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.

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.

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.

Phylogenetic debugging of a complete human biosynthetic pathway transplanted into yeast.

Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.

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.

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.

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.

Quantifying the Relationship between Conformational Dynamics and Enzymatic Activity in Ribonuclease HI Homologues.

Ribonuclease HI (RNHI), a ubiquitous, non-sequence-specific endonuclease, cleaves the RNA strand in RNA/DNA hybrids. RNHI functions in replication and genome maintenance, and retroviral reverse transcriptases contain an essential ribonuclease H domain. Nuclear magnetic resonance (NMR) spectroscopy combined with molecular dynamics (MD) simulations suggests a model in which the extended handle region domain of Escherichia coli RNHI populates (substrate-binding-competent) "open" and (substrate-binding-incompetent) "closed" states, while the thermophilic Thermus thermophilus RNHI mainly populates the closed state at 300 K [Stafford, K. A., Robustelli, P., and Palmer, A. G., III (2013) PLoS Comput. Biol. 9, 1-10]. In addition, an in silico-designed mutant E. coli Val98Ala RNHI was predicted to populate primarily the closed state. The work presented here validates this model and confirms the predicted properties of the designed mutant. MD simulations suggest that the conformational preferences of the handle region correlate with the conformations of Trp85, Thr92, and Val101. NMR residual dipolar coupling constants, three-bond scalar coupling constants, and chemical shifts experimentally define the conformational states of these residues and hence of the handle domain. These NMR parameters correlate with the Michaelis constants for RNHI homologues, confirming the important role of the handle region in the modulation of substrate recognition and illustrating the power of NMR spectroscopy in dissecting the conformational preferences underlying enzyme function.

Anopheline and human drivers of malaria risk in northern coastal, Ecuador: a pilot study.

BACKGROUND: Understanding local anopheline vector species and their bionomic traits, as well as related human factors, can help combat gaps in protection. METHODS: In San José de Chamanga, Esmeraldas, at the Ecuadorian Pacific coast, anopheline mosquitoes were sampled by both human landing collections (HLCs) and indoor-resting aspirations (IAs) and identified using both morphological and molecular methods. Human behaviour observations (HBOs) (including temporal location and bed net use) were documented during HLCs as well as through community surveys to determine exposure to mosquito bites. A cross-sectional evaluation of Plasmodium falciparum and Plasmodium vivax infections was conducted alongside a malaria questionnaire. RESULTS: Among 222 anopheline specimens captured, based on molecular analysis, 218 were Nyssorhynchus albimanus, 3 Anopheles calderoni (n = 3), and one remains unidentified. Anopheline mean human-biting rate (HBR) outdoors was (13.69), and indoors (3.38) (p = 0.006). No anophelines were documented resting on walls during IAs. HBO-adjusted human landing rates suggested that the highest risk of being bitten was outdoors between 18.00 and 20.00 h. Human behaviour-adjusted biting rates suggest that overall, long-lasting insecticidal bed nets (LLINs) only protected against 13.2% of exposure to bites, with 86.8% of exposure during the night spent outside of bed net protection. The malaria survey found 2/398 individuals positive for asymptomatic P. falciparum infections. The questionnaire reported high (73.4%) bed net use, with low knowledge of malaria. CONCLUSION: The exophagic feeding of anopheline vectors in San Jose de Chamanga, when analysed in conjunction with human behaviour, indicates a clear gap in protection even with high LLIN coverage. The lack of indoor-resting anophelines suggests that indoor residual spraying (IRS) may have limited effect. The presence of asymptomatic infections implies the presence of a human reservoir that may maintain transmission.

Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae.

As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.

Looking back on wartime service: Reminiscence by James G. Van Straten.

In this article we highlight the reminiscence of a distinguished Veteran of the Vietnam War. The narrative describes his wartime service in Vietnam and the legacy of these experiences across his military career and subsequent civilian life. The individual profiled is not intended to represent all Veterans who served in the Vietnam War. In fact, this is a very unique individual; a highly educated, career officer and committed Catholic who served in various roles within the United States Army Medical Department. This narrative represents memories and reflections from his military career and in his subsequent post-military civilian life.

Impacts of biomass production at civil airports on grassland bird conservation and aviation strike risk.

Growing concerns about climate change, foreign oil dependency, and environmental quality have fostered interest in perennial native grasses (e.g., switchgrass [Panicum virgatum]) for bioenergy production while also maintaining biodiversity and ecosystem function. However, biomass cultivation in marginal landscapes such as airport grasslands may have detrimental effects on aviation safety as well as conservation efforts for grassland birds. In 2011-2013, we investigated effects of vegetation composition and harvest frequency on seasonal species richness and habitat use of grassland birds and modeled relative abundance, aviation risk, and conservation value of birds associated with biomass crops. Avian relative abundance was greater in switchgrass monoculture plots during the winter months, whereas Native Warm-Season Grass (NWSG) mixed species plantings were favored by species during the breeding season. Conversely, treatment differences in aviation risk and conservation value were not biologically significant. Only 2.6% of observations included avian species of high hazard to aircraft, providing support for semi-natural grasslands as a feasible landcover option at civil airports. Additionally, varied harvest frequencies across a mosaic of switchgrass monocultures and NWSG plots allows for biomass production with multiple vegetation structure options for grassland birds to increase seasonal avian biodiversity and habitat use.

DAMPs Synergize with Cytokines or Fibronectin Fragment on Inducing Chondrolysis but Lose Effect When Acting Alone.

OBJECTIVE AND DESIGN: To investigate whether endogenous damage-associated molecular patterns (DAMPs) or alarmins originated from mitochondria or nucleus stimulates inflammatory response in articular chondrocytes to cause chondrolysis which leads to cartilage degradation featured in posttraumatic osteoarthritis (PTOA). MATERIALS: Primary cultures of bovine or human chondrocytes isolated from cartilage of weight-bearing joints. TREATMENT: Chondrocytes were subjected to mitochondrial DAMPs (MTDs) or HMGB1, a nuclear DAMP (NuD), with or without the presence of an N-terminal 29 kDa fibronectin fragment (Fn-f) or proinflammatory cytokines (IL-1ß and TNF-α). Injured cartilage-conditioned culturing medium containing a mixture of DAMPs was employed as a control. After 24 hrs, the protein expression of cartilage degrading metalloproteinases and iNOS in culture medium or cell lysates was examined with Western blotting, respectively. RESULTS: HMGB1 was synergized with IL-1ß in upregulating expression of MMP-3, MMP-13, ADAMTS-5, ADAM-8, and iNOS. Moreover, a moderate synergistic effect was detected between HMGB1 and Fn-f or between MTDs and TNF-α on MMP-3 expression. However, when acting alone, MTDs or HMGB1 did not upregulate cartilage degrading enzymes or iNOS. CONCLUSION: MTDs or HMGB1 could only stimulate inflammatory response in chondrocytes with the presence of cytokines or Fn-f.

The use of autologous adult, allogenic juvenile, and combined juvenile-adult cartilage fragments for the repair of chondral defects.

PURPOSE: The goal of the study was to evaluate the repair of chondral lesions treated with combined autologous adult/allogenic juvenile cartilage fragments, compared with isolated adult and isolated juvenile cartilage fragments. METHODS: Fifty-eight adult (>16 week old) and five juvenile (<6 week old) New Zealand White female rabbits were used. A large osteochondral defect was created in the center of the femoral trochlea of adult rabbits. The rabbits were divided in four groups: Group 1 = untreated defects (controls); Group 2 = adult cartilage fragments; Group 3 = juvenile cartilage fragments; and Group 4 = adult + juvenile cartilage fragments. Killings were performed at 3 and 6 months. The defects were evaluated with ICRS macroscopic score, modified O'Driscoll score, and Collagen type II immunostaining. RESULTS: At 3 months, Group 4 performed better than Group 1, in terms of modified O'Driscoll score (p = 0.001) and Collagen type II immunostaining (p = 0.015). At 6 months, Group 4 showed higher modified O'Driscoll score (p = 0.003) and Collagen type II immunostaining score (p < 0.001) than Group 1. Histologically, also Group 3 performed better than Group 1 (p = 0.03), and Group 4 performed better than Group 2 (p = 0.004). CONCLUSIONS: Mixing adult and juvenile cartilage fragments improved cartilage repair in a rabbit model. In the clinical setting, a new "one-stage" procedure combining the two cartilage sources can be hypothesized, with the advantages of improved chondral repair and large defect coverage, because of the use of an off-the-shelf juvenile allograft. Further studies on larger animals and clinical trials are required to confirm these results.

The Willingness of Military Members to Seek Help: The Role of Social Involvement and Social Responsibility.

Anchored in the social organization theory of action and change, we use data from a large sample of active-duty Air Force members to examine the direct and indirect influence of social involvement and social responsibility on willingness to seek help in times of need via trust in formal systems and informal supports. Group comparisons are conducted between junior male, junior female, senior male, and senior female service members. The key mediational path in the model for all groups is the connection between social involvement and willingness to seek help via trust in formal systems. These results can inform both unit- and community-level interventions intended to increase the likelihood that active-duty AF members will seek help in times of need.

Behavioral traits and airport type affect mammal incidents with U.S. civil aircraft.

Wildlife incidents with aircraft cost the United States (U.S.) civil aviation industry >US$1.4 billion in estimated damages and loss of revenue from 1990 to 2009. Although terrestrial mammals represented only 2.3 % of wildlife incidents, damage to aircraft occurred in 59 % of mammal incidents. We examined mammal incidents (excluding bats) at all airports in the Federal Aviation Administration (FAA) National Wildlife Strike Database from 1990 to 2010 to characterize these incidents by airport type: Part-139 certified (certificated) and general aviation (GA). We also calculated relative hazard scores for species most frequently involved in incidents. We found certificated airports had more than twice as many incidents as GA airports. Incidents were most frequent in October (n = 215 of 1,764 total) at certificated airports and November (n = 111 of 741 total) at GA airports. Most (63.2 %) incidents at all airports (n = 1,523) occurred at night but the greatest incident rate occurred at dusk (177.3 incidents/hr). More incidents with damage (n = 1,594) occurred at GA airports (38.6 %) than certificated airports (19.0 %). Artiodactyla (even-toed ungulates) incidents incurred greatest (92.4 %) damage costs (n = 326; US$51.8 million) overall and mule deer (Odocoileus hemionus) was the most hazardous species. Overall, relative hazard score increased with increasing log body mass. Frequency of incidents was influenced by species relative seasonal abundance and behavior. We recommend airport wildlife officials evaluate the risks mammal species pose to aircraft based on the hazard information we provide and consider prioritizing management strategies that emphasize reducing their occurrence on airport property.

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.

Chondrogenic progenitor cells respond to cartilage injury.

OBJECTIVE: Hypocellularity resulting from chondrocyte death in the aftermath of mechanical injury is thought to contribute to posttraumatic osteoarthritis. However, we observed that nonviable areas in cartilage injured by blunt impact were repopulated within 7-14 days by cells that appeared to migrate from the surrounding matrix. The aim of this study was to assess our hypothesis that the migrating cell population included chondrogenic progenitor cells that were drawn to injured cartilage by alarmins. METHODS: Osteochondral explants obtained from mature cattle were injured by blunt impact or scratching, resulting in localized chondrocyte death. Injured sites were serially imaged by confocal microscopy, and migrating cells were evaluated for chondrogenic progenitor characteristics. Chemotaxis assays were used to measure the responses to chemokines, injury-conditioned medium, dead cell debris, and high mobility group box chromosomal protein 1 (HMGB-1). RESULTS: Migrating cells were highly clonogenic and multipotent and expressed markers associated with chondrogenic progenitor cells. Compared with chondrocytes, these cells overexpressed genes involved in proliferation and migration and underexpressed cartilage matrix genes. They were more active than chondrocytes in chemotaxis assays and responded to cell lysates, conditioned medium, and HMGB-1. Glycyrrhizin, a chelator of HMGB-1 and a blocking antibody to receptor for advanced glycation end products (RAGE), inhibited responses to cell debris and conditioned medium and reduced the numbers of migrating cells on injured explants. CONCLUSION: Injuries that caused chondrocyte death stimulated the emergence and homing of chondrogenic progenitor cells, in part via HMGB-1 release and RAGE-mediated chemotaxis. Their repopulation of the matrix could promote the repair of chondral damage that might otherwise contribute to progressive cartilage loss.

Evaluation of cell viability and functionality in vessel-like bioprintable cell-laden tubular channels.

Organ printing is a novel concept recently introduced in developing artificial three-dimensional organs to bridge the gap between transplantation needs and organ shortage. One of the major challenges is inclusion of blood-vessellike channels between layers to support cell viability, postprinting functionality in terms of nutrient transport, and waste removal. In this research, we developed a novel and effective method to print tubular channels encapsulating cells in alginate to mimic the natural vascular system. An experimental investigation into the influence on cartilage progenitor cell (CPCs) survival, and the function of printing parameters during and after the printing process were presented. CPC functionality was evaluated by checking tissue-specific genetic marker expression and extracellular matrix production. Our results demonstrated the capability of direct fabrication of cell-laden tubular channels by our newly designed coaxial nozzle assembly and revealed that the bioprinting process could induce quantifiable cell death due to changes in dispensing pressure, coaxial nozzle geometry, and biomaterial concentration. Cells were able to recover during incubation, as well as to undergo differentiation with high-level cartilage-associated gene expression. These findings may not only help optimize our system but also can be applied to biomanufacturing of 3D functional cellular tissue engineering constructs for various organ systems.

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.