DNA binding analysis of rare variants in homeodomains reveals homeodomain specificity-determining residues.

Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) among eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease-associated mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyze 92 human HD mutants, including disease-associated variants and variants of uncertain significance (VUS), for their effects on DNA binding activity. Many of the variants alter DNA binding affinity and/or specificity. Detailed biochemical analysis and structural modeling identifies 14 previously unknown specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs often exhibits different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and prioritize dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies.

A terminal metabolite of niacin promotes vascular inflammation and contributes to cardiovascular disease risk.

Despite intensive preventive cardiovascular disease (CVD) efforts, substantial residual CVD risk remains even for individuals receiving all guideline-recommended interventions. Niacin is an essential micronutrient fortified in food staples, but its role in CVD is not well understood. In this study, untargeted metabolomics analysis of fasting plasma from stable cardiac patients in a prospective discovery cohort (n = 1,162 total, n = 422 females) suggested that niacin metabolism was associated with incident major adverse cardiovascular events (MACE). Serum levels of the terminal metabolites of excess niacin, N1-methyl-2-pyridone-5-carboxamide (2PY) and N1-methyl-4-pyridone-3-carboxamide (4PY), were associated with increased 3-year MACE risk in two validation cohorts (US n = 2,331 total, n = 774 females; European n = 832 total, n = 249 females) (adjusted hazard ratio (HR) (95% confidence interval) for 2PY: 1.64 (1.10-2.42) and 2.02 (1.29-3.18), respectively; for 4PY: 1.89 (1.26-2.84) and 1.99 (1.26-3.14), respectively). Phenome-wide association analysis of the genetic variant rs10496731, which was significantly associated with both 2PY and 4PY levels, revealed an association of this variant with levels of soluble vascular adhesion molecule 1 (sVCAM-1). Further meta-analysis confirmed association of rs10496731 with sVCAM-1 (n = 106,000 total, n = 53,075 females, P = 3.6 × 10-18). Moreover, sVCAM-1 levels were significantly correlated with both 2PY and 4PY in a validation cohort (n = 974 total, n = 333 females) (2PY: rho = 0.13, P = 7.7 × 10-5; 4PY: rho = 0.18, P = 1.1 × 10-8). Lastly, treatment with physiological levels of 4PY, but not its structural isomer 2PY, induced expression of VCAM-1 and leukocyte adherence to vascular endothelium in mice. Collectively, these results indicate that the terminal breakdown products of excess niacin, 2PY and 4PY, are both associated with residual CVD risk. They also suggest an inflammation-dependent mechanism underlying the clinical association between 4PY and MACE.

Chromosome-specific maturation of the epigenome in the Drosophila male germline.

Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome, and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, we profiled active chromatin features in the testes from wildtype and meiotic arrest mutants and integrate this with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, we detail widespread patterns associated with regulation of the sex chromosomes. Our results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome.

Protein supplementation changes gut microbial diversity and derived metabolites in subjects with type 2 diabetes.

High-protein diets are promoted for individuals with type 2 diabetes (T2D). However, effects of dietary protein interventions on (gut-derived) metabolites in T2D remains understudied. We therefore performed a multi-center, randomized-controlled, isocaloric protein intervention with 151 participants following either 12-week high-protein (HP; 30Energy %, N = 78) vs. low-protein (LP; 10 Energy%, N = 73) diet. Primary objectives were dietary effects on glycemic control which were determined via glycemic excursions, continuous glucose monitors and HbA1c. Secondary objectives were impact of diet on gut microbiota composition and -derived metabolites which were determined by shotgun-metagenomics and mass spectrometry. Analyses were performed using delta changes adjusting for center, baseline, and kidney function when appropriate. This study found that a short-term 12-week isocaloric protein modulation does not affect glycemic parameters or weight in metformin-treated T2D. However, the HP diet slightly worsened kidney function, increased alpha-diversity, and production of potentially harmful microbiota-dependent metabolites, which may affect host metabolism upon prolonged exposure.

Two distinct gut microbial pathways contribute to meta-organismal production of phenylacetylglutamine with links to cardiovascular disease.

Recent studies show gut microbiota-dependent metabolism of dietary phenylalanine into phenylacetic acid (PAA) is critical in phenylacetylglutamine (PAGln) production, a metabolite linked to atherosclerotic cardiovascular disease (ASCVD). Accordingly, microbial enzymes involved in this transformation are of interest. Using genetic manipulation in selected microbes and monocolonization experiments in gnotobiotic mice, we identify two distinct gut microbial pathways for PAA formation; one is catalyzed by phenylpyruvate:ferredoxin oxidoreductase (PPFOR) and the other by phenylpyruvate decarboxylase (PPDC). PPFOR and PPDC play key roles in gut bacterial PAA production via oxidative and non-oxidative phenylpyruvate decarboxylation, respectively. Metagenomic analyses revealed a significantly higher abundance of both pathways in gut microbiomes of ASCVD patients compared with controls. The present studies show a role for these two divergent microbial catalytic strategies in the meta-organismal production of PAGln. Given the numerous links between PAGln and ASCVD, these findings will assist future efforts to therapeutically target PAGln formation in vivo.

PREVALENCE OF RANAVIRUS IN SPOTTED SALAMANDER (AMBYSTOMA MACULATUM) LARVAE FROM CREATED VERNAL POOLS IN WEST VIRGINIA, USA.

Ranavirosis is a disease of high concern for amphibians due to widespread documentation of its lethal and sublethal impacts and its high transmission potential across populations and species. We investigated whether spotted salamander (Ambystoma maculatum) ranavirus prevalence and viral load were associated with habitat characteristics, genetic diversity, corticosterone levels, and body size. In 2015 and 2016, we sampled 34 recently created vernal pools in the Monongahela National Forest, West Virginia, USA. We collected tail clippings from 1,128 spotted salamander larvae and waterborne hormone samples from 436 of those larvae, along with eight environmental characteristics of the pools. Over the 2-yr period, we detected ranavirus in 62% of pools, with prevalence ranging from 0% to 63% (mean, 7.68%). Spotted salamander size was positively correlated with ranavirus presence and viral load; however, we did not find associations between ranavirus prevalence or viral load and habitat characteristics, spotted salamander genetic diversity, relatedness, effective number of breeders, or corticosterone levels. The widespread occurrence of ranavirus in the vernal pools illustrates the potential for rapid natural introduction of the pathogen to created wetlands. Managers could consider monitoring local distributions of ranavirus before creation of new vernal pools to guide strategic placement of the wetlands to minimize occurrence and prevalence of this pathogen.

Climate, hydrology, and human disturbance drive long-term (1988-2018) macrophyte patterns in water diversion lakes.

Macrophytes are affected by many natural and human stressors globally but their long-term responses to these multiple stressors are not often quantified. We employed remote sensing and statistical tools to analyze datasets from both short-term (2017-2018) field investigations to explore seasonal patterns, and long-term (1988-2018) Landsat remote-sensing images to detect annual patterns of macrophyte distributions and study their responses to changes in climate, hydrology, and anthropogenic activities in a chain of water diversion lakes in eastern China. We found: 1) biomass and species richness of macrophytes peaked in summer with dominant species of submerged macrophytes Ceratophyllum demersum, Potamogeton pectinatus, and Potamogeton maackianus and floating macrophytes Trapa bispinosa, and non-native species Cabomba caroliniana spread in midstream Luoma Lake and Nansi Lake in summer, while Potamogeton crispus was dominant in all the lakes in spring; 2) water physicochemical parameters (chloride and water depth), lake characteristics (area and water storage), climate factors (air temperature and precipitation), and anthropogenic activities (commercial fishery and urban development) were significantly correlated to the seasonal distribution of macrophytes; 3) long-term data showed a significantly negative correlation between coverage of floating macrophytes and precipitation where the wettest year of 2003 had the lowest coverage of floating macrophytes; and 4) climate (air temperature) and hydrology (water level) were positively correlated with total macrophyte coverage, but human disturbance indexed by the gross domestic product was negatively driving long-term coverage of macrophytes. Our study has important implications for understanding the long-term succession of macrophytes under both natural and human stressors, and for future environmental management and ecological restoration of freshwater lakes.

Metatranscriptomic Sequencing of Winter and Spring Planktonic Communities from Lake Erie, a Laurentian Great Lake.

Previous reports suggest planktonic and under-ice winter microbial communities in Lake Erie are dominated by diatoms. Here, we report the assembled metatranscriptomes of 79 Lake Erie surface water microbial communities spanning both the winter (28 samples) and spring (51 samples) months over spatial, temporal, and climatic gradients in 2019 through 2020.

Demographic implications of lead poisoning for eagles across North America.

Lead poisoning occurs worldwide in populations of predatory birds, but exposure rates and population impacts are known only from regional studies. We evaluated the lead exposure of 1210 bald and golden eagles from 38 US states across North America, including 620 live eagles. We detected unexpectedly high frequencies of lead poisoning of eagles, both chronic (46 to 47% of bald and golden eagles, as measured in bone) and acute (27 to 33% of bald eagles and 7 to 35% of golden eagles, as measured in liver, blood, and feathers). Frequency of lead poisoning was influenced by age and, for bald eagles, by region and season. Continent-wide demographic modeling suggests that poisoning at this level suppresses population growth rates for bald eagles by 3.8% (95% confidence interval: 2.5%, 5.4%) and for golden eagles by 0.8% (0.7%, 0.9%). Lead poisoning is an underappreciated but important constraint on continent-wide populations of these iconic protected species.

Novel hair snare and genetic methods for non-invasive bobcat detection.

Over the past 20 years, the use of non-invasive hair snare surveys in wildlife research and management has become more prevalent. While these tools have been used to answer important research questions, these techniques often fail to gather information on elusive carnivores, such as bobcats (Lynx rufus). Due to the limited success of previous bobcat studies using hair snares which required active rubbing, this technique has largely fallen out of use, in favor of camera trapping. The goal of our study was to construct a novel, passive bobcat hair snare that could be deployed regardless of terrain or vegetation features, which would be effective for use in capture-recapture population estimation at a large spatial scale. This new hair snare was deployed in 1500 10-km2 cells across West Virginia (USA) between two sampling seasons (2015-2016). Collected hair samples were analyzed with newly developed mitochondrial DNA primers specifically for felids and qPCR to determine species of origin, with enough sensitivity to identify samples as small as two bobcat hairs. Over the two years of the study, a total of 378 bobcat detections were recorded from 42,000 trap nights of sampling, for an overall rate of 0.9 detections/100 trap nights-nearly 2-6 times greater than any previous bobcat hair snare study. While the overall number of recaptured animals was low (n = 9), continued development of this platform should increase its usefulness in capture-recapture studies.

Gut microbe-targeted choline trimethylamine lyase inhibition improves obesity via rewiring of host circadian rhythms.

Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here, we show that gut microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (Lepob/ob). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake but is instead associated with alterations in the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that gut microbial CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have potential as anti-obesity therapeutics.

Gut microbial trimethylamine is elevated in alcohol-associated hepatitis and contributes to ethanol-induced liver injury in mice.

There is mounting evidence that microbes residing in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. We used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. We show the gut microbial choline metabolite TMA is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. The microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells.

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.

Influence of landscape condition on relative abundance and body condition of two generalist freshwater turtle species.

Anthropogenic land use changes have broad impacts on biological diversity, often resulting in shifts in community composition. While many studies have documented negative impacts on occurrence and abundance of species, less attention has been given to native species that potentially benefit from anthropogenic land use changes. For many species reaching high densities in human-dominated landscapes, it is unclear whether these environments represent higher quality habitat than more natural environments. We examined the influence of landscape ecological integrity on relative abundance and body condition of two native generalist freshwater turtle species that are prevalent in anthropogenic systems, the painted turtle (Chrysemys picta) and red-eared slider (Trachemys scripta elegans). Relative abundance was negatively associated with ecological integrity for both species, but the relationship was not strongly supported for painted turtles. Body condition was positively associated with ecological integrity for painted turtles, with no strong association for red-eared sliders. Our study suggests that both species benefitted at the population level from reduced ecological integrity, but individual-level habitat quality was reduced for painted turtles. The differing responses between these two habitat generalists could partially explain why red-eared sliders have become a widespread exotic invasive species, while painted turtles have not.

Differences in distress: Variance and production of American Crocodile (Crocodylus acutus) distress calls in Belize.

Acoustic communication of American Crocodiles (Crocodylus acutus) is relatively understudied. Our overall aim was to determine the acoustic structure of wild American Crocodile distress calls, distinguish call differences among size classes (hatchling, juvenile, sub-adult, and adult), and investigate call production on a gradient of human disturbance. American Crocodile distress calls have strong frequency modulation and are comprised of multiple harmonics in a downsweeping pattern. Measured parameters (total duration, first quartile duration, maximal frequency, first quartile frequency, end frequency, slope of first quartile, slope of last quartiles) differed significantly among size classes (p < .05). Hatchling distress calls are higher in frequency and strongly modulated, whereas calls produced by sub-adults and adults showed little modulation, are lower in frequency, and have greater overall duration. Proportion of crocodiles that produced distress calls during capture differed by size class and sampling location, particularly adult distress calls which are reported here to be produced with undocumented frequency. We determined that American Crocodiles of all size classes produce distress calls at varying rates among study sites. Our results demonstrate that American crocodiles produce distress call more frequently at sites with higher anthropogenic activity. Measured call parameters of juveniles and hatchling American crocodiles also varied among sites in relation to human disturbance. Calls recorded at sites of high anthropogenic impact have increased duration and less modulation which may adversely affect response to emitted distress calls. Proportional and call parameter variances suggest anthropogenic activity as a driver for increased call production and alteration of call parameters at high human-impacted sites.

A comparison of wetland characteristics between Agricultural Conservation Easement Program and public lands wetlands in West Virginia, USA.

In West Virginia, USA, there are 24 conservation easement program wetlands enrolled in the Agricultural Conservation Easement Program (ACEP). These wetlands are located on private agricultural land and are passively managed. Due to their location within fragmented agricultural areas, wetlands enrolled in ACEP in West Virginia have the potential to add wetland ecosystem services in areas that are lacking these features. We evaluated ACEP wetlands compared to reference wetlands on public land in West Virginia by using surrounding land cover, vegetative cover, and wetland features and stressors such as the presence or absence of erosion, upland inclusion, algal mats, and evidence of impacts from the surrounding landscape as surrogate measurements of wetland function on 13 ACEP wetlands and 10 reference wetlands. ACEP wetlands had higher percentages of tree coverage and a higher proportion of agricultural land in the areas immediately surrounding the wetland. Reference wetlands had higher percent coverage of emergent vegetation and had a higher proportion of forest in the immediate landscape. Our findings suggest that ACEP wetlands provide valuable early successional and forested wetland cover in a state that is largely forested. Because of this, it is important to maintain and even expand ACEP in West Virginia to continue providing a valuable source of early successional wetland habitat.

Feeding Ecology Drives Lead Exposure of Facultative and Obligate Avian Scavengers in the Eastern United States.

Lead poisoning of scavenging birds is a global issue. However, the drivers of lead exposure of avian scavengers have been understood from the perspective of individual species, not cross-taxa assemblages. We analyzed blood (n = 285) and liver (n = 226) lead concentrations of 5 facultative (American crows [Corvus brachyrhynchos], bald eagles [Haliaeetus leucocephalus], golden eagles [Aquila chrysaetos], red-shouldered hawks [Buteo lineatus], and red-tailed hawks [Buteo jamaicensis]) and 2 obligate (black vultures [Coragyps atratus] and turkey vultures [Cathartes aura] avian scavenger species to identify lead exposure patterns. Species and age were significant (α < 0.05) predictors of blood lead exposure of facultative scavengers; species, but not age, was a significant predictor of their liver lead exposure. We detected temporal variations in lead concentrations of facultative scavengers (blood: median = 4.41 µg/dL in spring and summer vs 13.08 µg/dL in autumn and winter; p = <0.001; liver: 0.32 ppm in spring and summer vs median = 4.25 ppm in autumn and winter; p = <0.001). At the species level, we detected between-period differences in blood lead concentrations of bald eagles (p = 0.01) and red-shouldered hawks during the winter (p = 0.001). During summer, obligate scavengers had higher liver lead concentrations than did facultative scavengers (median = 1.76 ppm vs 0.22 ppm; p = <0.001). These data suggest that the feeding ecology of avian scavengers is a determinant of the degree to which they are lead exposed, and they highlight the importance of dietary and behavioral variation in determining lead exposure. Environ Toxicol Chem 2020;39:882-892. © 2020 SETAC.

Context and number of noncanonical repeat variable diresidues impede the design of TALE proteins with improved DNA targeting.

Transcription activator-like effector (TALE) proteins have been used extensively for targeted binding of fusion proteins to loci of interest in (epi)genome engineering. Such approaches typically utilize four canonical TALE repeat variable diresidue (RVD) types, corresponding to the identities of two key amino acids, to target each nucleotide. Alternate RVDs with improved specificity are desired. Here, we focused on seven noncanonical RVDs that have been suggested to have improved specificity for their target nucleotides. We used custom protein binding microarrays to characterize the DNA-binding activity of 65 TALEs containing these alternate or corresponding canonical RVDs at multiple positions to ~5,000 unique DNA sequences per protein. We found that none of the noncanonical thymine-targeting RVDs displayed stronger preference for thymine than did the canonical RVD. Of the noncanonical RVDs with putatively improved specificity for guanine, only EN and NH showed greater discrimination of guanine over adenine. This improved specificity, however, comes at a cost: more substitutions of a noncanonical RVD for a canonical RVD generally decreased the protein's DNA-binding activity. Our results highlight the need to investigate RVD-nucleotide specificities in multiple protein contexts and suggest that a balance between canonical and noncanonical RVDs is needed to build TALEs with improved specificity.

Lead Exposure of Red-Shouldered Hawks During the Breeding Season in the Central Appalachians, USA.

Lead is toxic to humans and wildlife. Most studies of lead exposure of raptors focus on the winter, non-breeding season when they scavenge heavily. We evaluated blood lead concentrations (BLCs) of red-shouldered hawks (Buteo lineatus) during the non-scavenging season in the eastern United States. BLCs of 53 of 70 hawks were above the limit of detection ([Formula: see text] = 9.25 µg/dL ± 19.81; ± SD). Adult hawks had higher BLCs ([Formula: see text] = 12.86 µg/dL ± 24.72) than did nestlings ([Formula: see text] = 3.25 µg/dL ± 2.62; p ≤ 0.001, χ2 = 13.2). There was no difference in BLCs of adult hawks among physiographic provinces but there were differences between urban and non-urban settings (p = 0.04, χ2 = 4.2). Soils and invertebrate hawk prey also had quantifiable lead concentrations. Our work shows that red-shouldered hawks are exposed to lead when not scavenging, and suggests pathways by which these birds may be exposed.