approximation generally overestimates the absorption by BC. Second, and more importantly, inadequate consideration of heterogeneity in particle-to-particle composition engenders substantial overestimation in absorption by the total particle population, with greater heterogeneity associated with larger model-measurement differences. We show that accounting for these two effects-variability in per-particle composition and deviations from the core-shell approximation-reconciles absorption enhancement predictions with laboratory and field observations and resolves the apparent discrepancy. Furthermore, our consistent model framework provides a path forward for improving predictions of BC's radiative effect on climate.

Comparative diets and prey resource competition between early-juvenile common snook Centropomus undecimalis and non-native pike killifish Belonesox belizanus.

Pike killifish Belonesox belizanus is an established non-native fish species in Florida, USA, that was first documented in south Florida in 1957 and then in Tampa Bay tributaries in 1994. Decreases in small-bodied fish abundances have been linked to the introduction of B. belizanus in both of these regions. Increases in the range and abundance of B. belizanus in the Tampa Bay area and overlap in habitat usage have led to concerns about potential competition with, and predation on, early-juvenile common snook Centropomus undecimalis [≤100 mm standard length (SL)]. Stomach contents of B. belizanus (N = 422; 14-127 mm SL) and early-juvenile C. undecimalis (N = 1132; 5-119 mm SL) were collected to examine the dietary overlap of these two species and potential differences in the diet of early-juvenile C. undecimalis from locations with and without B. belizanus co-occurring. Prey resources were collected by seine to assess prey resource limitation and prey selectivity. Stomach content analysis indicated that there was low overlap in the diet of early-juvenile C. undecimalis and B. belizanus (C ≤ 0.40). Early-juvenile C. undecimalis had a wider diet breadth, consuming many organisms that are not consumed by B. belizanus and which make up a large portion of the early-juvenile C. undecimalis diet. Analysis of prey resources indicated that some prey groups may have lower abundances in locations where B. belizanus are present, with some of these differences reflected in the diet of early-juvenile C. undecimalis. Despite these differences, there was minimal difference in the diet overlap of early-juvenile C. undecimalis from locations with and without B. belizanus co-occurring. Currently B. belizanus appear to be competing minimally with early-juvenile C. undecimalis for prey resources, with no substantial impacts being detected.