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Caenagnathidae is a clade of derived, Late Cretaceous oviraptorosaurian theropods from Asia and North America. Because their remains are rare and often fragmentary, caenagnathid diversity is poorly understood. Anzu wyliei is the only caenagnathid species currently described from the late Maastrichtian Hell Creek Formation of the USA and is also among the largest and most completely preserved North American caenagnathids. Smaller, less complete caenagnathid material has long been known from the Hell Creek Formation, but it is unclear whether these are juvenile representatives of Anzu or if they represent distinct, unnamed taxa. Here, we describe a relatively small caenagnathid hindlimb from the Hell Creek Formation, and conduct osteohistological analysis to assess its maturity. Histological data and morphological differences from Anzu wyliei and other caenagnathids allow us to conclude that this specimen represents a new species of caenagnathid from the Hell Creek Formation, with a smaller adult body size than Anzu. This new taxon is also distinct from other small caenagnathid material previously described from the area, potentially indicating the coexistence of three distinct caenagnathid species in the Hell Creek Formation. These results show that caenagnathid diversity in the Hell Creek ecosystem has been underestimated.

Deep learning shows promise for automating detection and classification of wildlife from digital aerial imagery to support cost-efficient remote sensing solutions for wildlife population monitoring. To support in-flight orthorectification and machine learning processing to detect and classify wildlife from imagery in near real-time, we evaluated deep learning methods that address hardware limitations and the need for processing efficiencies to support the envisioned in-flight workflow. We developed an annotated dataset for a suite of marine birds from high-resolution digital aerial imagery collected over open water environments to train the models. The proposed 3-stage workflow for automated, in-flight data processing includes: 1) image filtering based on the probability of any bird occurrence, 2) bird instance detection, and 3) bird instance classification. For image filtering, we compared the performance of a binary classifier with Mask Region-based Convolutional Neural Network (Mask R-CNN) as a means of sub-setting large volumes of imagery based on the probability of at least one bird occurrence in an image. On both the validation and test datasets, the binary classifier achieved higher performance than Mask R-CNN for predicting bird occurrence at the image-level. We recommend the binary classifier over Mask R-CNN for workflow first-stage filtering. For bird instance detection, we leveraged Mask R-CNN as our detection framework and proposed an iterative refinement method to bootstrap our predicted detections from loose ground-truth annotations. We also discuss future work to address the taxonomic classification phase of the envisioned workflow.

Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing near-membrane cellular structures and processes, including imaging of local Ca2+ transients with single-channel resolution. TIRF is most commonly implemented in epi-fluorescence mode, whereby laser excitation light is introduced at a spot near the periphery of the back focal plane of a high numerical aperture objective lens. However, this approach results in an irregular illumination field, owing to interference fringes and scattering and shadowing by cellular structures. We describe a simple system to circumvent these limitations, utilizing a pair of galvanometer-driven mirrors to rapidly spin the laser spot in a circle at the back focal plane of the objective lens, so that irregularities average out during each camera exposure to produce an effectively uniform field. Computer control of the mirrors enables precise scanning at 200 Hz (5ms camera exposure times) or faster, and the scan radius can be altered on a frame-by-frame basis to achieve near-simultaneous imaging in TIRF, widefield and 'skimming plane' imaging modes. We demonstrate the utility of the system for dynamic recording of local inositol trisphosphate-mediated Ca2+ signals and for imaging the redistribution of STIM and Orai proteins during store-operated Ca2+ entry. We further anticipate that it will be readily applicable for numerous other near-membrane studies, especially those involving fast dynamic processes.

Membrane transporters carry key metabolites across the cell membrane and, from a resource standpoint, are hypothesized to be produced when necessary. The expression of membrane transporters in metabolic pathways is often upregulated by the transporter substrate. In E. coli, such systems include for example the lacY, araFGH, and xylFGH genes, which encode for lactose, arabinose, and xylose transporters, respectively. As a case study of a minimal system, we build a generalizable physical model of the xapABR genetic circuit, which features a regulatory feedback loop via membrane transport (positive feedback) and enzymatic degradation (negative feedback) of an inducer. Dynamical systems analysis and stochastic simulations show that the membrane transport makes the model system bistable in certain parameter regimes. Thus, it serves as a genetic "on-off" switch, enabling the cell to only produce a set of metabolic enzymes when the corresponding metabolite is present in large amounts. We find that the negative feedback from the degradation enzyme does not significantly disturb the positive feedback from the membrane transporter. We investigate hysteresis in the switching and discuss the role of cooperativity and multiple binding sites in the model circuit. Fundamentally, this work explores how a stable genetic switch for a set of enzymes is obtained from transcriptional auto-activation of a membrane transporter through its substrate.

Reptiles show varying degrees of facultative parthenogenesis. Here we use genetic methods to determine that an isolated, captive female Asian water dragon produced at least nine offspring via parthenogenesis. We identified microsatellites for the species from shotgun genomic sequences, selected and optimized primer sets, and tested all of the offspring for a set of seven microsatellites that were heterozygous in the mother. We verified that the seven loci showed high levels of polymorphism in four wild Asian water dragons from Vietnam. In all cases, the offspring (unhatched, but developed eggs, or hatched young) had only a single allele at each locus, and contained only alleles present in the mother's genotype (i.e., were homozygous or hemizygous). The probability that our findings resulted from the female mating with one or more males is extremely small, indicating that the offspring were derived from a single female gamete (either alone or via duplication and/or fusion) and implicating parthenogenesis. This is the first documented case of parthenogenesis in the Squamate family Agamidae.

The mitochondrial permeability transition pore (mPTP) is a key regulator of mitochondrial function that has been implicated in the pathogenesis of metabolic disease. Cyclophilin D (CypD) is a critical regulator that directly binds to mPTP constituents to facilitate the pore opening. We previously found that global CypD knockout mice (KO) are protected from diet-induced glucose intolerance; however, the tissue-specific function of CypD and mPTP, particularly in the control of glucose homeostasis, has not been ascertained. To this end, we performed calcium retention capacity (CRC) assay to compare the importance of CypD in the liver versus skeletal muscle. We found that liver mitochondria are more dependent on CypD for mPTP opening than skeletal muscle mitochondria. To ascertain the tissue-specific role of CypD in metabolic homeostasis, we generated liver-specific and muscle-specific CypD knockout mice (LKO and MKO, respectively) and fed them either a chow diet or 45% high-fat diet (HFD) for 14 weeks. MKO mice displayed similar body weight gain and glucose intolerance compared with wild type littermates (WT), whereas LKO mice developed greater visceral obesity, glucose intolerance and pyruvate intolerance compared with WT mice. These findings demonstrate that loss of muscle CypD is not sufficient to alter whole body glucose metabolism, while the loss of liver CypD exacerbates obesity and whole-body metabolic dysfunction in mice fed HFD.

Nonsteroidal anti-inflammatory drugs have been shown to reduce the incidence of gastrointestinal cancers, but the propensity of these drugs to cause ulcers and bleeding limits their use. H2S has been shown to be a powerful cytoprotective and anti-inflammatory substance in the digestive system. This study explored the possibility that a H2S-releasing nonsteroidal anti-inflammatory drug (ATB-346) would be effective in a murine model of hereditary intestinal cancer (APCMin+ mouse) and investigated potential mechanisms of action via transcriptomics analysis. Daily treatment with ATB-346 was significantly more effective at preventing intestinal polyp formation than naproxen. Significant beneficial effects were seen with a treatment period of only 3-7 days, and reversal of existing polyps was observed in the colon. ATB-346, but not naproxen, significantly decreased expression of intestinal cancer-associated signaling molecules (cMyc, β-catenin). Transcriptomic analysis identified 20 genes that were up-regulated in APCMin+ mice, 18 of which were reduced to wild-type levels by one week of treatment with ATB-346. ATB-346 is a novel, gastrointestinal-sparing anti-inflammatory drug that potently and rapidly prevents and reverses the development of pre-cancerous lesions in a mouse model of hereditary intestinal tumorigenesis. These effects may be related to the combined effects of suppression of cyclooxygenase and release of H2S, and correction of most of the APCMin+-associated alterations in the transcriptome. ATB-346 may represent a promising agent for chemoprevention of tumorigenesis in the GI tract and elsewhere.

Hepatitis C is a chronic liver disease that contributes to progressive metabolic dysfunction. Infection of hepatocytes by hepatitis C virus (HCV) results in reprogramming of hepatic and serum lipids. However, the specific contribution of these distinct pools of lipids to HCV infection remains ill defined. In this study, we investigated the role of hepatic lipogenesis in HCV infection by targeting the rate-limiting step in this pathway, which is catalyzed by the acetyl-CoA carboxylase (ACC) enzymes. Using two structurally unrelated ACC inhibitors, we determined that blockade of lipogenesis resulted in reduced viral replication, assembly, and release. Supplementing exogenous lipids to cells treated with ACC inhibitors rescued HCV assembly with no effect on viral replication and release. Intriguingly, loss of viral RNA was not recapitulated at the protein level and addition of 2-bromopalmitate, a competitive inhibitor of protein palmitoylation, mirrored the effects of ACC inhibitors on reduced viral RNA without a concurrent loss in protein expression. These correlative results suggest that newly synthesized lipids may have a role in protein palmitoylation during HCV infection.

Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing near-membrane cellular structures and processes, including imaging of local Ca2+ transients with single-channel resolution. TIRF is most commonly implemented in epi-fluorescence mode, whereby laser excitation light is introduced at a spot near the periphery of the back focal plane of a high numerical aperture objective lens. However, this approach results in an irregular illumination field, owing to interference fringes and scattering and shadowing by cellular structures. We describe a simple system to circumvent these limitations, utilizing a pair of galvanometer-driven mirrors to rapidly spin the laser spot in a circle at the back focal plane of the objective lens, so that irregularities average out during each camera exposure to produce an effectively uniform field. Computer control of the mirrors enables precise scanning at 200 Hz (5ms camera exposure times) or faster, and the scan radius can be altered on a frame-by-frame basis to achieve near-simultaneous imaging in TIRF, widefield and 'skimming plane' imaging modes. We demonstrate the utility of the system for dynamic recording of local inositol trisphosphate-mediated Ca2+ signals and for imaging the redistribution of STIM and Orai proteins during store-operated Ca2+ entry. We further anticipate that it will be readily applicable for numerous other near-membrane studies, especially those involving fast dynamic processes.

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.

Hydrogen sulfide (H₂S) is produced throughout the gastrointestinal tract, and it contributes to maintenance of mucosal integrity, resolution of inflammation, and repair of damaged tissue. H₂S synthesis is elevated in inflamed and damaged colonic tissue, but the enzymatic sources of that synthesis are not completely understood. In the present study, the contributions of three enzymatic pathways to colonic H₂S synthesis were determined, with tissues taken from healthy rats and rats with colitis. The ability of the colonic tissue to inactivate H₂S was also determined. Colonic tissue from rats with hapten-induced colitis produced significantly more H₂S than tissue from healthy controls. The largest source of the H₂S synthesis was the pathway involving cysteine amino transferase and 3-mercaptopyruvate sulfurtransferase (an α-ketoglutarate-dependent pathway). Elevated H₂S synthesis occurred specifically at sites of mucosal ulceration, and was not related to the extent of granulocyte infiltration into the tissue. Inactivation of H₂S by colonic tissue occurred rapidly, and was significantly reduced at sites of mucosal ulceration. This correlated with a marked decrease in the expression of sulfide quinone reductase in these regions. Together, the increased production and decreased inactivation of H₂S at sites of mucosal ulceration would result in higher H₂S levels at these sites, which promotes of resolution of inflammation and repair of damaged tissue.

Reactive oxygen species (ROS) are a common byproduct of mitochondrial energy metabolism, and can also be induced by exogenous sources, including UV light, radiation, and environmental toxins. ROS generation is essential for maintaining homeostasis by triggering cellular signaling pathways and host defense mechanisms. However, an imbalance of ROS induces oxidative stress and cellular death and is associated with human disease, including age-related locomotor impairment. To identify genes affecting sensitivity and resistance to ROS-induced locomotor decline, we assessed locomotion of aged flies of the sequenced, wild-derived lines from the Drosophila melanogaster Genetics Reference Panel on standard medium and following chronic exposure to medium supplemented with 3 mM menadione sodium bisulfite (MSB). We found substantial genetic variation in sensitivity to oxidative stress with respect to locomotor phenotypes. We performed genome-wide association analyses to identify candidate genes associated with variation in sensitivity to ROS-induced decline in locomotor performance, and confirmed the effects for 13 of 16 mutations tested in these candidate genes. Candidate genes associated with variation in sensitivity to MSB-induced oxidative stress form networks of genes involved in neural development, immunity, and signal transduction. Many of these genes have human orthologs, highlighting the utility of genome-wide association in Drosophila for studying complex human disease.

Nonsteroidal anti-inflammatory drugs have been shown to reduce the incidence of gastrointestinal cancers, but the propensity of these drugs to cause ulcers and bleeding limits their use. H2S has been shown to be a powerful cytoprotective and anti-inflammatory substance in the digestive system. This study explored the possibility that a H2S-releasing nonsteroidal anti-inflammatory drug (ATB-346) would be effective in a murine model of hereditary intestinal cancer (APCMin+ mouse) and investigated potential mechanisms of action via transcriptomics analysis. Daily treatment with ATB-346 was significantly more effective at preventing intestinal polyp formation than naproxen. Significant beneficial effects were seen with a treatment period of only 3–7 days, and reversal of existing polyps was observed in the colon. ATB-346, but not naproxen, significantly decreased expression of intestinal cancer-associated signaling molecules (cMyc, β-catenin). Transcriptomic analysis identified 20 genes that were up-regulated in APCMin+ mice, 18 of which were reduced to wild-type levels by one week of treatment with ATB-346. ATB-346 is a novel, gastrointestinal-sparing anti-inflammatory drug that potently and rapidly prevents and reverses the development of pre-cancerous lesions in a mouse model of hereditary intestinal tumorigenesis. These effects may be related to the combined effects of suppression of cyclooxygenase and release of H2S, and correction of most of the APCMin+-associated alterations in the transcriptome. ATB-346 may represent a promising agent for chemoprevention of tumorigenesis in the GI tract and elsewhere. [ABSTRACT FROM AUTHOR]

Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses. [ABSTRACT FROM AUTHOR]

Current USEPA ecological risk assessments for pesticide registration include a determination of potential risks to bees. Toxicity data are submitted to support these assessments and the USEPA maintains a large database containing acute and chronic toxicity data on adult and larval honey bees (Apis mellifera), which USEPA considers a surrogate for Apis and non-Apis bees. We compared these toxicity data to explore possible trends. This analysis indicated a significant correlation between acute contact and oral median lethal dose (LD50) values for adult honey bees (ρ = 0.74, p <0.0001). Using default EPA modeling assumptions, where exposure for an individual bee is roughly 12x lower through contact than through ingestion, the analysis indicates that the oral LD50 is similarly if not more protective of the contact LD50 for the majority of pesticides and modes of action evaluated. The analysis also provided evidence that compounds with a lower acute toxicity for adults through contact and oral exposure pathways may still be acutely toxic for larvae. The acute toxicity of herbicides and fungicides was higher for larvae relative to oral and contact toxicity for adult honey bees for the same compounds and the no observed adverse effect level (NOAEL) from chronic toxicity studies were lower for larvae relative to adults, indicating increased sensitivity of larvae. When comparing 8-day LD50 values between single dose larval acute studies to those derived from repeat dose 22-day larval chronic toxicity studies, the LD50 values derived from chronic studies were significantly lower than those from acute toxicity tests (Z = -37, p = 0.03)., Competing Interests: The authors have declared that no competing interests exist.

The recent rise in obesity is widely attributed to changes in the dietary environment (e.g., increased availability of energy-dense foods and larger portion sizes). However, a critical feature of our "obesogenic environment" may have been overlooked - the dramatic increase in "dietary variability" (the tendency for specific mass-produced foods to be available in numerous varieties that differ in energy content). In this study we tested the hypothesis that dietary variability compromises the control of food intake in humans. Specifically, we examined the effects of dietary variability in pepperoni pizza on two key outcome variables; i) compensation for calories in pepperoni pizza and ii) expectations about the satiating properties of pepperoni pizza (expected satiation). We reasoned that dietary variability might generate uncertainty about the postingestive effects of a food. An internet-based questionnaire was completed by 199 adults. This revealed substantial variation in exposure to different varieties of pepperoni pizza. In a follow-up study (n= 66; 65% female), high pizza variability was associated with i) poorer compensation for calories in pepperoni pizza and ii) lower expected satiation for pepperoni pizza. Furthermore, the effect of uncertainty on caloric compensation was moderated by individual differences in decision making (loss aversion). For the first time, these findings highlight a process by which dietary variability may compromise food-intake control in humans. This is important because it exposes a new feature of Western diets (processed foods in particular) that might contribute to overeating and obesity.