US Government Employees Research Articles

Peter Buxtun: US government employee who exposed racist and unethical Tuskegee syphilis study

Peter Buxtun: US government employee who exposed racist and unethical Tuskegee syphilis study

Wildlife ecological risk assessment in the 21st century: Promising technologies to assess toxicological effects.

Despite advances in toxicity testing and the development of new approach methodologies (NAMs) for hazard assessment, the ecological risk assessment (ERA) framework for terrestrial wildlife (i.e., air-breathing amphibians, reptiles, birds, and mammals) has remained unchanged for decades. While survival, growth, and reproductive endpoints derived from whole-animal toxicity tests are central to hazard assessment, nonstandard measures of biological effects at multiple levels of biological organization (e.g., molecular, cellular, tissue, organ, organism, population, community, ecosystem) have the potential to enhance the relevance of prospective and retrospective wildlife ERAs. Other factors (e.g., indirect effects of contaminants on food supplies and infectious disease processes) are influenced by toxicants at individual, population, and community levels, and need to be factored into chemically based risk assessments to enhance the "eco" component of ERAs. Regulatory and logistical challenges often relegate such nonstandard endpoints and indirect effects to postregistration evaluations of pesticides and industrial chemicals and contaminated site evaluations. While NAMs are being developed, to date, their applications in ERAs focused on wildlife have been limited. No single magic tool or model will address all uncertainties in hazard assessment. Modernizing wildlife ERAs will likely entail combinations of laboratory- and field-derived data at multiple levels of biological organization, knowledge collection solutions (e.g., systematic review, adverse outcome pathway frameworks), and inferential methods that facilitate integrations and risk estimations focused on species, populations, interspecific extrapolations, and ecosystem services modeling, with less dependence on whole-animal data and simple hazard ratios. Integr Environ Assess Manag 2024;20:725-748. © 2023 His Majesty the King in Right of Canada and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Evaluation of Glutaminolysis in T Cells.

The activity of living cells is necessarily dependent on the amount of available bioenergy. In T cells, the latter is mainly derived from ATP, a molecular energy "coin" generated by one of several metabolic processes that differ in their ability to satisfy energy demand. Thus, whereas naïve or quiescent T cells efficiently utilize oxidative phosphorylation to generate ATP, T cells subjected to antigenic stimulation followed by clonal expansion and cytokine production meet their increased need for energy by supplementing ATP generation by oxidative phosphorylation with ATP generation by glycolysis. Yet additional need for ATP can be met by other basic biologic sources of energy such as glutamine, an amino acid that is metabolized through a process called glutaminolysis to result in end products that flows into the TCA cycle and augment ATP generation by oxidative phosphorylation. It is now possible to track the dominant energy supplying processes (i.e., the ATP generation process) in differentiating or activated T cells in a real-time manner. Here, we provide one element of such tracking by describing protocols for the assessment of the contribution of glutaminolysis to overall ATP production within different T cell subsets. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Evaluation of the role of glutaminolysis during T cell activation/differentiation Basic Protocol 2: Evaluation of the role of glutaminolysis in T cell responses utilizing glutaminolysis inhibitors Basic Protocol 3: Evaluation of the effect of glutaminolysis on cellular oxidative phosphorylation/glycolysis.

Co‐Culture of Human Primary Hepatocytes and Nonparenchymal Liver Cells in the Emulate® Liver‐Chip for the Study of Drug‐Induced Liver Injury

Drug-induced liver injury (DILI) is a significant public health issue, but standard animal tests and clinical trials sometimes fail to predict DILI due to species differences and the relatively low number of human subjects involved in preapproval studies of a new drug, respectively. In vitro models have long been used to aid DILI prediction, with primary human hepatocytes (PHHs) being generally considered the gold standard. However, despite many efforts and decades of work, traditional culture methods have been unsuccessful in either fully preserving essential liver functions after isolation of PHHs or in emulating interactions between PHHs and hepatic nonparenchymal cells (NPCs), both of which are essential for the development of DILI under in vivo conditions. Recently, various liver-on-a-chip (Liver-Chip) systems have been developed to co-culture hepatocytes and NPCs in a three-dimensional environment on microfluidic channels, enabling better maintenance of primary liver cells and thus improved DILI prediction. The Emulate® Liver-Chip is a commercially available system that can recapitulate some in vivo DILI responses associated with certain compounds whose liver safety profile cannot be accurately evaluated using conventional approaches involving PHHs or animal models due to a lack of innate immune responses or species-dependent toxicity, respectively. Here, we describe detailed procedures for the use of Emulate® Liver-Chips for co-culturing PHHs and NPCs for the purpose of DILI evaluation. First, we describe the procedures for preparing the Liver-Chip. We then outline the steps needed for sequential seeding of PHHs and NPCs in the prepared Liver-Chips. Lastly, we provide a protocol for utilizing cells maintained in perfusion culture in the Liver-Chips to evaluate DILI, using acetaminophen as an example. In all, use of this system and the procedures described here allow better preservation of the functions of human primary liver cells, resulting in an improved in vitro model for DILI assessment. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Liver-Chip preparation Basic Protocol 2: Seeding primary human hepatocytes and nonparenchymal cells on Liver-Chips Basic Protocol 3: Perfusion culture for the study of acetaminophen-induced liver injury.

The ECOTOXicology Knowledgebase: A Curated Database of Ecologically Relevant Toxicity Tests to Support Environmental Research and Risk Assessment.

The need for assembled existing and new toxicity data has accelerated as the amount of chemicals introduced into commerce continues to grow and regulatory mandates require safety assessments for a greater number of chemicals. To address this evolving need, the ECOTOXicology Knowledgebase (ECOTOX) was developed starting in the 1980s and is currently the world's largest compilation of curated ecotoxicity data, providing support for assessments of chemical safety and ecological research through systematic and transparent literature review procedures. The recently released version of ECOTOX (Ver 5, www.epa.gov/ecotox) provides single-chemical ecotoxicity data for over 12,000 chemicals and ecological species with over one million test results from over 50,000 references. Presented is an overview of ECOTOX, detailing the literature review and data curation processes within the context of current systematic review practices and discussing how recent updates improve the accessibility and reusability of data to support the assessment, management, and research of environmental chemicals. Relevant and acceptable toxicity results are identified from studies in the scientific literature, with pertinent methodological details and results extracted following well-established controlled vocabularies and newly extracted toxicity data added quarterly to the public website. Release of ECOTOX, Ver 5, included an entirely redesigned user interface with enhanced data queries and retrieval options, visualizations to aid in data exploration, customizable outputs for export and use in external applications, and interoperability with chemical and toxicity databases and tools. This is a reliable source of curated ecological toxicity data for chemical assessments and research and continues to evolve with accessible and transparent state-of-the-art practices in literature data curation and increased interoperability to other relevant resources. Environ Toxicol Chem 2022;41:1520-1539. © 2022 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Interlaboratory Comparison of Three Sediment Bioaccumulation Tests

Standard bioaccumulation tests are commonly conducted using Macoma nasuta (clam), and Alitta virens (polychaete) for marine tests, and Lumbriculus variegatus (an oligochaete) for freshwater tests. Because the interlaboratory variability associated with these tests is unknown, four experienced laboratories conducted standard 28‐day bioaccumulation tests with the above species using sediments contaminated with polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). Chemical analysis of tissue samples was performed by a single laboratory. The intralaboratory variance among replicates was relatively low for PCB tissue concentrations, with coefficients of variation (CVs) ranging from 9% to 28% for all laboratories and species, with the exception of one laboratory reporting higher variability for L. variegatus (CV = 51%). Intralaboratory variance for PCB tissue concentrations was higher than interlaboratory variance for A. virens and L. variegatus, and the magnitude of difference (MOD) for laboratory means ranged from 1.4 to 2.0 across species. Intralaboratory variability was also low for lipid content, and lipid normalization of PCB and PAH body residues generally had little impact on variability. In addition to variability across bioassay laboratories, analytical variability was evaluated by different laboratories measuring the concentration of PCBs and total lipids in a subsample of tissue homogenate of sediment‐exposed test organisms. Variability associated with tissue analysis was higher than bioassay laboratory variability only in tests with L. variegatus. Statistical differences between samples may be observed due to the low intralaboratory variability; however, the biological significance of these differences may be limited because the MOD is low. Considering the MOD when comparing bioaccumulation across treatments accounts for uncertainty related to inherent variability of the test in the interpretation of statistically significant results. Environ Toxicol Chem 2022;41:1260–1275. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Site- and Individual-Level Contaminations Affect Infection Prevalence of an Emerging Infectious Disease of Amphibians.

Emerging infectious disease outbreaks are one of multiple stressors responsible for amphibian declines globally. In the northeastern United States, ranaviral diseases are prevalent in amphibians and other ectothermic species, but there is still uncertainty as to whether their presence is leading to population-level effects. Further, there is also uncertainty surrounding the potential interactions among disease infection prevalence in free-ranging animals and habitat degradation (co-occurrence of chemical stressors). The present study was designed to provide field-based estimates of the relationship between amphibian disease and chemical stressors. We visited 40 wetlands across three protected areas, estimated the prevalence of ranavirus among populations of larval wood frogs and spotted salamanders, and assessed chemical and biological stressors in wetland habitats and larval amphibians using a suite of selected bioassays, screening tools, and chemical analyses. Ranavirus was detected on larval amphibians from each protected area with an estimated occupancy ranging from 0.27 to 0.55. Considerable variation in ranavirus occupancy was also observed within and among each protected area. Of the stressors evaluated, ranavirus prevalence was strongly and positively related to concentrations of metalloestrogens (metals with the potential to bind to estrogen receptors) and total metals in wetland sediments and weakly and negatively related to total pesticide concentrations in larval amphibians. These results can be used by land managers to refine habitat assessments to include such environmental factors with the potential to influence disease susceptibility. Environ Toxicol Chem 2022;41:781-791. © 2022 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Population modeling to inform management and recovery efforts for lake sturgeon, Acipenser fulvescens.

Lake sturgeon (Acipenser fulvescens) populations have significantly declined across their historic range, in large part due to anthropogenic impacts that have likely been exacerbated by the life-history traits of this slow-growing and long-lived species. We developed a population model to explore how Contaminants of Emerging Concern (CECs) impact lake sturgeon populations. We explored how different physiological modes of action (pMoAs) of CECs impacted population abundance and recovery and how different simulated management actions could enable recovery. We first estimated the impacts on population abundance and recovery by comparing the trajectory of an unexposed population to a population that had been exposed to a CEC with a specific pMoA after the end of the exposure. We then predicted how different management actions would impact population recovery by comparing the trajectories of an unexposed population to an exposed population for which a management action started at a fixed time without discontinuation of the exposure. Our results predicted that the individual-level pMoA of CECs has an important impact on population-level effects because different stressor's pMoA impacts the life-history traits of sturgeon differently. For example, the feeding and reproduction pMoAs caused the strongest and weakest population declines, respectively. For the same reason, pMoA also impacted recovery. For example, recovery was delayed when the pMoA was growth, maintenance, or feeding, but it was immediate when the pMoA was reproduction. We found that management actions that increased the egg survival rate or the stocking of fingerlings resulted in faster and stronger recovery than management actions that increased the juvenile or adult survival rate. This result occurred because the first two management actions immediately impacted recruitment, whereas the impact was delayed for the last two. Finally, there was greater potential for recovery when management action targeted eggs and fingerlings because these life stages have lower natural survival rates. Integr Environ Assess Manag 2022;18:1597-1608. © 2022 Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

A Sensitive and Quantitative Assay to Enumerate and Measure Mycobacterium leprae Viability in Clinical and Experimental Specimens.

Mycobacterium leprae, the etiologic agent of leprosy, cannot be cultured on artificial media. This characteristic, coupled with its long generation time, presents a number of unique challenges to studying this pathogen. One of the difficulties facing both researchers and clinicians is the absence of a rapid test to measure the viability of M. leprae in clinical or experimental specimens. The lack of such a tool limits the understanding of M. leprae immunopathogenesis and makes determining the efficacy of drug treatments difficult. With this in mind, we developed a robust two-step molecular viability assay (MVA) that first enumerates the M. leprae in the tissue; then, this data is used to normalize bacterial RNA quantities for the second step, in which the expression of M. leprae esxA and hsp18 are measured. This assay is specific and sensitive enough to be used on most clinical samples. This protocol describes the steps required to extract DNA and RNA from M. leprae-infected tissue, enumerate M. leprae, and measure M. leprae viability based on the normalized expression of two M. leprae-specific genes (hsp18 and esxA). This protocol also outlines an optimal laboratory design and workflow for performing this assay. © 2022 The Leprosy Mission Nepal. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: DNA and RNA P purification from M. leprae-infected tissue Basic Protocol 2: Enumeration of M. leprae by RLEP qPCR on the DNA fraction Basic Protocol 3: Calculation of M. leprae per tissue and normalization of RNA Basic Protocol 4: Reverse-transcription of normalized RNA to generate cDNA Basic Protocol 5: Determination of M. leprae viability using HSP18 and ESXA qPCR on the cDNA Support Protocol 1: M. leprae qPCR primer/probe stock preparation Support Protocol 2: Preparation of plasmid stocks and standard curves.

Risk Factors and Consequences of Lower Extremity Fracture Nonunions in Veterans With Spinal Cord Injury

ABSTRACTWe used Veterans Health Administration (VHA) national administrative data files to identify a cohort (fiscal years 2005–2014) of veterans with spinal cord injuries and disorders (SCID) to determine risk factors for and consequences of lower extremity fracture nonunions. Odds ratios (OR) for fracture nonunion were computed using multivariable‐adjusted logistic regression models. We identified three risk factors for nonunion: (i) older age (OR = 2.29; 95% confidence interval [CI] 1.21–4.33), (ii) longer duration of SCID (OR = 1.02; 95% CI 1.00–1.04), and (iii) fracture site (distal femur), with OR (comparison distal femur) including distal tibia/fibula (OR = 0.14; 95% CI 0.09–0.24), proximal tibia/fibula (OR = 0.19; 95% CI 0.09–0.38), proximal femur (OR = 0.10; 95% CI 0.04–0.21), and hip (OR = 0.13; 95% CI 0.07–0.26). Nonunions resulted in multiple complications, with upwards of 1/3 developing a pressure injury, 13% osteomyelitis, and almost 25% requiring a subsequent amputation. Our data have identified a high‐risk population for fracture nonunion of older veterans with a long duration of SCID who sustain a distal femur fracture. In view of the serious complications of these nonunions, targeted interventions in these high‐risk individuals who have any signs of delayed union should be considered. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Use of Arabinonucleosides for Development and Implementation of a Novel 2'-O-Protecting Group for Efficient Solid-Phase Synthesis and 2'-O-Deprotection of RNA Sequences.

The implementation of protecting groups for 2'-hydroxyl function of ribonucleosides is very demanding in that synthetic RNA sequences must be highly pure to ensure the safety and efficacy of nucleic acid-based drugs for treatment of human diseases. A synthetic approach consisting of a condensation reaction between 2'-O-aminoribonucleosides with ethyl pyruvate has been employed to provide stable 2'-O-imino-2-methyl propanoic acid ethyl esters. Conversion of these esters to fully protected ribonucleoside phosphoramidite monomers has allowed rapid and efficient incorporation of 2'-O-protected ribonucleosides into RNA sequences while minimizing the formation of process-related impurities during solid-phase synthesis. Two chimeric 20-mer RNA sequences have been synthesized and then exposed to a solution of sodium hydroxide to saponify the 2'-O-imino-2-methyl propanoic acid ethyl ester protecting groups to their sodium salts. When subjected to ion-exchange conditions at 65°C and near neutral pH, fully deprotected RNA sequences are isolated without production of alkylating side-products and/or formation of mutagenic nucleobase adducts. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Synthesis of uridine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 2: Synthesis of N6 -protected adenosine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 3: Synthesis of N4 -protected cytidine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 4: Synthesis of N2 -protected guanosine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 5: Automated solid-phase RNA synthesis and deprotection using 2'-O-imino-2-proponate-protected phosphoramidites.

Multiple lines of evidence for identifying potential hazards to fish from contaminants of emerging concern in Great Lakes tributaries.

Contaminants of emerging concern (CECs; e.g., pharmaceuticals, flame retardants, pesticides, and industrial chemicals) are omnipresent throughout tributaries to the Great Lakes. Furthermore, CECs are often present at concentrations that are potentially hazardous to aquatic species. Since 2010, we characterized the presence of CECs at 309 sites within 47 Great Lakes tributaries and characterized responses of fathead minnow (Pimephales promelas) exposed to river water at a subset of 26 sites within four tributaries. Our work resulted in three independent lines of evidence related to the potential hazards of CEC exposure to fish. First, vulnerability (where vulnerability refers to likelihood) of surface waters to CEC presence was predicted using select watershed characteristics. Second, hazard to fish (where hazard means the potential for adverse biological responses) was predicted using screening values for a subset of CECs. Third, biological responses of fathead minnow exposed to river water in streamside exposures were measured. We assessed the congruence of these three lines of evidence for identifying sites with elevated hazards to CEC exposure. Predicted vulnerability and hazards agreed at 66% of all sites. Where the two indices did not agree, vulnerability often underestimated predicted hazard. When compared with measured biological responses from streamside exposures, predicted hazards agreed for 42% of samples. Furthermore, when predicted hazards for specific effect categories were compared with similar measured biomarkers, 26% and 46% of samples agreed for reproductive and physiological effect categories, respectively. Overall, vulnerability and hazard predictions tended to overestimate the measured biological responses, providing a protective estimate of the potential hazards of CEC exposure to fish. When used together, these three approaches can help resource managers prioritize management activities in minimizing hazards of CEC exposure and can be used by researchers to prioritize studies focused on understanding the hazards of CEC exposure to fish. Integr Environ Assess Manag 2022;18:1246–1259. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Nutritional status significantly affects toxicological endpoints in the CDC bottle bioassay.

The CDC Bottle Bioassay serves as an inexpensive and effective way to screen field-caught mosquitoes against a wide variety of insecticidal active ingredients and commercial formulations, with the goal of detecting resistant individuals. For this study, we assessed how sucrose-water (10% w/v) feeding status impacted the response of Aedes aegypti mosquitoes to select insecticides. Starvation for 24 or 48 h decreased permethrin and malathion mean survival time by about 40%, with little difference in the two starvation times. Similar findings were also observed in a pyrethroid-resistant Puerto Rico strain challenged with permethrin, but these effects were less pronounced. To test the impact of mosquito weight, we measured weight under different 48-h nutritional conditions and found that sugar-water-fed and sugar-only-fed individuals were approximately the same weight (ANOVA, Bonferroni post-test P value >0.999) and that individuals fed water only were considerably lighter than either sugar-water-fed (ANOVA, Bonferroni post-test P value=0.034) or sugar-only-fed individuals (ANOVA, Bonferroni post-test P value=0.027) but equal in weight to starved mosquitoes (ANOVA, Bonferroni post-test P value >0.99). Of the nutritional challenges, water-only-fed mosquitoes were the most insecticide tolerant (ANOVA, Bonferroni post-test P values to all other treatments <0.01). The results indicate insect nutritional status is an important experimental variable, particularly the hydration status of mosquitoes shortly before insecticide exposure. Moreover, as significant differences were observed between starved and component-fed (water, sugar, or sugar and water) mosquitoes in a pyrethroid-resistant strain, some caution is appropriate when interpreting resistance/susceptibility diagnoses with this bioassay. © 2021 Society of Chemical Industry. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Ectopic Calcification and Hypophosphatemic Rickets: Natural History of ENPP1 and ABCC6 Deficiencies.

ABSTRACTGeneralized arterial calcification of infancy (GACI) is a rare disorder caused by ENPP1 or ABCC6 variants. GACI is characterized by low pyrophosphate, arterial calcification, and high mortality during the first year of life, but the natural course and possible differences between the causative genes remain unknown. In all, 247 individual records for patients with GACI (from birth to 58.3 years of age) across 19 countries were reviewed. Overall mortality was 54.7% (13.4% in utero or stillborn), with a 50.4% probability of death before the age of 6 months (critical period). Contrary to previous publications, we found that bisphosphonate treatment had no survival benefit based on a start‐time matched analysis and inconclusive results when initiated within 2 weeks of birth. Despite a similar prevalence of GACI phenotypes between ENPP1 and ABCC6 deficiencies, including arterial calcification (77.2% and 89.5%, respectively), organ calcification (65.8% and 84.2%, respectively), and cardiovascular complications (58.4% and 78.9%, respectively), mortality was higher for ENPP1 versus ABCC6 variants (40.5% versus 10.5%, respectively; p = 0.0157). Higher prevalence of rickets was reported in 70.8% of surviving affected individuals with ENPP1 compared with that of ABCC6 (11.8%; p = 0.0001). Eleven affected individuals presenting with rickets and without a GACI diagnosis, termed autosomal recessive hypophosphatemic rickets type 2 (ARHR2), all had confirmed ENPP1 variants. Approximately 70% of these patients demonstrated evidence of ectopic calcification or complications similar to those seen in individuals with GACI, which shows that ARHR2 is not a distinct condition from GACI but represents part of the spectrum of ENPP1 deficiency. Overall, this study identified an early mortality risk in GACI patients despite attempts to treat with bisphosphonates, high prevalence of rickets almost exclusive to ENPP1 deficiency, and a spectrum of heterogenous calcification and multiple organ complications with both ENPP1 and ABCC6 variants, which suggests an overlapping pathology. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Linking Trace Organic Contaminants in On-Site Wastewater-Treatment Discharge with Biological Effects.

Around the globe, on-site wastewater-treatment systems (OWTSs) are critical for rural communities without access to a municipal sewer system. However, their treatment efficiency does not match that of modern wastewater-treatment plants. The impact of OWTS discharge on nearby aquatic ecosystems and their resident fish species is poorly understood. In the present study, larval and adult fathead minnows (Pimephales promelas) and adult sunfish (Lepomis macrochirus) were exposed for 21 days to two trace organic contaminant (TOrC) mixtures replicating water chemistry derived from a previous environmental study. Larval fathead minnows were assessed for survival, growth, predator avoidance, and feeding efficiency. Adult fathead minnows and sunfish were assessed for a suite of physiological endpoints (condition indices, vitellogenin, glucose), histological changes, and fecundity. The only observed effect of TOrC mixture exposure on larval fathead minnows was a decrease in feeding efficiency. Effects were mixed in exposed adult fishes, except for male sunfish which realized a significant induction of vitellogenin (p < 0.05). The consequences of TOrC mixture exposure in the present controlled laboratory study match effects observed in wild-caught sunfish in a corresponding field study. The present study begins to bridge the gap by connecting nonpoint OWTS pollution with biological effects observed in resident lake fish species. Given the effects observed despite the brevity of the laboratory mixture exposure, longer-term studies are warranted to understand the full impacts of OWTS discharge to nearby aquatic ecosystems. Environ Toxicol Chem 2021;40:3193-3204. © 2021 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Protocols for Characterization of Cdk5 Kinase Activity.

Cyclin-dependent kinases (Cdks) are generally known to be involved in controlling the cell cycle, but Cdk5 is a unique member of this protein family for being most active in post-mitotic neurons. Cdk5 is developmentally important in regulating neuronal migration, neurite outgrowth, and axon guidance. Cdk5 is enriched in synaptic membranes and is known to modulate synaptic activity. Postnatally, Cdk5 can also affect neuronal processes such as dopaminergic signaling and pain sensitivity. Dysregulated Cdk5, in contrast, has been linked to neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Despite primarily being implicated in neuronal development and activity, Cdk5 has lately been linked to non-neuronal functions including cancer cell growth, immune responses, and diabetes. Since Cdk5 activity is tightly regulated, a method for measuring its kinase activity is needed to fully understand the precise role of Cdk5 in developmental and disease processes. This article includes methods for detecting Cdk5 kinase activity in cultured cells or tissues, identifying new substrates, and screening for new kinase inhibitors. Furthermore, since Cdk5 shares homology and substrate specificity with Cdk1 and Cdk2, the Cdk5 kinase assay can be used, with modification, to measure the activity of other Cdks as well. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Measuring Cdk5 activity from protein lysates Support Protocol 1: Immunoprecipitation of Cdk5 using Dynabeads Alternate Protocol: Non-radioactive protocols to measure Cdk5 kinase activity Support Protocol 2: Western blot analysis for the detection of Cdk5, p35, and p39 Support Protocol 3: Immunodetection analysis for Cdk5, p35, and p39 Support Protocol 4: Genetically engineered mice (+ and - controls) Basic Protocol 2: Identifying new Cdk5 substrates and kinase inhibitors.

Relations Between Bone Quantity, Microarchitecture, and Collagen Cross-links on Mechanics Following In Vivo Irradiation in Mice.

ABSTRACTHumans are exposed to ionizing radiation via spaceflight or cancer radiotherapy, and exposure from radiotherapy is known to increase risk of skeletal fractures. Although irradiation can reduce trabecular bone mass, alter trabecular microarchitecture, and increase collagen cross‐linking, the relative contributions of these effects to any loss of mechanical integrity remain unclear. To provide insight, while addressing both the monotonic strength and cyclic‐loading fatigue life, we conducted total‐body, acute, gamma‐irradiation experiments on skeletally mature (17‐week‐old) C57BL/6J male mice (n = 84). Mice were administered doses of either 0 Gy (sham), 1 Gy (motivated by cumulative exposures from a Mars mission), or 5 Gy (motivated by clinical therapy regimens) with retrieval of the lumbar vertebrae at either a short‐term (11‐day) or long‐term (12‐week) time point after exposure. Micro‐computed tomography was used to assess trabecular and cortical quantity and architecture, biochemical composition assays were used to assess collagen quality, and mechanical testing was performed to evaluate vertebral compressive strength and fatigue life. At 11 days post‐exposure, 5 Gy irradiation significantly reduced trabecular mass (p < 0.001), altered microarchitecture (eg, connectivity density p < 0.001), and increased collagen cross‐links (p < 0.001). Despite these changes, vertebral strength (p = 0.745) and fatigue life (p = 0.332) remained unaltered. At 12 weeks after 5 Gy exposure, the trends in trabecular bone persisted; in addition, regardless of irradiation, cortical thickness (p < 0.01) and fatigue life (p < 0.01) decreased. These results demonstrate that the highly significant effects of 5 Gy total‐body irradiation on the trabecular bone morphology and collagen cross‐links did not translate into detectable effects on vertebral mechanics. The only mechanical deficits observed were associated with aging. Together, these vertebral results suggest that for spaceflight, irradiation alone will likely not alter failure properties, and for radiotherapy, more investigations that include post‐exposure time as a positive control and testing of both failure modalities are needed to determine the cause of increased fracture risk. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Modeling the Bioavailability of Nickel and Zinc to Ceriodaphnia dubia and Neocloeon triangulifer in Toxicity Tests with Natural Waters

We studied biotic ligand model (BLM) predictions of the toxicity of nickel (Ni) and zinc (Zn) in natural waters from Illinois and Minnesota, USA, which had combinations of pH, hardness, and dissolved organic carbon (DOC) more extreme than 99.7% of waters in a nationwide database. We conducted 7-day chronic tests with Ceriodaphnia dubia and 96-hour acute and 14-day chronic tests with Neocloeon triangulifer and estimated median lethal concentrations and 20% effect concentrations for both species. Toxicity of Ni and Zn to both species differed among test waters by factors from 8 (Zn tests with C. dubia) to 35 (Zn tests with N. triangulifer). For both species and metals, tests with Minnesota waters (low pH and hardness, high DOC) showed lower toxicity than Illinois waters (high pH and high hardness, low DOC). Recalibration of the Ni BLM to be more responsive to pH-related changes improved predictions of Ni toxicity, especially for C. dubia. For the Zn BLM, we compared several input data scenarios, which generally had minor effects on model performance scores (MPS). A scenario that included inputs of modeled dissolved inorganic carbon and measured Al and Fe(III) produced the highest MPS values for tests with both C. dubia and N. triangulifer. Overall, the BLM framework successfully modeled variation in toxicity for both Zn and Ni across wide ranges of water chemistry in tests with both standard and novel test organisms. Environ Toxicol Chem 2021;40:3049-3062. © 2021 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Coding and Noncoding Variation in LRRK2 and Parkinson's Disease Risk.

BackgroundThe leucine‐rich repeat kinase 2 (LRRK2) gene harbors both rare highly damaging missense variants (eg, p.G2019S) and common noncoding variants (eg, rs76904798) with lower effect sizes that are associated with Parkinson's disease (PD) risk.ObjectivesThis study aimed to investigate in a large meta‐analysis whether the LRRK2 Genome‐Wide Association Study (GWAS) signal represented by rs76904798 is independently associated with PD risk from LRRK2 coding variation and whether complex linkage disequilibrium structures with p.G2019S and the 5′ noncoding haplotype account for the association of LRRK2 coding variants.MethodsWe performed a meta‐analysis using imputed genotypes from 17,838 patients, 13,404 proxy patients, and 173,639 healthy controls of European ancestry. We excluded carriers of p.G2019S and/or rs76904798 to clarify the role of LRRK2 coding variation in mediating disease risk and excluded carriers of relatively rare LRRK2 coding variants to assess the independence of rs76904798. We also investigated the co‐inheritance of LRRK2 coding variants with p.G2019S, rs76904798, and p.N2081D.Results LRRK2 rs76904798 remained significantly associated with PD after excluding the carriers of relatively rare LRRK2 coding variants. LRRK2 p.R1514Q and p.N2081D were frequently co‐inherited with rs76904798, and the allele distribution of p.S1647T significantly changed among patients after removing rs76904798 carriers.ConclusionsThese data suggest that the LRRK2 coding variants previously related to PD (p.N551K, p.R1398H, p.M1646T, and p.N2081D) do not drive the 5′ noncoding GWAS signal. These data, however, do not preclude the independent association of the haplotype p.N551K‐p.R1398H and p.M1646T with altered disease risk. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Rooting Out Genetic Structure of Invasive Wild Pigs in Texas

ABSTRACTInvasive wild pigs (Sus scrofa), also called feral swine or wild hogs, are recognized as among the most destructive invasive species in the world. Throughout the United States, invasive wild pigs have expanded rapidly over the past 40 years with populations now established in 38 states. Of the estimated 6.9 million wild pigs distributed throughout the United States, Texas supports approximately 40% of the population and similarly bears disproportionate ecological and economic costs. Genetic analyses are an effective tool for understanding invasion pathways and tracking dispersal of invasive species such as wild pigs and have been used recently in California and Florida, USA, which have similarly long‐established populations and high densities of wild pigs. Our goals were to use molecular approaches to elucidate invasion and migration processes shaping wild pig populations throughout Texas, compare our results with patterns of genetic structure observed in California and Florida, and provide insights for effective management of this invasive species. We used a high‐density single nucleotide polymorphism (SNP) array to evaluate population genetic structure. Genetic clusters of wild pigs throughout Texas demonstrate 2 distinct patterns: weakly resolved, spatially dispersed clusters and well‐resolved, spatially localized clusters. The disparity in patterns of genetic structure suggests disparate processes are differentially shaping wild pig populations in various localities throughout the state. Our results differed from the patterns of genetic structure observed in California and Florida, which were characterized by localized genetic clusters. These differences suggest distinct biological and perhaps anthropogenic processes are shaping genetic structure in Texas. Further, these disparities demonstrate the need for location‐specific management strategies for controlling wild pig populations and mitigating associated ecological and economic costs. © 2021 The Wildlife Society. This article has been contributed to by US Government employees and their work is in the public domain in the USA.