Use of Lateral Flow Immunoassay to Characterize SARS-CoV-2 RBD-Specific Antibodies and Their Ability to React with the UK, SA and BR P.1 Variant RBDs.

Identifying anti-spike antibodies that exhibit strong neutralizing activity against current dominant circulating variants, and antibodies that are escaped by these variants, has important implications in the development of therapeutic and diagnostic solutions and in improving understanding of the humoral response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We characterized seven anti-SARS-CoV-2 receptor binding domain (RBD) antibodies for binding activity, pairing capability, and neutralization activity to SARS-CoV-2 and three variant RBDs via lateral flow immunoassays. The results allowed us to group these antibodies into three distinct epitope bins. Our studies showed that two antibodies had broadly potent neutralizing activity against SARS-CoV-2 and these variant RBDs and that one antibody did not neutralize the South African (SA) and Brazilian P.1 (BR P.1) RBDs. The antibody escaped by the SA and BR P.1 RBDs retained binding activity to SA and BR P.1 RBDs but was unable to induce neutralization. We demonstrated that lateral flow immunoassay could be a rapid and effective tool for antibody characterization, including epitope classification and antibody neutralization kinetics. The potential contributions of the mutations (N501Y, E484K, and K417N/T) contained in these variants' RBDs to the antibody pairing capability, neutralization activity, and therapeutic antibody targeting strategy are discussed.

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.

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.

Lead in piscivorous raptors during breeding season in the Chesapeake Bay region of Maryland and Virginia, USA.

Sources of lead exposure of many bird species are poorly understood. We analyzed blood lead concentrations from osprey (n = 244; Pandion haliaetus) and bald eagles (n = 68; Haliaeetus leucocephalus) and documented potential sources of lead they may encounter. Adult bald eagles had higher blood lead concentrations than did adult osprey. However, blood lead concentrations of nestlings were similar for both species. Although 62% of osprey had detectable lead concentrations ( x¯ = 1.99 ± 4.02 µg/dL, mean ± standard deviation [SD]), there was no difference in the detection frequency or lead concentrations between osprey adults and nestlings. Likewise, we found no differences in the detection frequency or lead concentrations in osprey adults and nestlings from high- and low-salinity areas. Of the bald eagle samples tested, 55% had detectable lead levels ( x¯ = 6.23 ± 10.74 µg/dL). Adult bald eagles had more detectable and higher lead concentrations than did nestlings or pre-adults. Among environmental samples, paint had the highest lead concentrations, followed by sediment, blue catfish (Ictalurus furcatus), and gizzard shad (Dorosoma cepedianum). There was no correlation between blood lead concentrations of osprey adults and their offspring. Our results indicate that, in the Chesapeake Bay region of Maryland and Virginia (USA), there are multiple sources by which piscivorous raptors may be exposed to lead. Environ Toxicol Chem 2019;38:862-871. © Published 2019 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Phosphosite-Specific Antibodies: A Brief Update on Generation and Applications.

Phosphate addition is a posttranslational modification of proteins, and this modification can affect the activity and other properties of intracellular proteins. Different animal species can be used to generate phosphosite-specific antibodies as either polyclonals or monoclonals, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is vital for their use in proteomics and profiling of disease.

Use of multiple modes of flight subsidy by a soaring terrestrial bird, the golden eagle Aquila chrysaetos, when on migration.

Large birds regularly use updrafts to subsidize flight. Although most research on soaring bird flight has focused on use of thermal updrafts, there is evidence suggesting that many species are likely to use multiple modes of subsidy. We tested the degree to which a large soaring species uses multiple modes of subsidy to provide insights into the decision-making that underlies flight behaviour. We statistically classified more than 22 000 global positioning satellite-global system for mobile communications telemetry points collected at 30-s intervals to identify the type of subsidized flight used by 32 migrating golden eagles during spring in eastern North America. Eagles used subsidized flight on 87% of their journey. They spent 41.9% ± 1.5 ([Formula: see text], range: 18-56%) of their subsidized northbound migration using thermal soaring, 45.2% ± 2.1 (12-65%) of time gliding between thermals, and 12.9% ± 2.2 (1-55%) of time using orographic updrafts. Golden eagles responded to the variable local-scale meteorological events they encountered by switching flight behaviour to take advantage of multiple modes of subsidy. Orographic soaring occurred more frequently in morning and evening, earlier in the migration season, and when crosswinds and tail winds were greatest. Switching between flight modes allowed migration for relatively longer periods each day and frequent switching behaviour has implications for a better understanding of avian flight behaviour and of the evolution of use of subsidy in flight.

Assessing risk to birds from industrial wind energy development via paired resource selection models.

When wildlife habitat overlaps with industrial development animals may be harmed. Because wildlife and people select resources to maximize biological fitness and economic return, respectively, we estimated risk, the probability of eagles encountering and being affected by turbines, by overlaying models of resource selection for each entity. This conceptual framework can be applied across multiple spatial scales to understand and mitigate impacts of industry on wildlife. We estimated risk to Golden Eagles (Aquila chrysaetos) from wind energy development in 3 topographically distinct regions of the central Appalachian Mountains of Pennsylvania (United States) based on models of resource selection of wind facilities (n = 43) and of northbound migrating eagles (n = 30). Risk to eagles from wind energy was greatest in the Ridge and Valley region; all 24 eagles that passed through that region used the highest risk landscapes at least once during low altitude flight. In contrast, only half of the birds that entered the Allegheny Plateau region used highest risk landscapes and none did in the Allegheny Mountains. Likewise, in the Allegheny Mountains, the majority of wind turbines (56%) were situated in poor eagle habitat; thus, risk to eagles is lower there than in the Ridge and Valley, where only 1% of turbines are in poor eagle habitat. Risk within individual facilities was extremely variable; on average, facilities had 11% (SD 23; range = 0-100%) of turbines in highest risk landscapes and 26% (SD 30; range = 0-85%) of turbines in the lowest risk landscapes. Our results provide a mechanism for relocating high-risk turbines, and they show the feasibility of this novel and highly adaptable framework for managing risk of harm to wildlife from industrial development.

Flight responses by a migratory soaring raptor to changing meteorological conditions.

Soaring birds that undertake long-distance migration should develop strategies to minimize the energetic costs of endurance flight. This is relevant because condition upon completion of migration has direct consequences for fecundity, fitness and thus, demography. Therefore, strong evolutionary pressures are expected for energy minimization tactics linked to weather and topography. Importantly, the minute-by-minute mechanisms birds use to subsidize migration in variable weather are largely unknown, in large part because of the technological limitations in studying detailed long-distance bird flight. Here, we show golden eagle (Aquila chrysaetos) migratory response to changing meteorological conditions as monitored by high-resolution telemetry. In contrast to expectations, responses to meteorological variability were stereotyped across the 10 individuals studied. Eagles reacted to increased wind speed by using more orographic lift and less thermal lift. Concomitantly, as use of thermals decreased, variation in flight speed and altitude also decreased. These results demonstrate how soaring migrant birds can minimize energetic expenditures, they show the context for avian decisions and choices of specific instantaneous flight mechanisms and they have important implications for design of bird-friendly wind energy.

Testing an emerging paradigm in migration ecology shows surprising differences in efficiency between flight modes.

To maximize fitness, flying animals should maximize flight speed while minimizing energetic expenditure. Soaring speeds of large-bodied birds are determined by flight routes and tradeoffs between minimizing time and energetic costs. Large raptors migrating in eastern North America predominantly glide between thermals that provide lift or soar along slopes or ridgelines using orographic lift (slope soaring). It is usually assumed that slope soaring is faster than thermal gliding because forward progress is constant compared to interrupted progress when birds pause to regain altitude in thermals. We tested this slope-soaring hypothesis using high-frequency GPS-GSM telemetry devices to track golden eagles during northbound migration. In contrast to expectations, flight speed was slower when slope soaring and eagles also were diverted from their migratory path, incurring possible energetic costs and reducing speed of progress towards a migratory endpoint. When gliding between thermals, eagles stayed on track and fast gliding speeds compensated for lack of progress during thermal soaring. When thermals were not available, eagles minimized migration time, not energy, by choosing energetically expensive slope soaring instead of waiting for thermals to develop. Sites suited to slope soaring include ridges preferred for wind-energy generation, thus avian risk of collision with wind turbines is associated with evolutionary trade-offs required to maximize fitness of time-minimizing migratory raptors.

Overview of the generation, validation, and application of phosphosite-specific antibodies.

Protein phosphorylation is a universal key posttranslational modification that affects the activity and other properties of intracellular proteins. Phosphosite-specific antibodies can be produced as polyclonals or monoclonals in different animal species, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is key for their use in proteomics and profiling of disease.

Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B.

Elongation of transcription by mammalian RNA polymerase II (RNAPII) is regulated by specific factors, including transcription factor IIS (TFIIS) and positive transcription elongation factor b (P-TEFb). We show that the E3 ubiquitin ligase UBR5 associates with the CDK9 subunit of positive transcription elongation factor b to mediate its polyubiquitination in human cells. TFIIS also binds UBR5 to stimulate CDK9 polyubiquitination. Co-localization of UBR5, CDK9, and TFIIS along specific regions of the γ fibrinogen (γFBG) gene indicates that a ternary complex involving these factors participates in the transcriptional regulation of this gene. In support of this notion, overexpression of TFIIS not only modifies the ubiquitination pattern of CDK9 in vivo but also increases the association of CDK9 with various regions of the γFBG gene. Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene. This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9. Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

Experiential, autonomic, and neural responses during threat anticipation vary as a function of threat intensity and neuroticism.

Anticipatory emotional responses play a crucial role in preparing individuals for impending challenges. They do this by triggering a coordinated set of changes in behavioral, autonomic, and neural response systems. In the present study, we examined the biobehavioral impact of varying levels of anticipatory anxiety, using a shock anticipation task in which unpredictable electric shocks were threatened and delivered to the wrist at variable intervals and intensities (safe, medium, strong). This permitted investigation of a dynamic range of anticipatory anxiety responses. In two studies, 95 and 51 healthy female participants, respectively, underwent this shock anticipation task while providing continuous ratings of anxiety experience and electrodermal responding (Study 1) and during fMRI BOLD neuroimaging (Study 2). Results indicated a step-wise pattern of responding in anxiety experience and electrodermal responses. Several brain regions showed robust responses to shock anticipation relative to safe trials, including the hypothalamus, periaqueductal gray, caudate, precentral gyrus, thalamus, insula, ventrolateral PFC, dorsomedial PFC, and ACC. A subset of these regions demonstrated a linear pattern of increased responding from safe to medium to strong trials, including the bilateral insula, ACC, and inferior frontal gyrus. These responses were modulated by individual differences in neuroticism, such that those high in neuroticism showed exaggerated anxiety experience across the entire task, and reduced brain activation from medium to strong trials in a subset of brain regions. These findings suggest that individual differences in neuroticism may influence sensitivity to anticipatory threat and provide new insights into the mechanism through which neuroticism may confer risk for developing anxiety disorders via dysregulated anticipatory responses.

Microarray gene expression profiling using core biopsies of renal neoplasia.

We investigate the feasibility of using microarray gene expression profiling technology to analyze core biopsies of renal tumors for classification of tumor histology. Core biopsies were obtained ex-vivo from 7 renal tumors-comprised of four histological subtypes-following radical nephrectomy using 18-gauge biopsy needles. RNA was isolated from these samples and, in the case of biopsy samples, amplified by in vitro transcription. Microarray analysis was then used to quantify the mRNA expression patterns in these samples relative to non-diseased renal tissue mRNA. Genes with significant variation across all non-biopsy tumor samples were identified, and the relationship between tumor and biopsy samples in terms of expression levels of these genes was then quantified in terms of Euclidean distance, and visualized by complete linkage clustering. Final pathologic assessment of kidney tumors demonstrated clear cell renal cell carcinoma (4), oncocytoma (1), angiomyolipoma (1) and adrenalcortical carcinoma (1). Five of the seven biopsy samples were most similar in terms of gene expression to the resected tumors from which they were derived in terms of Euclidean distance. All seven biopsies were assigned to the correct histological class by hierarchical clustering. We demonstrate the feasibility of gene expression profiling of core biopsies of renal tumors to classify tumor histology.

Spit (Smokeless)-Tobacco Use by Baseball Players Entering the Professional Ranks.

OBJECTIVE: To describe the prevalence and characteristics of spit (smokeless)-tobacco (ST) use in rookie baseball players as they enter the professional ranks and to identify factors associated with use before entering professional baseball. DESIGN AND SETTING: This cross-sectional study was an anonymous questionnaire survey administered during the 1999 baseball season by professional baseball athletic trainers at 30 professional baseball clubs. SUBJECTS: The target group was all rookie professional baseball players entering professional baseball. Of 862 eligible players in the summer of 1999, 616 participated in the survey. MEASUREMENTS: The questionnaire assessed tobacco use, demographic variables relevant to rookie professional baseball players, and factors hypothesized to be associated with current ST use (ie, other forms of tobacco use, social norms, environmental cues, and risky behavior intentions). Univariate associations with ST use were described by relative risks with 95% confidence intervals. For continuous variables, mean values of ST users and nonusers were compared. RESULTS: Overall, 67% of the players had tried ST and 31% were current users. Significant risk factors for ST use were being a current cigarette or cigar smoker, having a family member who used ST, and perceiving ST use by peers and role models. CONCLUSIONS: Almost one third of rookie baseball players in the 1999 season were regular ST users on entering professional baseball. Interventions for prevention and cessation of the use of ST targeting young baseball players are needed.