Surveillance for Selected Pathogens and Parasites of Northern Bobwhite (Colinus virginianus) from Western Oklahoma, USA, 2018-20.

The Northern Bobwhite (Colinus virginianus) has been undergoing a range-wide population decline. Potential causes for declines across its historic range have been investigated for decades and include habitat loss and fragmentation and a variety of parasitic and infectious diseases. Although there have been studies on bobwhite ecology in Oklahoma, USA, relatively little is known about parasites and pathogens in the region. We evaluated the health of free-ranging bobwhites from nine sites in western Oklahoma. From 2018 to 2020, 206 bobwhites were evaluated for gross and microscopic lesions and tested for selected pathogens. In general, bobwhites were in good nutritional condition with ample muscle mass and fat stores. No significant gross lesions were observed in any bobwhite and no significant histologic lesions were detected in a subset. There was no evidence of infection with or exposure to reticuloendotheliosis virus, West Nile virus, respiratory Mycoplasmataceae species, Pasteurella multocida, intestinal Eimeria spp., or oral Trichomonas spp. Several pathogens of potential concern were detected, including avian adenovirus (8.6%), Toxoplasma gondii (2.3%), and haemosporidians (a Haemoproteus sp. (1.5%), Leucocytozoon schoutedeni (1.5%), and Plasmodium homopolare haplotype 2 [lineage LAIRI01; 3.6%]). Physaloptera sp. (12%) and Sarcocystis sp. (1%) were detected in the breast muscle. Low intraspecific genetic diversity was noted for Physaloptera sp., and sequences were most similar to Physaloptera sequences from bobwhites and grasshoppers (Orthoptera) in Texas. Low intensities of chewing lice, chiggers, and ticks were observed. A subset of bobwhites had evidence of exposure to selected toxicants and heavy metals; a small number had low levels of iron, manganese, zinc, molybdenum, and copper, which were not considered diagnostically relevant. In general, bobwhites from western Oklahoma appeared to be in good health with a low diversity of pathogens detected, but future work is needed to understand potentially changing disease risks for this population.

Buffering Mitigates Chondrocyte Oxidative Stress, Metabolic Dysfunction, and Death Induced by Normal Saline: Formulation of a Novel Arthroscopic Irrigant.

For decades, surgeons have utilized 0.9% normal saline (NS) for joint irrigation to improve visualization during arthroscopic procedures. This continues despite mounting evidence that NS exposure impairs chondrocyte metabolism and compromises articular cartilage function. We hypothesized that chondrocyte oxidative stress induced by low pH is the dominant factor driving NS toxicity, and that buffering NS to increase its pH would mitigate these effects. Effects on chondrocyte viability, reactive oxygen species (ROS) production, and overall metabolic function were assessed. Even brief exposure to NS caused cell death, ROS overproduction, and disruption of glycolysis, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways. NS also stimulated ROS overproduction in synovial cells that could adversely alter the synovial function and subsequently the entire joint health. Buffering NS with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) significantly increased chondrocyte viability, reduced ROS production, and returned metabolite levels to near control levels while also reducing ROS production in synovial cells. These results confirm that chondrocytes and synoviocytes are vulnerable to insult from the acidic pH of NS and demonstrate that adding a buffering agent to NS averts many of its most harmful effects.

Health impacts of gastrointestinal and ocular parasites in northern bobwhite (Colinus virginianus) in western Oklahoma, USA.

The northern bobwhite (Colinus virginianus) is a popular upland game bird that is suffering from severe and ongoing population decline. In this study, we investigated the potential health impacts of gastrointestinal and periorbital parasites in bobwhite in western Oklahoma, USA. A sample of 206 bobwhites from 2018 to 2020 indicated a low prevalence and diversity of parasites. However, at least one gastrointestinal or ocular parasite species was detected in 112 bobwhite (54.4%). A total of three gastrointestinal parasite species were detected, including Aulonocephalus pennula (54% prevalence, mean intensity 71.6 ± 99.8), Raillietina spp. (7%, 4.2 ± 1.9), and a single immature Mediorhynchus sp. acanthocephalan (0.5%). Burdens of A. pennula infections were negatively associated with fat stores in their bobwhite host. Low intensities (range 1-10, mean 3.9 ± 2.9) of eyeworms (Oxyspirura petrowi) were observed in 12.6% (26/206) of bobwhite sampled and were not associated with fat stores. No significant histologic lesions were associated with O. petrowi worms in ocular and surrounding tissues of 68 quail eyes examined, of which 26 (38%) were positive for eyeworms. Overall, the prevalence and intensity of parasites in bobwhite in Oklahoma were lower than in previous studies in Texas in similar physiographic regions. However, continued studies on the impacts of these parasites on quail health are needed as environmental and climate changes could alter the ecology and significance of these parasites.

Storage time and temperature of filter paper strips affect anti-West Nile virus antibody detection in 2 galliform species.

The effects of filter paper strip (FPS) storage time and temperature on antibody detection are poorly understood despite widespread use in wildlife research. We collected sera and FPSs from 23 wild turkeys (Meleagris gallopavo) and 20 northern bobwhites (Colinus virginianus) experimentally infected with West Nile virus (WNV) to compare FPS storage methods with WNV plaque reduction neutralization test (PRNT) sensitivity. FPS storage methods included: immediate elution after drying, and storage at -20°C for 3 mo, -20°C for 6 mo, room temperature (RT) for 3 mo, and RT for 6 mo prior to elution. FPS eluates and sera were co-titrated to determine endpoint antibody titers, which were compared between FPS sera eluted immediately and sera, and among FPS eluates that underwent different storage conditions. Antibody titers were ~4-fold less in FPS sera eluted immediately versus sera, and dropped more frequently below PRNT detection threshold in northern bobwhites, which had ~10-fold lower serum antibody titers than wild turkeys. Antibody titers were lower in FPS samples stored at RT and for 6 mo. WNV serologic surveys may result in falsely low seroprevalence estimates if FPSs are stored at RT for ≥ 3 mo before elution.

Intraoperative Methadone Reduces Postoperative Opioid Requirements in Nuss Procedure for Pectus Excavatum.

INTRODUCTION: Surgical correction of pectus excavatum by Nuss procedure, commonly referred to as minimally invasive repair of pectus excavatum (MIRPE), often results in significant postoperative pain. This study investigated whether adding intraoperative methadone would reduce the postoperative opioid requirement during admission for patients undergoing MIRPE. METHODS: A retrospective cohort chart review was conducted for 40 MIRPE patients between 2018 and 2020. Patients were stratified into 2 groups: those who received multimodal anesthesia (MM, n = 20) and those who received multimodal anesthesia with the addition of intraoperative methadone (MM + M, n = 20). Data collected included total opioid consumption during hospital stay (morphine milligram equivalents [MMEs]), hospital length of stay (LOS), pain scores, time to ambulation, and time to tolerating solid food. RESULTS: Addition of intraoperative methadone for patients undergoing MIRPE significantly reduced postoperative opioid requirements (MME/kg) during admission (P = .007). On average, patients in the MM group received 1.61 ± .55 MME/kg while patients in the MM + M group received 1.16 ± .44 MME/kg. Hospital opioid (non-methadone) total was also significantly reduced between the MM (1.87 ± .54) and MM + M group (1.37 ± .46), P = .003. There was no significant difference in hospital opioid total MME/kg administered between the groups. There were no significant differences observed in hospital LOS, pain scores, time to ambulation, or time to toleration of solid food. DISCUSSION: Incorporating intraoperative methadone for patients undergoing MIRPE reduced postoperative opioid requirements and hospital opioid (non-methadone) totals without a significant change in pain scores. Patients undergoing the Nuss procedure may benefit from the administration of intraoperative methadone.

Susceptibility of wild turkeys (Meleagris gallopavo) to experimental West Nile virus infection.

Since the introduction of West Nile virus (WNV) to North America in 1999, WNV is estimated to have contributed to population-level declines in numerous avian species. However, the potential impacts of this virus on many free-ranging upland game bird species, including the wild turkey (Meleagris gallopavo), which is undergoing regional population declines, remain unknown. Herein, two age groups (∼5 to 6 weeks and ∼15 to 16 weeks post-hatch) of juvenile wild turkeys were subcutaneously inoculated with WNV, sampled daily from 1 to 7 days post-inoculation (dpi), and euthanized on 14 dpi. No clinical signs and minimal gross lesions were attributable to WNV infection. Peak viraemia titres were similar between age groups (<101.7 to 104.6 plaque-forming units [PFU]/ml), but the duration of viraemia was longer in the old group (3-4 days) than in the young group (0-3 days). Intermittent oral and/or cloacal viral shedding from 2 to 7 dpi was detected in both age groups. No infectious virus was detected in the heart, brain, kidney, skeletal muscle, spleen, and feathers from WNV-inoculated turkeys euthanized on 14 dpi. All WNV-inoculated birds seroconverted by 14 dpi, as well as two co-housed sham-inoculated birds. The most consistent microscopic lesions among all WNV-inoculated birds were mild lymphoplasmacytic myocarditis and encephalitis. Minimal immunohistochemical labelling was detected in tissues in addition to scant macrophages within the blood, spleen, and bone marrow. These data suggest WNV is unlikely to pose a significant risk to wild turkey populations, although the possibility remains that WNV may indirectly decrease fitness or predispose wild turkeys to other health stressors.RESEARCH HIGHLIGHTS Clinical disease was not observed in wild turkeys experimentally infected with WNV.Pathology attributed to WNV was mild and included brain and heart inflammation.Viraemias suggest WNV-infected wild turkeys do not play a role in WNV transmission.No age-associated differences in WNV clinical disease or pathology were observed.

Force-Bioreactor for Assessing Pharmacological Therapies for Mechanobiological Targets.

Tissue fibrosis is a major health issue that impacts millions of people and is costly to treat. However, few effective anti-fibrotic treatments are available. Due to their central role in fibrotic tissue deposition, fibroblasts and myofibroblasts are the target of many therapeutic strategies centered primarily on either inducing apoptosis or blocking mechanical or biochemical stimulation that leads to excessive collagen production. Part of the development of these drugs for clinical use involves in vitro prescreening. 2D screens, however, are not ideal for discovering mechanobiologically significant compounds that impact functions like force generation and other cell activities related to tissue remodeling that are highly dependent on the conditions of the microenvironment. Thus, higher fidelity models are needed to better simulate in vivo conditions and relate drug activity to quantifiable functional outcomes. To provide guidance on effective drug dosing strategies for mechanoresponsive drugs, we describe a custom force-bioreactor that uses a fibroblast-seeded fibrin gels as a relatively simple mimic of the provisional matrix of a healing wound. As cells generate traction forces, the volume of the gel reduces, and a calibrated and embedded Nitinol wire deflects in proportion to the generated forces over the course of 6 days while overhead images of the gel are acquired hourly. This system is a useful in vitro tool for quantifying myofibroblast dose-dependent responses to candidate biomolecules, such as blebbistatin. Administration of 50 µM blebbistatin reliably reduced fibroblast force generation approximately 40% and lasted at least 40 h, which in turn resulted in qualitatively less collagen production as determined via fluorescent labeling of collagen.

Influence of weather on gobbling activity of male wild turkeys.

Gobbling activity of Eastern wild turkeys (Meleagris gallopavo silvestris; hereafter, turkeys) has been widely studied, focusing on drivers of daily variation. Weather variables are widely believed to influence gobbling activity, but results across studies are contradictory and often equivocal, leading to uncertainty in the relative contribution of weather variables to daily fluctuations in gobbling activity. Previous works relied on road-based auditory surveys to collect gobbling data, which limits data consistency, duration, and quantity due to logistical difficulties associated with human observers and restricted sampling frames. Development of new methods using autonomous recording units (ARUs) allows researchers to collect continuous data in more locations for longer periods of time, providing the opportunity to delve into factors influencing daily gobbling activity. We used ARUs from 1 March to 31 May to detail gobbling activity across multiple study sites in the southeastern United States during 2014-2018. We used state-space modeling to investigate the effects of weather variables on daily gobbling activity. Our findings suggest rainfall, greater wind speeds, and greater temperatures negatively affected gobbling activity, whereas increasing barometric pressure positively affected gobbling activity. Therefore, when using daily gobbling activity to make inferences relative to gobbling chronology, reproductive phenology, and hunting season frameworks, stakeholders should recognize and consider the potential influences of extended periods of inclement weather.

Comparisons of Ribonuclease HI Homologs and Mutants Uncover a Multistate Model for Substrate Recognition.

Ribonuclease HI (RNHI) nonspecifically cleaves the RNA strand of RNA:DNA hybrid duplexes in a myriad of biological processes. Several RNHI homologs contain an extended domain, termed the handle region, which is critical to substrate binding. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have suggested a kinetic model in which the handle region can exist in open (substrate-binding competent) or closed (substrate-binding incompetent) states in homologs containing arginine or lysine at position 88 (using sequence numbering of E. coli RNHI), while the handle region populates states intermediate between the open and closed conformers in homologs with asparagine at residue 88 [Stafford, K. A., et al., PLoS Comput. Biol. 2013, 9, 1-10]. NMR parameters characterizing handle region dynamics are highly correlated with enzymatic activity for RNHI homologs with two-state (open/closed) handle regions [Martin, J. A., et al., Biochemistry 2020, 59, 3201-3205]. The work presented herein shows that homologs containing asparagine 88 display distinct structural features compared with their counterparts containing arginine or lysine 88. Comparisons of RNHI homologs and site-directed mutants with asparagine 88 support a kinetic model for handle region dynamics that includes 12 unique transitions between eight conformations. Overall, these findings present an example of the structure-function relationships of enzymes and spotlight the use of NMR spectroscopy and MD simulations in uncovering fine details of conformational preferences.

Objective evaluation of chondrocyte density & cloning after joint injury using convolutional neural networks.

Variations in chondrocyte density and organization in cartilage histology sections are associated with osteoarthritis progression. Rapid, accurate quantification of these two features can facilitate the evaluation of cartilage health and advance the understanding of their significance. The goal of this work was to adapt deep-learning-based methods to detect articular chondrocytes and chondrocyte clones from safranin-O-stained cartilage to evaluate chondrocyte cellularity and organization. The U-net and "you-only-look-once" (YOLO) models were trained and validated for identifying chondrocytes and chondrocyte clones, respectively. Validated models were then used to quantify chondrocyte and clone density in talar cartilage from Yucatan minipigs sacrificed 1 week, 3, 6, and 12 months after fixation of an intra-articular fracture of the hock joint. There was excellent/good agreement between expert researchers and the developed models in identifying chondrocytes/clones (U-net: R2 = 0.93, y = 0.90x-0.69; median F1 score: 0.87/YOLO: R2 = 0.79, y = 0.95x; median F1 score: 0.67). Average chondrocyte density increased 1 week after fracture (from 774 to 856 cells/mm2 ), decreased substantially 3 months after fracture (610 cells/mm2 ), and slowly increased 6 and 12 months after fracture (638 and 683 cells/mm2 , respectively). Average detected clone density 3, 6, and 12 months after fracture (11, 11, 9 clones/mm2 ) was higher than the 4-5 clones/mm2 detected in normal tissue or 1 week after fracture and show local increases in clone density that varied across the joint surface with time. The accurate evaluation of cartilage cellularity and organization provided by this deep learning approach will increase objectivity of cartilage injury and regeneration assessments.

Exosome-Based Cell Homing and Angiogenic Differentiation for Dental Pulp Regeneration.

Exosomes have attracted attention due to their ability to promote intercellular communication leading to enhanced cell recruitment, lineage-specific differentiation, and tissue regeneration. The object of this study was to determine the effect of exosomes on cell homing and angiogenic differentiation for pulp regeneration. Exosomes (DPSC-Exos) were isolated from rabbit dental pulp stem cells cultured under a growth (Exo-G) or angiogenic differentiation (Exo-A) condition. The characterization of exosomes was confirmed by nanoparticle tracking analysis and an antibody array. DPSC-Exos significantly promoted cell proliferation and migration when treated with 5 × 108/mL exosomes. In gene expression analysis, DPSC-Exos enhanced the expression of angiogenic markers including vascular endothelial growth factor A (VEGFA), Fms-related tyrosine kinase 1 (FLT1), and platelet and endothelial cell adhesion molecule 1 (PECAM1). Moreover, we identified key exosomal microRNAs in Exo-A for cell homing and angiogenesis. In conclusion, the exosome-based cell homing and angiogenic differentiation strategy has significant therapeutic potential for pulp regeneration.

HPLC-UV Method Validation for Amobarbital and Pharmaceutical Stability Evaluation When Dispersed in a Hyaluronic Acid Hydrogel: A New Concept for Post-Traumatic Osteoarthritis Prevention.

A mitochondrial electron transport chain member complex I inhibitor, amobarbital, can reduce oxidative damage and chondrocyte death, eventually preventing post-traumatic osteoarthritis (PTOA). Viscosupplementation using a crosslinked hyaluronic acid (HA) hydrogel is currently applied clinically for knee OA pain relief. In this work, we utilized the HA hydrogel as a drug delivery vehicle to improve the long-term efficacy of amobarbital. Here we evaluated the pharmaceutic stability of amobarbital when dispersed in a crosslinked HA hydrogel formulated in proportions intended for clinical use. We validated a high-performance liquid chromatography with an ultraviolet detector (HPLC-UV) method following International Conference for Harmonization Q2(R1) guidelines to ensure its suitability for amobarbital detection. The feasibility of this formulation's drug delivery capability was proven by measuring the release, solubility, and drug uniformity. The amobarbital/HA hydrogel showed comparable amobarbital stability in different biological fluids compared to amobarbital solution. In addition, the amobarbital/HA hydrogel imparted significantly greater drug stability when stored at 70°C for 24 hours. In conclusion, we confirmed the pharmaceutical stability of the amobarbital/HA hydrogel in various conditions and biological fluids using a validated HPLC-UV method. This data provides essential evidence in support of the use of this amobarbital/HA formulation in future clinical trials for PTOA treatment.

Intra-Articular Adeno-Associated Virus-Mediated Proteoglycan 4 Gene Therapy for Preventing Posttraumatic Osteoarthritis.

Lubricin, a glycoprotein encoded by the proteoglycan 4 (PRG4) gene, is an essential boundary lubricant that reduces friction between articular cartilage surfaces. The loss of lubricin subsequent to joint injury plays a role in the pathogenesis of posttraumatic osteoarthritis. In this study, we describe the development and evaluation of an adeno-associated virus (AAV)-based PRG4 gene therapy intended to restore lubricin in injured joints. The green fluorescent protein (GFP) gene was inserted the PRG4 gene to facilitate tracing the distribution of the transgene product (AAV-PRG4-GFP) in vivo. Transduction efficiency of AAV-PRG4-GFP was evaluated in joint cells, and the conditioned medium containing secreted PRG4-GFP was used for shear loading/friction and viability tests. In vivo transduction of joint tissues following intra-articular injection of AAV-PRG4-GFP was confirmed in the mouse stifle joint in a surgical model of destabilization of the medial meniscus (DMM), and chondroprotective activity was tested in a rabbit anterior cruciate ligament transection (ACLT) model. In vitro studies showed that PRG4-GFP has lubricin-like cartilage-binding and antifriction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP before cyclic shear loading (n = 3). Polymerase chain reaction and confocal microscopy confirmed the presence of PRG4-GFP DNA and protein, respectively, in a mouse DMM (n = 3 per group). In the rabbit ACLT model, AAV-PRG4-GFP gene therapy enhanced lubricin expression (p = 0.001 vs. AAV-GFP: n = 7-14) and protected the cartilage from degeneration (p = 0.014 vs. AAV-GFP: n = 9-10) when treatments were administered immediately postoperation, but efficacy was lost when treatment was delayed for 2 weeks. AAV-PRG4-GFP gene therapy protected cartilage from degeneration in a rabbit ACLT model; however, data from the ACLT model suggest that early intervention is essential for efficacy.

Temporal and scalar variations affect resource use of northern bobwhite broods.

Disparate resource use originating from phenology of biotic resources, abiotic conditions, and life cycles of exploiting organisms underscores the importance of research across time and space to guide management practices. Our goal was to evaluate resource use of northern bobwhite (Colinus virginianus; bobwhite) at two spatial scales and across three age classes, from hatching through a period of the postjuvenile molt. Our study was conducted at Tall Timbers Research Station, Tallahassee, FL, USA-situated in a landscape subjected to small scale (<20 ha) prescribed fires on a 2-year fire rotation. We predicted prescribed fire, disking, and supplemental feeding would dictate resource use, but effects would depend on time since fire, brood age, and time of day. We predicted vegetation and temperature would govern roost use by broods, but these effects would also depend on age. We radio-tracked 62 broods 21-35 times / week during May-October 2018 and 2019. Broods were less likely to use areas with large proportions of hardwood drains but favored sites with greater proportions of burned uplands, regardless of the time of day. Broods were less likely to use areas at greater distances from supplemental feed; this relationship had no interaction with age but was stronger later in the nesting season (>July 15). Broods were more likely to use areas with greater proportions of fallow fields during the day than for roosting. Broods used roosts with more woody cover and visual obscurity than at available sites. Roosts consisted of less grass and bare ground. However, these effects interacted with age; broods used sparser cover at older ages. Neonate broods were more likely to use cooler roosts with greater thermal stability, but this effect was reversed for juveniles. Broods may alter resource use with changes in vulnerabilities to threats such as thermal risks and predation.

Migrating Progenitor Cells Derived From Injured Cartilage Surface Respond to Damage-Associated Molecular Patterns.

OBJECTIVE: To delineate the response of migrating chondrogenic progenitor cells (CPCs) that arose from the surface of mechanically injured articular cartilage to proinflammatory damage-associated-molecular-patterns (DAMPs). DESIGN: Bovine CPCs and non-CPC chondrocytes isolated from either impacted or scratched articular cartilage were studied. Those 2 types of cells were treated with mitochondrial DAMPs (MTDs; 10 nM fMLF and 10 µg/mL CpG DNA), or 10 nM HMGB1, or 10 ng/mL IL-1b for 24 hours. At the end of experiments, conditioned media and cell lysates were collected for analysis of expression levels of matrix metalloproteinases (MMPs), chemokines, and cytokines that are associated with cartilage degeneration with Western blotting and quantitative polymerase chain reaction. The difference of expression levels was compared by Welch's t-test. RESULTS: Our data indicated that HMGB1 and MTDs remarkably upregulated pro-MMP-13 expression in CPCs. Compared with non-CPCs, CPCs expressed significantly more baseline mRNAs of MMP-13, CXCL12, and IL-6. MTDs greatly increased the expression of MMP-13 and IL-6 in CPCs by over 100-fold (P < 0.001). MTDs also significantly increased IL-8 expression in CPCs to a similar extent (P < 0.001). However, when IL-1b was present, CPCs expressed less MMP-3 and active MMP-13 proteins as well as less CCL2 and IL-6 than did non-CPCs. CONCLUSIONS: We concluded that CPCs were more sensitive than non-CPCs in response to DAMPs, especially MTDs. The proinflammatory nature of CPCs implied their critical role in the early phase of posttraumatic osteoarthritis development.

Top-down effects of raptor predation on northern bobwhite.

Understanding how predators affect prey species is a central endeavor in applied ecology. Game birds are a culturally and economically important group of birds throughout the world. Specifically, northern bobwhite (Colinus virginianus) is an imperiled game bird native to North America that has declined precipitously over the past 65 years. Concurrently, raptor populations increased substantially as a result of pesticide bans and legal protections. However, relationships between raptors and bobwhites are not well-understood because of limited long-term data. We analyzed long-term raptor survey and bobwhite survival datasets from 2008 to 2018 to determine if oscillations in raptor abundance affected bobwhite survival. We used a novel open multi-species dynamics hierarchical distance sampling model to estimate the abundance of raptors. We used a known-fate survival model to determine if variation in raptor abundance affected bobwhite survival. We had multiple working hypotheses regarding biological relationships between raptor abundance and bobwhite survival. Raptors affected bobwhites in every biological season but were more influential in the breeding season and late winter supporting the notion of bobwhite behavior and raptor migration were driving observed patterns. Our results suggest that even in areas with abundant habitat, predators exert top-down influences on vital rates suggesting similar or greater influences on populations under poor habitat conditions.

Optimizing conservation in species-specific agricultural landscapes.

Recovery of grassland birds in agricultural landscapes is a global imperative. Agricultural landscapes are complex, and the value of resource patches may vary substantially among species. The spatial extent at which landscape features affect populations (i.e., scale of effect) may also differ among species. There is a need for regional-scale conservation planning that considers landscape-scale and species-specific responses of grassland birds to environmental change. We developed a spatially explicit approach to optimizing grassland conservation in the context of species-specific landscapes and prioritization of species recovery and applied it to a conservation program in Kentucky (USA). We used a hierarchical distance-sampling model with an embedded scale of effect predictor to estimate the relationship between landscape structure and abundance of eastern meadowlarks (Sturnella magna), field sparrows (Spizella pusilla), and northern bobwhites (Colinus virginianus). We used a novel spatially explicit optimization procedure rooted in multi-attribute utility theory to design alternative conservation strategies (e.g., prioritize only northern bobwhite recovery or assign equal weight to each species' recovery). Eastern meadowlarks and field sparrows were more likely to respond to landscape-scale resource patch adjacencies than landscape-scale patch densities. Northern bobwhite responded to both landscape-scale resource patch adjacencies and densities and responded strongly to increased grassland density. Effects of landscape features on local abundance decreased as distance increased and had negligible influence at 0.8 km for eastern meadowlarks (0.7-1.2 km 95% Bayesian credibility intervals [BCI]), 2.5 km for field sparrows (1.5-5.8 km 95% BCI), and 8.4 km for bobwhite (6.4-26 km 95% BCI). Northern bobwhites were predicted to benefit greatly from future grassland conservation regardless of conservation priorities, but eastern meadowlark and field sparrow were not. Our results suggest similar species can respond differently to broad-scale conservation practices because of species-specific, distance-dependent relationships with landscape structure. Our framework is quantitative, conceptually simple, customizable, and predictive and can be used to optimize conservation in heterogeneous ecosystems while considering landscape-scale processes and explicit prioritization of species recovery.

La recuperación de las aves de pastizal en los paisajes agrícolas es una obligación mundial. Los paisajes agrícolas son complejos y el valor de los fragmentos con recursos puede variar sustancialmente entre especies. La magnitud espacial a la que las características del paisaje afectan a las poblaciones (es decir, la escala del efecto) también puede diferir entre especies. Existe la necesidad de una planeación de la conservación a escala regional que considere la escala de paisaje y las respuestas específicas de especie de aves de pastizal al cambio ambiental. Desarrollamos una estrategia espacialmente explícita para optimizar la conservación de pastizales en el contexto de los paisajes de especies específicas y la priorización de la recuperación de especies y la aplicamos a un programa de conservación en Kentucky (E.U.A.). Usamos un modelo jerárquico de muestreo a distancia con una escala integrada del efecto pronosticador para estimar la relación entre la estructura del paisaje y la abundancia de la alondra oriental de pradera (Sturnella magna), el gorrión de campo (Spizella pusilla) y la codorniz norteña (Colinus virginianus). Usamos un novedoso procedimiento de optimización espacialmente explícito basado en la teoría de utilidad multicaracterística para diseñar estrategias de conservación alternativas (p. ej.: priorizar solamente la recuperación de la codorniz norteña o asignar una importancia idéntica a la recuperación de cada especie). La alondra y el gorrión tuvieron una mayor probabilidad de responder a la proximidad de fragmentos con recursos a escala de paisaje que a la densidad de fragmentos a escala de paisaje. La codorniz respondió tanto a la proximidad de fragmentos con recursos a escala de paisaje como a la densidad y también respondió fuertemente al incremento en la densidad del pastizal. Los efectos de las características del paisaje sobre la abundancia local disminuyeron conforme incrementó la distancia, representando una influencia insignificante a los 0.8 km para la alondra (0.7-1.2 km 95% de intervalos de credibilidad bayesiana [ICB]), a los 2.5 km para el gorrión (1.5-5.8 km 95% ICB) y a los 8.4 km para la codorniz (6.4-26 km 95% ICB). Se pronosticó que la codorniz se beneficiaría enormemente con la conservación futura de los pastizales sin importar las prioridades de conservación, pero no fue el caso para la alondra y el gorrión. Nuestros resultados sugieren que especies similares pueden responder de manera diferente a las prácticas de conservación a escalas generalizadas debido a las relaciones específicas de especie y dependientes de la distancia con la estructura del paisaje. Nuestro marco de trabajo es cuantitativo, conceptualmente simple, adaptable y predictivo y puede usarse para optimizar la conservación en los ecosistemas heterogéneos a la vez que considera los procesos a escala de paisaje y la priorización explícita de la recuperación de las especies.

De novo assembly and delivery to mouse cells of a 101 kb functional human gene.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

Targeting oxidative stress with amobarbital to prevent intervertebral disc degeneration: Part I. in vitro and ex vivo studies.

BACKGROUND: Mounting evidence that oxidative stress contributes to the pathogenesis of intervertebral disc (IVD) degeneration (IDD) suggests that therapies targeting oxidative stress may slow or prevent disease progression. PURPOSE: The objective of this study was to investigate the inhibitory effects of amobarbital (Amo) on the mitochondria of nucleus pulposus (NP) cells under tert-butyl hydrogen peroxide (tBHP)-induced oxidative stress or in NP tissues under oxidative stress from tissue injury as a means of identifying therapeutic targets for IDD. STUDY DESIGN/SETTING: We tested the effects inhibiting mitochondria, a major source of oxidants, with Amo in NP cells subjected to two different forms of insult: exposure to tBHP, and physical injury induced by disc transection. N-acetylcysteine (NAC), an antioxidant known to protect NP cells, was compared to the complex I inhibitor, Amo. METHODS: NP cells were pre-treated for 2 hours with Amo, NAC, or both, and then exposed to tBHP for 1 hour. Apoptosis, necrosis, and reactive oxygen species (ROS) production were assessed using confocal microscopy and fluorescent probes (Annexin V, propidium iodide, and MitoSox Red, respectively). The activation of mitogen-activated protein kinases (MAPKs) involved in oxidative stress responses were interrogated by confocal imaging of immunofluorescence stains using phospho-specific antibodies to extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38. Mitochondrial function was assessed by imaging JC-1 staining, a probe for membrane potential. RESULTS: Amo was modestly more protective than NAC by some measures, while both agents improved mitochondrial function and lowered tBHP-induced apoptosis, necrosis, and ROS production. Activation of MAPK by tBHP was significantly suppressed by both drugs. Physically injured IVDs were treated immediately after transection with Amo or NAC for 24 hours, and then stained with dihydroethidium (DHE), a fluorescent probe for ROS production. Immunofluorescence was used to track the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor that induces the expression of antioxidant genes. Amo and NAC significantly reduced ROS production and increased Nrf2 expression. CONCLUSION: These findings suggest that the progression of IDD may be forestalled by Amo via protection of NP cells from oxidative stress following IVD injury. CLINICAL SIGNIFICANCE: This study will define the extent to which a novel, minimally invasive procedure targeting oxidative stress in NP cells can augment surgical interventions intended to retard IVD degeneration.

Accumulation of Contaminants by Wild Turkeys and Potential for Consumer Exposure.

Despite their popularity as a game species across North America, little is known about contaminant burdens in wild turkeys (Meleagris gallopavo) inhabiting areas of environmental contamination, and their potential to expose human and wildlife consumers. We compared trace element concentrations and radionuclide activity in muscle and liver tissues of turkeys inhabiting the Savannah River Site (USA), an area with known anthropogenic contaminant inputs, with turkeys from uncontaminated areas offsite. In addition, we tested breast feathers from a subset of individuals for mercury (Hg) to assess the viability of nonlethal sampling for quantifying Hg concentrations. Finally, we quantified arsenic, Hg, and selenium (Se) in blood of live-captured turkeys inhabiting a coal ash basin on the Savannah River Site. Compared with reference samples, we found that turkeys inhabiting the Savannah River Site contained elevated Hg levels in both muscle and liver tissues, and lower concentrations of chromium. Turkeys from the ash basin also had elevated levels of Se. We found a positive correlation between breast muscle and feather Hg concentrations (F1,59 = 267.5, R2 = 0.82, p < 0.001), suggesting that feathers can potentially be used as a nonlethal sampling technique. All elements analyzed were below reference limits set by the Centers for Disease Control and Prevention for safe consumption. Thus, our data suggest that turkeys likely are not an important pathway of contaminant exposure on the Savannah River Site or other areas with similar contaminant distributions. Environ Toxicol Chem 2021;40:1222-1231. © 2020 SETAC.