Plant pathogens provide clues to the potential origin of bat white-nose syndromePseudogymnoascus destructans

White-nose syndrome has killed millions of bats, yet both the origins and infection strategy of the causative fungus, Pseudogymnoascus destructans, remain elusive. We provide evidence for a novel hypothesis that P. destructans emerged from plant-associated fungi and retained invasion strategies affiliated with fungal pathogens of plants. We demonstrate that P. destructans invades bat skin in successive biotrophic and necrotrophic stages (hemibiotrophic infection), a mechanism previously only described in plant fungal pathogens. Further, the convergence of hyphae at hair follicles suggests nutrient tropism. Tropism, biotrophy, and necrotrophy are often associated with structures termed appressoria in plant fungal pathogens; the penetrating hyphae produced by P. destructans resemble appressoria. Finally, we conducted a phylogenomic analysis of a taxonomically diverse collection of fungi. Despite gaps in genetic sampling of prehistoric and contemporary fungal species, we estimate an 88% probability the ancestral state of the clade containing P. destructans was a plant-associated fungus

A GC-MS Method for the Determination of Isoxsuprine in Biological Fluids of the Horse Utilizing Electron Impact Ionization

Isoxsuprine is used to treat navicular disease and other lower-limb problems in the horse. Isoxsuprine is regulated as a class 4 compound by the Association of Racing Commissioners, International (ARCI) and, thus, requires regulatory monitoring. A gas chromatography-mass spectrometry method utilizing electron impact ionization was developed and validated for the quantitation of isoxsuprine in equine plasma or equine urine. The method utilized robotic solid-phase extraction and tri-methyl silyl ether products of derivatization. Products were bis-trimethylsilyl (TMS) isoxsuprine and tris-TMS ritodrine, which released intense quantifier ions m/z 178 for isoxsuprine and m/z 236 for ritodrine that were products of C-C cleavage. To our knowledge, this procedure is faster and more sensitive than other methods in the literature. Concentrations in urine and plasma of isoxsuprine were determined from a calibrator curve that was generated along with unknowns. Ritodrine was used as an internal standard and was, therefore, present in all samples, standards, and blanks. Validation data was also collected. The limit of detection of isoxsuprine in plasma was determined to be 2 ng/mL, the limit of quantitation of isoxsuprine in plasma was determined to be \u3c 5 ng/mL. The mean coefficient of determination for the calibrator curves for plasma was 0.9925 ± 0.0052 and for calibrator curves for urine 0.9904 ± 0.0075. The recovery efficiencies at concentrations of 50, 200, and 300 ng/mL were 76%, 73%, and 76%, respectively, in plasma and 92%, 89% and 91% in urine

Discovery and Follow-up of Rotating Radio Transients with the Green Bank and LOFAR Telescopes

We have discovered 21 Rotating Radio Transients (RRATs) in data from the Green Bank Telescope (GBT) 350-MHz Drift-scan and the Green Bank North Celestial Cap pulsar surveys using a new candidate sifting algorithm. RRATs are pulsars with sporadic emission that are detected through their bright single pulses rather than Fourier domain searches. We have developed {\tt RRATtrap}, a single-pulse sifting algorithm that can be integrated into pulsar survey data analysis pipelines in order to find RRATs and Fast Radio Bursts. We have conducted follow-up observations of our newly discovered sources at several radio frequencies using the GBT and Low Frequency Array (LOFAR), yielding improved positions and measurements of their periods, dispersion measures, and burst rates, as well as phase-coherent timing solutions for four of them. The new RRATs have dispersion measures (DMs) ranging from 15 to 97 pc cm$^{-3}$, periods of 240 ms to 3.4 s, and estimated burst rates of 20 to 400 pulses hr$^{-1}$ at 350 MHz. We use this new sample of RRATs to perform statistical comparisons between RRATs and canonical pulsars in order to shed light on the relationship between the two populations. We find that the DM and spatial distributions of the RRATs agree with those of the pulsars found in the same survey. We find evidence that slower pulsars (i.e. $P>200$ ms) are preferentially more likely to emit bright single pulses than are faster pulsars ($P<200$ ms), although this conclusion is tentative. Our results are consistent with the proposed link between RRATs, transient pulsars, and canonical pulsars as sources in various parts of the pulse activity spectrum.Comment: 18 pages, 13 figures, 5 tables, published in Ap

Detection and Confirmation of Ractopamine and Its Metabolites in Horse Urine after Paylean® Administration

We have investigated the detection, confirmation, and metabolism of the beta-adrenergic agonist ractopamine administered as Paylean to the horse. A Testing Components Corporation enzyme-linked imunosorbent assay (ELISA) kit for ractopamine displayed linear response between 1.0 and 100 ng/ml, with an 1-50 of 10 ng/ml, and an effective screening limit of detection of 50 ng/mL. The kit was readily able to detect ractopamine equivalents in unhydrolyzed urine up to 24 h following a 300-mg oral dose. Gas chromatography-mass spectrometry (GC-MS) confirmation comprised glucuronidase treatment, solid-phase extraction, and trimethylsilyl derivatization, with selected-ion monitoring of ractopamine-tris(trimethylsilane) (TMS) m/z 267, 250, 179, and 502 ions. Quantitation was elaborated in comparison to a 445 Mw isoxsuprine-bis(TMS) internal standard monitored simultaneously. The instrumental limit of detection, defined as that number of ng on column for which signal-to-noise ratios for one or more diagnostic ions fell below a value of three, was 0.1 ng, corresponding to roughly 5 ng/mL in matrix. Based on the quantitation ions for ractopamine standards extracted from urine, standard curves showed a linear response for ractopamine concentrations between 10 and 100 ng/mL with a correlation coefficient r \u3e 0.99, whereas standards in the concentration range of 10-1000 ng/mL were fit to a second-order regression curve with r \u3e 0.99. The lower limit of detection for ractopamine in urine, defined as the lowest concentration at which the identity of ractopamine could be confirmed by comparison of diagnostic MS ion ratios, ranged between 25 and 50 ng/mL. Urine concentration of parent ractopamine 24 h post-dose was measured at 360 ng/mL by GC-MS after oral administration of 300 mg. Urinary metabolites were identified by electrospray ionization (+) tandem quadrupole mass spectrometry and were shown to include glucuronide, methyl, and mixed methyl-glucuronide conjugates. We also considered the possibility that an unusual conjugate added 113 amu to give an observed m/z 415 [M+H] species or two times 113 amu to give an m/z 528 [M+H] species with a daughter ion mass spectrum related to the previous one. Sulfate and mixed methyl-sulfate conjugates were revealed following glucuronidase treatment, suggesting that sulfation occurs in combination with glucuronidation. We noted a paired chromatographic peak phenomenon of apparent ractopamine metabolites appearing as doublets of equivalent intensity with nearly identical mass spectra on GC-MS and concluded that this phenomenon is consistent with Paylean being a mixture of RR, RS, SR, and SS diastereomers of ractopamine. The results suggest that ELISA-based screening followed by glucuronide hydrolysis, parent drug recovery, and TMS derivatization provide an effective pathway for detection and GC-MS confirmation of ractopamine in equine urine

Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine

Amitraz (N′-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino] methyl]-N-methyl-methanimidamide) is an alpha-2 adrenergic agonist used in veterinary medicine primarily as a scabicide- or acaricide-type insecticide. As an alpha-2 adrenergic agonist, it also has sedative/tranquilizing properties and is, therefore, listed as an Association of Racing Commissioners International Class 3 Foreign Substance, indicating its potential to influence the outcome of horse races. We identified the principal equine metabolite of amitraz as N-2,4-dimethylphenyl-N′-methylformamidine by electrospray ionization(+)-mass spectrometry and developed a gas chromatographic-mass spectrometric (GC-MS) method for its detection, quantitation, and confirmation in performance horse regulation. The GC-MS method involves derivatization with t-butyldimethylsilyl groups; selected ion monitoring (SIM) of m/z 205 (quantifier ion), 278, 261, and 219 (qualifier ions); and elaboration of a calibration curve based on ion area ratios involving simultaneous SIM acquisition of an internal standard m/z 208 quantifier ion based on an in-house synthesized d6 deuterated metabolite. The limit of detection of the method is approximately 5 ng/mL in urine and is sufficiently sensitive to detect the peak urinary metabolite at 1 h post dose, following administration of amitraz at a 75-mg/horse intraveneous dose

Meta-analysis identifies pleiotropic loci controlling phenotypic trade-offs in sorghum

Community association populations are composed of phenotypically and genetically diverse accessions. Once these populations are genotyped, the resulting marker data can be reused by different groups investigating the genetic basis of different traits. Because the same genotypes are observed and scored for a wide range of traits in different environments, these populations represent a unique resource to investigate pleiotropy. Here, we assembled a set of 234 separate trait datasets for the Sorghum Association Panel, a group of 406 sorghum genotypes widely employed by the sorghum genetics community. Comparison of genome-wide association studies (GWAS) conducted with two independently generated marker sets for this population demonstrate that existing genetic marker sets do not saturate the genome and likely capture only 35–43% of potentially detectable loci controlling variation for traits scored in this population. While limited evidence for pleiotropy was apparent in cross-GWAS comparisons, a multivariate adaptive shrinkage approach recovered both known pleiotropic effects of existing loci and new pleiotropic effects, particularly significant impacts of known dwarfing genes on root architecture. In addition, we identified new loci with pleiotropic effects consistent with known trade-offs in sorghum development. These results demonstrate the potential for mining existing trait datasets from widely used community association populations to enable new discoveries from existing trait datasets as new, denser genetic marker datasets are generated for existing community association populations

PROPEL: implementation of an evidence based pelvic floor muscle training intervention for women with pelvic organ prolapse: a realist evaluation and outcomes study protocol

Abstract Background Pelvic Organ Prolapse (POP) is estimated to affect 41%–50% of women aged over 40. Findings from the multi-centre randomised controlled “Pelvic Organ Prolapse PhysiotherapY” (POPPY) trial showed that individualised pelvic floor muscle training (PFMT) was effective in reducing symptoms of prolapse, improved quality of life and showed clear potential to be cost-effective. However, provision of PFMT for prolapse continues to vary across the UK, with limited numbers of women’s health physiotherapists specialising in its delivery. Implementation of this robust evidence from the POPPY trial will require attention to different models of delivery (e.g. staff skill mix) to fit with differing care environments. Methods A Realist Evaluation (RE) of implementation and outcomes of PFMT delivery in contrasting NHS settings will be conducted using multiple case study sites. Involving substantial local stakeholder engagement will permit a detailed exploration of how local sites make decisions on how to deliver PFMT and how these lead to service change. The RE will track how implementation is working; identify what influences outcomes; and, guided by the RE-AIM framework, will collect robust outcomes data. This will require mixed methods data collection and analysis. Qualitative data will be collected at four time-points across each site to understand local contexts and decisions regarding options for intervention delivery and to monitor implementation, uptake, adherence and outcomes. Patient outcome data will be collected at baseline, six months and one year follow-up for 120 women. Primary outcome will be the Pelvic Organ Prolapse Symptom Score (POP-SS). An economic evaluation will assess the costs and benefits associated with different delivery models taking account of further health care resource use by the women. Cost data will be combined with the primary outcome in a cost effectiveness analysis, and the EQ-5D-5L data in a cost utility analysis for each of the different models of delivery. Discussion Study of the implementation of varying models of service delivery of PFMT across contrasting sites combined with outcomes data and a cost effectiveness analysis will provide insight into the implementation and value of different models of PFMT service delivery and the cost benefits to the NHS in the longer term

A millisecond pulsar in a stellar triple system

Gravitationally bound three-body systems have been studied for hundreds of years and are common in our Galaxy. They show complex orbital interactions, which can constrain the compositions, masses, and interior structures of the bodies and test theories of gravity, if sufficiently precise measurements are available. A triple system containing a radio pulsar could provide such measurements, but the only previously known such system, B1620-26 (with a millisecond pulsar, a white dwarf, and a planetary-mass object in an orbit of several decades), shows only weak interactions. Here we report precision timing and multi-wavelength observations of PSR J0337+1715, a millisecond pulsar in a hierarchical triple system with two other stars. Strong gravitational interactions are apparent and provide the masses of the pulsar (1.4378(13) Msun, where Msun is the solar mass and the parentheses contain the uncertainty in the final decimal places) and the two white dwarf companions (0.19751(15) Msun and 0.4101(3) Msun), as well as the inclinations of the orbits (both approximately 39.2 degrees). The unexpectedly coplanar and nearly circular orbits indicate a complex and exotic evolutionary past that differs from those of known stellar systems. The gravitational field of the outer white dwarf strongly accelerates the inner binary containing the neutron star, and the system will thus provide an ideal laboratory in which to test the strong equivalence principle of general relativity.Comment: 17 pages, 3 figures, 1 table. Published online by Nature on 5 Jan 2014. Extremely minor differences with published version may exis

Remifentanil in the Horse: Identification and Detection of its Major Urinary Metabolite

Remifentanil (4-methoxycarbonyl-4-[(1-oxopropyl)phyenylamino]-1- piperidinepropionic acid methyl ester) is a μ-opioid receptor agonist with considerable abuse potential in racing horses. The identification of its major equine urinary metabolite, 4-methoxycarbonyl-4-[(1- oxopropyl)phenylamino]-1-piperidinepropionic acid, an ester hydrolysis product of remifentanil is reported. Administration of remifentanil HCl (5 mg, intravenous) produced clear-cut locomotor responses, establishing the clinical efficacy of this dose. ELISA analysis of postadministration urine samples readily detected fentanyl equivalents in these samples. Mass spectrometric analysis, using solid-phase extraction and trimethylsilyl (TMS) derivatization, showed the urine samples contained parent remifentanil in low concentrations, peaking at 1 h. More significantly, a major peak was identified as representing 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1- piperidinepropionic acid, arising from ester hydrolysis of remifentanil. This metabolite reached its maximal urinary concentrations at 1 h and was present at up to 10-fold greater concentrations than parent remifentanil. Base hydrolysis of remifentanil yielded a carboxylic acid with the same mass spectral characteristics as those of the equine metabolite. In summary, these data indicate that remifentanil administration results in the appearance of readily detectable amounts of 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]- 1-piperidinepropionic acid in urine. On this basis, screening and confirmation tests for this equine urinary metabolite should be optimized for forensic control of remifentanil