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Anatoxin-a is a globally occurring, yet understudied, chiral cyanobacterial toxin that threatens public health and the environment. It has led to numerous dog. livestock and bird poisonings and although it has been studied in rodent models, comparatively little research has occurred in aquatic species. To advance a comparative toxicology understanding of this toxin in alternative vertebrate models, developing zebrafish and fathead minnow were exposed to environmentally relevant and elevated levels (13–4400 μg/L) of (+) anatoxin-a to examine potential mortality and sublethal effects, including photolocomotor behavior and gene expression responses. We observed significantly higher mortality (p 0.05) transcriptional changes were observed in the fathead minnow model. These differential responses between commonly employed species employed as alternative vertebrate models in toxicology research and chemicals risk assessments highlight the need for more comparative studies to understand sensitivities and variations in organismal response. Furthermore, we identified higher mortality, refractory behavioral effects, and gene expression in (+) anatoxin-a exposed fish when compared to previously reported (±) anatoxin-a (racemic 50:50 enantiomer mixture) studies, which is frequently used as a surrogate chemical for experimental work. Our findings identify the importance of understanding species and enantiomer specific effects of natural toxins.

Abstract Protein complexes are essential for proteins' folding and biological function. Currently, native analysis of large multimeric protein complexes remains challenging. Structural biology techniques are time‐consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis‐mass spectrometry (CE–MS) method is reported to characterize, under near‐physiological conditions, the conformational rearrangements of ∽1 MDa GroEL upon complexation with binding partners involved in a protein folding cycle. The developed CE–MS method is fast (30 min per run), highly sensitive (low‐amol level), and requires ∽10 000‐fold fewer samples compared to biochemical/biophysical techniques. The method successfully separates GroEL14 (∽800 kDa), GroEL7 (∽400 kDa), GroES7 (∽73 kDa), and NanA4 (∽130 kDa) oligomers. The non‐covalent binding of natural substrate proteins with GroEL14 can be detected and quantified. The technique allows monitoring of GroEL14 conformational changes upon complexation with (ATPγS)4–14 and GroES7 (∽876 kDa). Native CE‐pseudo‐MS3 analyses of wild‐type (WT) GroEL and two GroEL mutants result in up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE–MS performance for multimeric complexes' characterization versus direct infusion ESI–MS. This study shows the CE–MS potential to provide information on binding stoichiometry and kinetics for various protein complexes.

Anatoxin-a and its analogues are potent neurotoxins produced by several genera of cyanobacteria. Due in part to its high toxicity and potential presence in drinking water, these toxins pose threats to public health, companion animals and the environment. It primarily exerts toxicity as a cholinergic agonist, with high affinity at neuromuscular junctions, but molecular mechanisms by which it elicits toxicological responses are not fully understood. To advance understanding of this cyanobacteria, proteomic characterization (DIA shotgun proteomics) of two common fish models (zebrafish and fathead minnow) was performed following (±) anatoxin-a exposure. Specifically, proteome changes were identified and quantified in larval fish exposed for 96 h (0.01–3 mg/L (±) anatoxin-a and caffeine (a methodological positive control) with environmentally relevant treatment levels examined based on environmental exposure distributions of surface water data. Proteomic concentration - response relationships revealed 48 and 29 proteins with concentration - response relationships curves for zebrafish and fathead minnow, respectively. In contrast, the highest number of differentially expressed proteins (DEPs) varied between zebrafish (n = 145) and fathead minnow (n = 300), with only fatheads displaying DEPs at all treatment levels. For both species, genes associated with reproduction were significantly downregulated, with pathways analysis that broadly clustered genes into groups associated with DNA repair mechanisms. Importantly, significant differences in proteome response between the species was also observed, consistent with prior observations of differences in response using both behavioral assays and gene expression, adding further support to model specific differences in organismal sensitivity and/or response. When DEPs were read across from humans to zebrafish, disease ontology enrichment identified diseases associated with cognition and muscle weakness consistent with the prior literature. Our observations highlight limited knowledge of how (±) anatoxin-a, a commonly used synthetic racemate surrogate, elicits responses at a molecular level and advances its toxicological understanding.

Robyn Kendall,1 Alan A Martin,2 Dhvani Shah,3 Soham Shukla,4 Chris Compton,5 Afisi S Ismaila4,6 1ICON Health Economics, ICON plc, Vancouver, BC, Canada; 2Value Evidence and Outcomes, GSK, London, UK; 3ICON Health Economics, ICON plc, New York, NY, USA; 4Value Evidence and Outcomes, GSK, Collegeville, PA, USA; 5Global Respiratory Franchise, GSK, London, UK; 6Department of Health Research Methods, Evidence, and Impact, McMaster University, Hamilton, ON, CanadaCorrespondence: Afisi S Ismaila, Value Evidence and Outcomes, GSK, 1250 South Collegeville Road, Collegeville, PA, 19426-0989, USA, Tel +1 919 315 8229, Email afisi.s.ismaila@gsk.comPurpose: For patients with chronic obstructive pulmonary disease (COPD) who remain symptomatic despite maintenance treatment, clinical management guidelines recommend a stepwise escalation from monotherapy to dual therapy, and from dual therapy to triple therapy. However, in clinical practice, patients are often escalated directly from monotherapy to triple therapy based on disease severity. This study evaluated the cost-effectiveness of once-daily, single-inhaler fluticasone furoate, umeclidinium, and vilanterol (FF/UMEC/VI) triple therapy compared with long-acting muscarinic antagonist monotherapy with once-daily tiotropium (TIO) in patients with symptomatic moderate-to-very severe COPD, from a UK National Health Service perspective.Patients and Methods: The validated GALAXY-COPD disease progression model was populated with patient baseline characteristics and treatment effect data from the 12-week GSK Study 207626 comparing FF/UMEC/VI with TIO in patients with moderate-to-very severe COPD. UK unit costs and drug costs (British Pound, 2021) were applied to healthcare resource utilization and treatments. The base case analysis was conducted over a lifetime horizon, and costs and health outcomes (except for life years [LYs]) were discounted at 3.5% per year. Model outputs included exacerbation rates, healthcare costs, LYs, quality-adjusted LYs (QALYs), and incremental cost-effectiveness ratios.Results: Overall, treatment with FF/UMEC/VI resulted in increased clinical benefit (reduction in total exacerbations and increased overall survival and QALYs), coupled with cost savings (derived from lower maintenance and exacerbation healthcare costs) compared with TIO monotherapy. In the base case analysis, FF/UMEC/VI provided an additional 0.393 LYs (95% range: 0.176, 0.655) and 0.443 QALYs (0.246, 0.648), at a cost saving of £ 880 (£ 54, £ 1608) versus TIO. FF/UMEC/VI remained the cost-effective (dominant) treatment option across sensitivity and scenario analyses.Conclusion: FF/UMEC/VI offers greater clinical benefits and is a cost-effective treatment option compared with TIO for the treatment of adult patients with COPD with persistent symptoms and/or who are at risk of exacerbation in the UK.Keywords: chronic obstructive pulmonary disease, health technology assessment, economic evaluation, real-world, single-inhaler triple therapy

Neuroblasts in Drosophila divide asymmetrically, sequentially expressing a series of intrinsic factors to generate a diversity of neuron types. These intrinsic factors known as temporal factors dictate timing of neuroblast transitions in response to steroid hormone signaling and specify early versus late temporal fates in neuroblast neuron progeny. After completing their temporal programs, neuroblasts differentiate or die, finalizing both neuron number and type within each neuroblast lineage. From a screen aimed at identifying genes required to terminate neuroblast divisions, we identified Notch and Notch pathway components. When Notch is knocked down, neuroblasts maintain early temporal factor expression longer, delay late temporal factor expression, and continue dividing into adulthood. We find that Delta, expressed in cortex glia, neuroblasts, and after division, their GMC progeny, regulates neuroblast Notch activity. We also find that Delta in neuroblasts is expressed high early, low late, and is controlled by the intrinsic temporal program: early factor Imp promotes Delta, late factors Syp/E93 reduce Delta. Thus, in addition to systemic steroid hormone cues, forward lineage progression is controlled by local cell-cell signaling between neuroblasts and their cortex glia/GMC neighbors: Delta transactivates Notch in neuroblasts bringing the early temporal program and early temporal factor expression to a close.

Abstract Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world‐wide survey to ISEV members to assess methods considered ‘best practices’ for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

Human pressures such as hunting and habitat destruction can generate a deep fear in animals and this fear can influence their diel activity patterns and use of space. However, whether these behavioural responses to human pressure are consistent among key functional groups has been poorly studied. For example, while mammal species tend to become more nocturnal in areas with high human pressure, it is unclear if co-occurring birds display similar or opposite patterns. Here we used information from camera trapping (367 camera stations and 16,939 camera/days) along a gradient of human pressure in the Colombian Llanos to assess diel activity changes in understory birds and mammals. We found that diel activity significantly changed with higher human pressure for 45% of the birds (five species) and 36% of the mammals (five species) assessed, with four of five birds becoming more diurnal and all five mammals becoming more nocturnal. The average increase in nocturnality for the mammals was 11.3% while the average increase in diurnality for the birds was 7%. There was high variation in body size and dietary guild within impacted species, and only some were directly persecuted or hunted, suggesting that there are different pathways through which human pressure can affect vertebrates’ activity patterns. The contrasting behavioural responses to humans among vertebrate functional groups has significant repercussions for the fields of community ecology, including intraguild predation and competition, and should be a significant ecosystem-level conservation consideration.

Human-induced forest loss has had devastating impacts on biodiversity. Mammal populations in the tropics have been hit particularly hard by the resulting habitat loss, fragmentation and degradation, as well as by overhunting which often goes hand-in-hand. While declines in these populations are generally well documented, few studies offer a pathway for their recovery. Here, we test the association between changes in forest habitat and occupancy trends of Sumatran megafauna (elephant and tiger) and key tiger prey species (wild boar and sambar) in the Leuser Ecosystem: a large forest landscape on the Indonesian island of Sumatra. For elephant and tiger, we develop additional occupancy models to predict their respective spatial distribution under different scenarios of forest loss and gain (through restoration and increased connectivity) to provide a blueprint for avoiding future species loss and assisting with their population recovery. From 2000 to 2019, 254,722 ha (6.7%) of natural forest was converted, primarily to plantations and shrubs. The species-specific responses over the study period revealed that the occurrence of elephant declined along the west, with a range shift to the northeast of Leuser, whereas wild boar underwent a dramatic widespread decline and although sambar experienced losses around the forest edge, it remained widespread in the interior forest, while tiger occupancy remained stable. Modelling habitat loss and fragmentation led to an unsurprising demise of Sumatran megafauna, whereas strategic investments that reconnected several forest patches provided disproportionately large benefits for their recovery through the recolonization of former parts of their range. Indonesia has achieved six consecutive years of declining forest loss rates, and our study’s findings can build off this conservation success by supporting improved provincial spatial planning and field-based restoration efforts that avoid declines of threatened megafauna species and act as a catalyst for rewilding a landscape of global importance.

David MG Halpin,1 Robyn Kendall,2 Soham Shukla,3 Alan Martin,4 Dhvani Shah,5 Dawn Midwinter,6 Kai M Beeh,7 Janwillem WH Kocks,8– 11 Paul W Jones,12 Chris Compton,12 Nancy A Risebrough,13 Afisi S Ismaila3,14 1University of Exeter Medical School, College of Medicine and Health, University of Exeter, Exeter, UK; 2ICON Health Economics, ICON plc, Vancouver, BC, Canada; 3Value Evidence and Outcomes, GSK, Collegeville, PA, USA; 4Value Evidence and Outcomes, GSK, Uxbridge, UK; 5ICON Health Economics, ICON plc, New York, NY, USA; 6Biostatistics, GSK, Brentford, UK; 7Insaf Respiratory Research Institute, Wiesbaden, Germany; 8General Practitioners Research Institute, Groningen, the Netherlands; 9Observational and Pragmatic Research Institute, Singapore; 10Groningen Research Institute Asthma and COPD (GRIAC), University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; 11Department of Pulmonology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; 12Global Respiratory Franchise, GSK, Brentford, UK; 13ICON Health Economics, ICON plc, Toronto, ON, Canada; 14Department of Health Research Methods, Evidence, and Impact, McMaster University, Hamilton, ON, CanadaCorrespondence: David MG Halpin, University of Exeter Medical School, College of Medicine and Health, University of Exeter, Exeter, EX1 2LU, UK, Tel +44 01392 201178, Email d.halpin@nhs.netPurpose: The 24-week INTREPID trial demonstrated the clinical benefits of once-daily single-inhaler triple therapy (SITT) with fluticasone furoate, umeclidinium, and vilanterol (FF/UMEC/VI) versus non-ELLIPTA multiple-inhaler triple therapy (MITT) in patients with symptomatic chronic obstructive pulmonary disease (COPD). This analysis assessed the cost-effectiveness of FF/UMEC/VI versus non-ELLIPTA MITT for the treatment of symptomatic COPD from a United Kingdom (UK) National Health Service (NHS) perspective.Patients and Methods: The analysis was conducted using the validated GALAXY COPD disease progression model. Baseline characteristics, treatment effect parameters (forced expiratory volume in 1 second and St. George’s Respiratory Questionnaire score [derived from exploratory COPD Assessment Test score mapping]), and discontinuation data from INTREPID were used to populate the model. UK healthcare resource and drug costs (2020 British pounds) were applied, and costs and outcomes were discounted at 3.5%. Analyses were conducted over a lifetime horizon from a UK NHS perspective. Model outputs included exacerbation rates, total costs, life years (LYs), quality-adjusted LYs (QALYs) and incremental cost-effectiveness ratio per QALY. Sensitivity analyses were conducted to assess the robustness of the results by varying parameter values and assumptions.Results: Over a lifetime horizon, FF/UMEC/VI provided an additional 0.174 (95% confidence interval [CI]: 0.024, 0.344) LYs (approximately 2 months), and 0.253 (95% CI: 0.167, 0.346) QALYs (approximately 3 months), at a cost saving of £ 1764 (95% CI: −£ 2600, −£ 678) per patient, compared with non-ELLIPTA MITT. FF/UMEC/VI remained the dominant treatment option, meaning greater benefits at lower costs, across all scenario and sensitivity analyses.Conclusion: Based on this analysis, in a UK setting, FF/UMEC/VI would improve health outcomes and reduce costs compared with non-ELLIPTA MITT for the treatment of patients with symptomatic COPD. SITT may help to reduce the clinical and economic burden of COPD and should be considered by physicians as a preferred treatment option.Keywords: chronic obstructive pulmonary disease, cost-effectiveness, health technology assessment, pragmatic, real-world, triple therapy

Longitudinal hybrid metagenomic analyses of a human stool sample reveal compositional and functional variation in response to six structurally-distinct carbohydrates, providing insight into how gut bacteria utilize various carbohydrate sources.

Zebrafish behavior is increasingly common in biomedical and environmental studies of chemical bioactivity. Multiple experimental arena sizes have been used to measure photolocomotion in zebrafish depending on age, endpoints observed, and instrumentation, among other factors. However, the extent to which methodological parameters may influence naïve behavioral performance and detection of behavioral changes is poorly understood. Here we measured photolocomotion and behavioral profiles of naïve larval zebrafish across arena sizes. We then performed concentration response studies with the model neurostimulant caffeine, again across various arena dimensions. We found total swimming distance of unexposed fish to increase logarithmically with arena size, which as related to circumference, area, and volume. Photomotor response during light/dark transitions also increased with arena size. Following caffeine exposure, total distance travelled was significantly (p

Abstract De novo purine biosynthesis is required for the incorporation of fixed nitrogen in ureide exporting nodules, as formed on soybean [Glycine max (L.) Merr.] roots. However, in many cases, the enzymes involved in this pathway have been deduced strictly from genome annotations with little direct genetic evidence, such as mutant studies, to confirm their biochemical function or importance to nodule development. While efforts to develop large mutant collections of soybean are underway, research on this plant is still hampered by the inability to obtain mutations in any specific gene of interest. Using a forward genetic approach, as well as CRISPR/Cas9 gene editing via Agrobacterium rhizogenes‐mediated hairy root transformation, we identified and characterized the role of GmUOX (Uricase) and GmXDH (Xanthine Dehydrogenase) in nitrogen fixation and nodule development in soybean. The gmuox knockout soybean mutants displayed nitrogen deficiency chlorosis and early nodule senescence, as exemplified by the reduced nitrogenase (acetylene reduction) activity in nodules, the internal greenish‐white internal appearance of nodules, and diminished leghemoglobin production. In addition, gmuox1 nodules showed collapsed infected cells with degraded cytoplasm, aggregated bacteroids with no discernable symbiosome membranes, and increased formation of poly‐β‐hydroxybutyrate granules. Similarly, knockout gmxdh mutant nodules, generated with the CRISPR/Cas9 system, also exhibited early nodule senescence. These genetic studies confirm the critical role of the de novo purine metabolisms pathway not only in the incorporation of fixed nitrogen but also in the successful development of a functional, nitrogen‐fixing nodule. Furthermore, these studies demonstrate the great utility of the CRISPR/Cas9 system for studying root‐associated gene traits when coupled with hairy root transformation.

Soham Shukla,1 Dhvani Shah,2 Alan Martin,3 Nancy A Risebrough,4 Robyn Kendall,5 Claus F Vogelmeier,6 Isabelle Boucot,3 Lee Tombs,7 Leif Bjermer,8 Paul W Jones,3 Edward Kerwin,9 Chris Compton,3 François Maltais,10 David A Lipson,11,12 Afisi S Ismaila1,13 1Value Evidence and Outcomes, GSK, Collegeville, PA, USA; 2ICON, New York, NY, USA; 3Value Evidence and Outcomes, GSK, Brentford, Middlesex, UK; 4Global Health Economics, and Outcomes Research and Epidemiology, ICON, Toronto, ON, Canada; 5Global Health Economics, and Outcomes Research and Epidemiology, ICON, Vancouver, BC, Canada; 6Department of Medicine, Pulmonary and Critical Care Medicine, University Medical Center Giessen and Marburg, Philipps-Universität Marburg, Member of the German Center for Lung Research (DZL), Marburg, Germany; 7Precise Approach Ltd, Contingent Worker on Assignment at GSK, Brentford, Middlesex, UK; 8Respiratory Medicine and Allergology, Lund University, Lund, Sweden; 9Altitude Clinical Consulting and Clinical Research Institute of Southern Oregon, Medford, OR, USA; 10Centre de Pneumologie, Institut universitaire de cardiologie et de pneumologie de Québec, Université Laval, Québec, QC, Canada; 11Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 12Respiratory Clinical Sciences, GSK, Collegeville, PA, USA; 13Department of Health Research Methods, Evidence and Impact, McMaster University, Hamilton, ON, CanadaCorrespondence: Afisi S IsmailaValue Evidence and Outcomes, GlaxoSmithKline 1250 South Collegeville Road, Collegeville, PA, 19426-0989, USATel +1 919 315 8229Email afisi.s.ismaila@gsk.comIntroduction: Dual bronchodilators are recommended as maintenance treatment for patients with symptomatic COPD in the UK; further evidence is needed to evaluate cost-effectiveness versus monotherapy. Cost-effectiveness of umeclidinium/vilanterol versus umeclidinium and salmeterol from a UK healthcare perspective in patients without exacerbations in the previous year was assessed using post hoc EMAX trial data.Methods: The validated GALAXY model was populated with baseline characteristics and treatment effects from the non-exacerbating subgroup of the symptomatic EMAX population (COPD assessment test score ≥ 10) and 2020 UK healthcare and drug costs. Outputs included estimated exacerbation rates, costs, life-years (LYs), and quality-adjusted LYs (QALYs); incremental cost-effectiveness ratio (ICER) was calculated as incremental cost/QALY gained. The base case (probabilistic model) used a 10-year time horizon, assumed no treatment discontinuation, and discounted future costs and QALYs by 3.5% annually. Sensitivity and scenario analyses assessed robustness of model results.Results: Umeclidinium/vilanterol treatment was dominant versus umeclidinium and salmeterol, providing an additional 0.090 LYs (95% range: 0.035, 0.158) and 0.055 QALYs (− 0.059, 0.168) with total cost savings of £ 690 (£ 231, £ 1306) versus umeclidinium, and 0.174 LYs (0.076, 0.286) and 0.204 QALYs (0.079, 0.326) with savings of £ 1336 (£ 1006, £ 2032) versus salmeterol. In scenario and sensitivity analyses, umeclidinium/vilanterol was dominant versus umeclidinium except over a 5-year time horizon (more QALYs at higher total cost; ICER=£ 4/QALY gained) and at the lowest estimate of the St George’s Respiratory Questionnaire treatment effect (fewer QALYs at lower total cost; ICER=£ 12,284/QALY gained); umeclidinium/vilanterol was consistently dominant versus salmeterol. At willingness-to-pay threshold of £ 20,000/QALY, probability that umeclidinium/vilanterol was cost-effective in this non-exacerbating subgroup was 95% versus umeclidinium and 100% versus salmeterol.Conclusion: Based on model predictions from a UK perspective, symptomatic patients with COPD and no exacerbations in the prior year receiving umeclidinium/vilanterol are expected to have better outcomes at lower costs versus umeclidinium and salmeterol.Keywords: COPD treatment, cost-effectiveness, umeclidinium, salmeterol, umeclidinium/vilanterol

Abstract Human activities such as urbanization, infrastructure and agriculture are driving global biodiversity declines. In an attempt to balance economic development goals with biodiversity conservation, governments and industry apply a decision‐making framework known as the mitigation hierarchy, with a goal of achieving no net loss or net gain outcomes for biodiversity. Successful application of the mitigation hierarchy requires biodiversity assessments and spatial planning to inform the design of mitigation policies, identify priority areas for biodiversity conservation and impact avoidance, assess the biodiversity impacts of developments, and identify appropriate mitigation measures including offsetting residual impacts. However, guidance on the necessary data and assessment techniques is often lacking, especially in countries where formal mitigation policies do not exist or are in their infancy. Here, we discuss and demonstrate analyses that can help answer some key questions for formulating effective mitigation policies and applying the mitigation hierarchy. We focus on data and analyses that can inform the avoidance and offset steps in particular, and demonstrate these techniques using a case study in Mozambique. While these analyses will not replace field‐based assessments for projects, they offer rapid, low‐cost approaches to support scoping and development of mitigation policy, planning and decision‐making, especially in relatively data‐poor regions.

The potential of parasites to affect host abundance has been a topic of heated contention within the scientific community for some time, with many maintaining that issues such as habitat loss are more important in regulating wildlife populations than diseases. This is in part due to the difficulty in detecting and quantifying the consequences of disease, such as parasitic infection, within wild systems. An example of this is found in the Northern bobwhite quail (Colinus virginanus), an iconic game bird that is one of the most extensively studied vertebrates on the planet. Yet, despite countless volumes dedicated to the study and management of this bird, bobwhite continue to disappear from fields, forest margins, and grasslands across the United States in what some have referred to as “our greatest wildlife tragedy”. Here, we will discuss the history of disease and wildlife conservation, some of the challenges wildlife disease studies face in the ever-changing world, and how a “weight of evidence” approach has been invaluable to evaluating the impact of parasites on bobwhite in the Rolling Plains of Texas. Through this, we highlight the potential of using “weight of the evidence” to better understand the complex effects of diseases on wildlife and urge a greater consideration of the importance of disease in wildlife conservation.

Isogenic pairs of cell lines are powerful tools but time-consuming to generate. Here the authors conduct genome-wide genetic interactions screens of ‘anchor’ genes with SaCas9 and SpCas9.

Peanut foliar diseases and disorders can be difficult to rapidly diagnose with little experience because some abiotic and biotic symptoms present similar symptoms. Developing algorithms for automated identification of peanut foliar diseases and disorders could potentially provide a quick, affordable, and easy method for diagnosing peanut symptoms. To examine this, images of peanut leaves were captured from various angles, distances, and lighting conditions using various cameras. Color space data from all images was subsequently extracted and subjected to logistic regression. Separate algorithms were developed for each symptom to include healthy, hopperburn, late leaf spot, Provost injury, tomato spotted wilt, paraquat injury, or surfactant injury. The majority of these symptoms are not included within currently available disease identification mobile apps. All of the algorithms developed for peanut foliar diagnostics were ≥ 86% accurate. These diagnostic algorithms have the potential to be a valuable tool for growers if made available via a web-accessible platform, which is the next step of this work.

As demand for commodities increases globally, resource-rich countries are focal areas for industrial expansion. The extractives sector is driving unprecedented growth of infrastructure (e.g., roads, railways and pipelines) into sparsely populated regions, which contain some of the world’s remaining intact forests. Yet, the extent of the threat posed by an increased footprint of extractive industries is not well understood. Here we provide a novel global assessment of the potential threat posed to tropical Intact Forest Landscapes (IFLs), continuous patches of forest that have no remotely sensed signs of human activity or forest fragmentation, from the extractives sector including mining, oil and gas projects, using one of the most extensive global spatial datasets on extractives concessions to date. We found ∼20% of the global tropical IFL estate is designated as extractive’s concessions, presenting an enormous threat to these forests. Oil and gas concessions overlap with 408,000 km2 of tropical IFLs (7.85% of total IFL area), with the biggest overlap in the Democratic Republic of the Congo and Papua New Guinea. Mining concessions overlap with 589,000 km2 (11.33%) of tropical IFL area, mainly in Brazil and Democratic Republic of Congo. As the majority of concessions overlapping IFLs are currently at the exploration stages for all types of extractives, now is the time to plan for reducing the potential threat through strategies such as identifying “no go” areas and applying the mitigation hierarchy. Nations and investors should undertake regional strategic environmental assessments to reduce impacts, including potential indirect and cumulative impacts of multiple projects and other threats in the landscape. We also call for banks that funders of extractives projects to apply global best practice industry environmental safeguards that recognize the value of intact forests and the risk of cumulative impacts to these unique sites.

A key obstacle to wildlife conservation is a scarcity of funding. A recent paper [Courchamp, F., Jaric, I., Albert, C., Meinard, Y., Ripple, W. J., and Chapron, G. (2018). The paradoxical extinction of the most charismatic animals. PLoS Biol. 16:e2003997. doi: 10.1371/journal.pbio.2003997] illustrates how for-profit businesses' widespread use of threatened wildlife imagery could create complacency in the public about their conservation. A wildlife imagery royalty, whereby businesses that use threatened wildlife in their marketing pay a small percentage of their sales to the conservation of those species could be revolutionary for conservation funding. However, businesses are not currently compelled to support the protection of the species espoused in their products. We build upon the arguments presented by recent publications [Good, C., Burnham, D., and Macdonald, D. W. (2017). A cultural conscience for conservation. Animals 7:52. doi: 10.3390/ani7070052; Courchamp, F., Jaric, I., Albert, C., Meinard, Y., Ripple, W. J., and Chapron, G. (2018). The paradoxical extinction of the most charismatic animals. PLoS Biol. 16:e2003997. doi: 10.1371/journal.pbio.2003997] to explore limitations and a number of key pathways that may help bring a wildlife imagery royalty to fruition.

Abstract Background Oxyspirura petrowi (Spirurida: Thelaziidae), a heteroxenous nematode of birds across the USA, may play a role in the decline of the northern bobwhite (Colinus virginianus) in the Rolling Plains Ecoregion of West Texas. Previous molecular studies suggest that crickets, grasshoppers and cockroaches serve as potential intermediate hosts of O. petrowi, although a complete study on the life-cycle of this nematode has not been conducted thus far. Consequently, this study aims to improve our understanding of the O. petrowi life-cycle by experimentally infecting house crickets (Acheta domesticus) with O. petrowi eggs, feeding infected crickets to bobwhite and assessing the life-cycle of this nematode in both the definitive and intermediate hosts. Methods Oxyspirura petrowi eggs were collected from gravid worms recovered from wild bobwhite and fed to house crickets. The development of O. petrowi within crickets was monitored by dissection of crickets at specified intervals. When infective larvae were found inside crickets, parasite-free pen-raised bobwhite were fed four infected crickets each. The maturation of O. petrowi in bobwhite was monitored through fecal floats and bobwhite necropsies at specified intervals. Results In this study, we were able to infect both crickets (n = 45) and bobwhite (n = 25) with O. petrowi at a rate of 96%. We successfully replicated and monitored the complete O. petrowi life-cycle in vivo, recovering embryonated O. petrowi eggs from the feces of bobwhite 51 days after consumption of infected crickets. All life-cycle stages of O. petrowi were confirmed in both the house cricket and the bobwhite using morphological and molecular techniques. Conclusions This study provides a better understanding of the infection mechanism and life-cycle of O. petrowi by tracking the developmental progress within both the intermediate and definitive host. To our knowledge, this study is the first to fully monitor the complete life-cycle of O. petrowi and may allow for better estimates into the potential for future epizootics of O. petrowi in bobwhite. Finally, this study provides a model for experimental infection that may be used in research examining the effects of O. petrowi infection in bobwhite.

The northern bobwhite quail (Colinus virginianus) is a popular gamebird in the Rolling Plains Ecoregion of West Texas. However, there has been a population decline in this area over recent decades. Consistent reports indicate a high prevalence of the eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula), which may be of major influence on the bobwhite population. While research has suggested pathological consequences and genetic relatedness to other pathologically significant parasites, little is known about the influence of climate on these parasites. In this study, we examined whether seasonal temperature and precipitation influences the intensity of these parasites in bobwhite. We also analyzed quantitative PCR results for bobwhite feces and cloacal swabs against temperature and precipitation to identify climatic impacts on parasite reproduction in this region. Multiple linear regression analyses were used for parasite intensity investigation while binary logistic regression analyses were used for parasite reproduction studies. Our analyses suggest that caecal worm intensity, caecal worm reproduction, and eyeworm reproduction are influenced by temperature and precipitation. Temperature data was collected 15, 30, and 60 days prior to the date of collection of individual bobwhite and compared to qPCR results to generate a temperature range that may influence future eyeworm reproduction. This is the first preliminary study investigating climatic influences with predictive statistics on eyeworm and caecal worm infection of northern bobwhite in the Rolling Plains. Keywords: Bobwhite, Caecal, Climate, Eyeworm, qPCR

Genome-wide libraries for CRISPR knockout, interference, and activation have allowed the systemic interrogation of gene function. Here, the authors evaluate the Brunello CRISPRko library and introduce Dolcetto and Calabrese for CRISPRi and CRISPRa, respectively.

Abstract We assess the magnitude and the extent of recent change of significant human footprint within protected areas, key biodiversity areas and the habitat range of 308 lowland forest specialist birds in Sundaland, a global hotspot of biodiversity in Southeast Asia. Using the most recent human footprint dataset, we find that 70% of Sundaland has been heavily modified by humans. This represents a 55% increase in areas under intense human pressure since 1993. Areas under intense human pressure covered on average 50% of the extent of key biodiversity areas, 78% of each protected area and 38% of the range of lowland forest specialist birds. The results imply that the actual level of protection by protected areas is only one‐third to half of that on paper once human footprint is accounted for. While all protected areas were impacted by human pressures, those managed strictly for biodiversity conservation presented the largest increases. These results highlight an exceptionally high human footprint across Sundaland and an impending further deepening of the biodiversity crisis across the region.

Introduction and aim. Adherence to hepatitis C (HCV) care was suboptimal in the interferon era among underserved African Americans (AA), but adherence data in the era of direct acting antivirals (DAA) is lacking in this population. We aimed to evaluate the impact of DAA on HCV care in underserved AA.Material and methods. Clinical records of AAs undergoing HCV evaluation attending a safety net health system liver clinic were reviewed from 2006 to 2011 (pre-DAA), and January 1, 2014 to December 31, 2016 (post-DAA).Results. 291 patients were identified (129 pre-DAA, and 162 post-DAA). Median age was 58, 66% were male, 91% had HCV genotype 1, and 70% had fibrosis ≥ stage 2. Post-DAA patients were older (60 vs. 53 years; p < 0.001), had higher rates of insurance (98% vs. 88%; p < 0.001), liver fibrosis ≥ stage 2 (77% vs. 61%; p = 0.048), ≥ 2 medical comorbidities (19 vs. 0.8%; p < 0.001), and median baseline log10 HCV RNA (6.07 vs. 5.81 IU/mL; p < 0.001), but lower median ALT (46 vs. 62 U/L; p < 0.001). Post-DAA, fewer patients were treatment ineligible (5.6% vs. 39%; p < 0.001) and more initiated therapy (71% vs. 8.5%; < 0.001), were adherent to HCV care (82% vs. 38%; p < 0.001), and achieved cure (95.7% vs. 63.6%, p < 0.001). Availability of DAA was independently associated with improved adherence to HCV care (OR 10.3, 95% CI 4.84-22.0).Conclusion. Availability of DAA is associated with increased treatment eligibility, initiation, adherence to HCV care, and cure in HCV-infected underserved AAs; highlighting the critical role of access to DAA in this population.

Aulonocephalus pennula is a nematode living in the caeca of the wild Northern bobwhite quail (Colinus virginianus) present throughout the Rolling Plains Ecoregion of Texas. The cytochrome oxidase 1 (COX 1) gene of the mitochondrial genome was used to screen A. pennula in wild quail. Through BLAST analysis, similarity of A. pennula to other nematode parasites was compared at the nucleotide level. Phylogenetic analysis of A. pennula COX1 indicated relationships to Subuluridae, Ascarididae, and Anisakidae. This study on molecular characterization of A. pennula provides new insight for the diagnosis of caecal worm infections of quail in the Rolling plains Ecoregion of Texas. Keywords: Aulonocephalus pennula, Caecal worm, Northern bobwhite, Phylogeny, PCR, COX1

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

Habitat loss and urbanization are primary components of human impact on the environment. Here, Venter et al.use global data on infrastructure, agriculture, and urbanization to show that the human footprint is growing slower than the human population, but footprints are increasing in biodiverse regions.

Decision making for the conservation and management of coral reef biodiversity requires an understanding of spatial variability and distribution of reef habitat types. Despite the existence of very high-resolution remote sensing technology for nearly two decades, comprehensive assessment of coral reef habitats at national to regional spatial scales and at very high spatial resolution is still scarce. Here, we develop benthic habitat maps at a sub-national scale by analyzing large multispectral QuickBird imagery dataset covering ~686 km2 of the main shallow coral fringing reef along the southern border with Tanzania (4.68°S, 39.18°E) to the reef end at Malindi, Kenya (3.2°S, 40.1°E). Mapping was conducted with a user approach constrained by ground-truth data, with detailed transect lines from the shore to the fore reef. First, maps were used to evaluate the present management system’s effectiveness at representing habitat diversity. Then, we developed three spatial prioritization scenarios based on differing objectives: (i) minimize lost fishing opportunity; (ii) redistribute fisheries away from currently overfished reefs; and (iii) minimize resource use conflicts. We further constrained the priority area in each prioritization selection scenario based on optionally protecting the least or the most climate exposed locations using a model of exposure to climate stress. We discovered that spatial priorities were very different based on the different objectives and on whether the aim was to protect the least or most climate-exposed habitats. Our analyses provide a spatially explicit foundation for large-scale conservation and management strategies that can account for ecosystem service benefits.

Loblolly pine residues have enormous potential to be the raw material for advanced biofuel production due to extensive sources and high cellulose content. Hot water (HW) pretreatment, while being a relatively economical and clean technology for the deconstruction of lignocellulosic biomass, could also inhibit the ensuing enzymatic hydrolysis process because of the production of inhibitors. In this study, we investigated the effect of oligosaccharide fractions purified from HW pre-hydrolyzate of pinewood using centrifugal partition chromatography (CPC) on three recombinant cellulolytic enzymes (E1, CBHI and CBHII), which were expressed in the transgenic corn grain system. The efficiency of recombinant enzymes was measured using either a 4-methylumbelliferyl-β-D-cellobioside (MUC) or a cellulose-dinitrosalicylic acid (DNS) assay system. The results showed that HW pre-hydrolyzate CPC fractions contain phenolics, furans, and monomeric and oligomeric sugars. Among CPC fractions, oligomers composed of xylan, galactan, and mannan were inhibitory to the three recombinant enzymes and to the commercial cellulase cocktail, reducing the enzymatic efficiency to as low as 10%.

We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer’s disease (AD), Parkinson’s disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained ~106 EVs/μL by NTA and VFC. Brain tumour and SAH CSF contained more EVs and RNA relative to normal, AD, and PD. RT-qPCR and RNASeq identified disease-related populations of microRNAs and messenger RNAs (mRNAs) relative to normal CSF, in both total and EV fractions. This work presents relevant measures selected to inform the design of subsequent replicative CSF studies. The range of neurological diseases highlights variations in total and EV RNA content due to disease or collection site, revealing critical considerations guiding the selection of appropriate approaches and controls for CSF studies.

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.

Plant cell walls are composed predominantly of cellulose, a range of non-cellulosic polysaccharides and lignin. The walls account for a large proportion not only of crop residues such as wheat straw and sugarcane bagasse, but also of residues of the timber industry and specialist grasses and other plants being grown specifically for biofuel production. The polysaccharide components of plant cell walls have long been recognized as an extraordinarily large source of fermentable sugars that might be used for the production of bioethanol and other renewable liquid transport fuels. Estimates place annual plant cellulose production from captured light energy in the order of hundreds of billions of tonnes. Lignin is synthesised in the same order of magnitude and, as a very large polymer of phenylpropanoid residues, lignin is also an abundant, high energy macromolecule. However, one of the major functions of these cell wall constituents in plants is to provide the extreme tensile and compressive strengths that enable plants to resist the forces of gravity and a broad range of other mechanical forces. Over millions of years these wall constituents have evolved under natural selection to generate extremely tough and resilient biomaterials. The rapid degradation of these tough cell wall composites to fermentable sugars is therefore a difficult task and has significantly slowed the development of a viable lignocellulose-based biofuels industry. However, good progress has been made in overcoming this so-called recalcitrance of lignocellulosic feedstocks for the biofuels industry, through modifications to the lignocellulose itself, innovative pre-treatments of the biomass, improved enzymes and the development of superior yeasts and other microorganisms for the fermentation process. Nevertheless, it has been argued that bioethanol might not be the best or only biofuel that can be generated from lignocellulosic biomass sources and that hydrocarbons with intrinsically higher energy densities might be produced using emerging and continuous flow systems that are capable of converting a broad range of plant and other biomasses to bio-oils through so-called ‘agnostic’ technologies such as hydrothermal liquefaction. Continued attention to regulatory frameworks and ongoing government support will be required for the next phase of development of internationally viable biofuels industries.