Proteomic Analysis of Excretory-Secretory Products of Heligmosomoides polygyrus Assessed with Next-Generation Sequencing Transcriptomic Information

The murine parasite Heligmosomoides polygyrus is a convenient experimental model to study immune responses and pathology associated with gastrointestinal nematode infections. The excretory-secretory products (ESP) produced by this parasite have potent immunomodulatory activity, but the protein(s) responsible has not been defined. Identification of the protein composition of ESP derived from H. polygyrus and other relevant nematode species has been hampered by the lack of genomic sequence information required for proteomic analysis based on database searches. To overcome this, a transcriptome next generation sequencing (RNA-seq) de novo assembly containing 33,641 transcripts was generated, annotated, and used to interrogate mass spectrometry (MS) data derived from 1D-SDS PAGE and LC-MS/MS analysis of ESP. Using the database generated from the 6 open reading frames deduced from the RNA-seq assembly and conventional identification programs, 209 proteins were identified in ESP including homologues of vitellogenins, retinol- and fatty acid-binding proteins, globins, and the allergen V5/Tpx-1-related family of proteins. Several potential immunomodulators, such as macrophage migration inhibitory factor, cysteine protease inhibitors, galectins, C-type lectins, peroxiredoxin, and glutathione S-transferase, were also identified. Comparative analysis of protein annotations based on the RNA-seq assembly and proteomics revealed processes and proteins that may contribute to the functional specialization of ESP, including proteins involved in signalling pathways and in nutrient transport and/or uptake. Together, these findings provide important information that will help to illuminate molecular, biochemical, and in particular immunomodulatory aspects of host-H. polygyrus biology. In addition, the methods and analyses presented here are applicable to study biochemical and molecular aspects of the host-parasite relationship in species for which sequence information is not available

Characterization of the Ca2+-gated and voltage-dependent k+-channel slo-1 of nematodes and its interaction with emodepside

The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class

World Association for the Advancement of Veterinary Parasitology (WAAVP) guideline: anthelmintic combination products targeting nematode infections of ruminants and horses

Increasing threats from anthelmintic resistant nematode populations warrant and motivate a reappraisal of chemotherapeutic strategies for nematode control in ruminant livestock and horses. The objective of this paper is to present a guideline for the evaluation of products containing two or more constituent anthelmintic actives in a single dosage form for the treatment of nematode infections in these animals. At present, regulatory policies on the approval of such products vary across jurisdictions, and this World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guideline should enable the harmonization of the requirements. This guideline makes clear recommendations on the minimal standards needed, but stresses that registration dossiers for combination anthelmintic products submitted for approval must conform to the standards and practices already established in existing guidelines for anthelmintics

The Effect of In Vitro Cultivation on the Transcriptome of Adult Brugia malayi

Infections with filarial worms cause serious physical impairment and affect tens of millions of people in tropical and subtropical countries. To better understand the biology and phar- macology of these parasites, Brugia malayi is often used as a model. This parasite can be maintained in the laboratory in Mongolian jirds, enabling researchers to test drugs in vivoand in vitro, among other studies. The effects of removing worms from their hosts and cul- turing them may affect many aspects of their physiology, including response to drugs, but the extent to which the worms undergo changes during culture has remained unknown. Using deep RNA sequencing and bioinformatics tools, we examined the global transcriptomic profile of B. malayi females at four different time points over 5 days in culture. Focusing on genes that are differentially expressed at various time points, we observed a general perturbation of the expression profile between dissection from the host and receipt after shipment. The expression of several genes remained changed at the end of the experi- ment, after 5 days under controlled conditions; in particular, genes encoding cuticle colla- gens were prominently represented and strongly overexpressed

The Effects of Ivermectin on Brugia malayi Females In Vitro: A Transcriptomic Approach

Lymphatic filariasis and onchocerciasis are tropical diseases caused by infections with parasitic nematodes. Resulting chronic diseases can be strongly blinding and disfiguring, and contribute to an entrenched cycle of poverty in affected populations. Ivermectin is one of the pivotal drugs used to control these infections. The mechanism of antifilarial action of the drug is incompletely resolved. It kills circulating larval stages (microfilariae), but only reversibly sterilizes adult worms without killing them. Our limited understanding of the involved mechanisms hampers treatment optimization and sustainability of the efficacy of the drug, and investigations into its pharmacology are of paramount importance. Working with Brugia malayi adult females, we employed RNA sequencing and bioinformatics analyses to identify genes for which expression levels changed as a result of exposure to the drug in vitro. Ivermectin exposure altered the expression of genes that are likely to func- tion in the B. malayi female reproductive system even at the lowest concentration tested. Through several biological pathways, genes involved in meiosis were particularly affected. These findings provide some insight into the mechanisms involved in ivermectin-induced reduction in microfilaria output and impaired fertility, embryogenesis, and larval development

A quantitative assessment of shoot flammability for 60 tree and shrub species supports rankings based on expert opinion

Fire is an important ecological disturbance in vegetated ecosystems across the globe, and also has considerable impacts on human infrastructure. Vegetation flammability is a key bottom-up control on fire regimes, and on the nature of individual fires. Although New Zealand (NZ) historically had low fire frequencies, anthropogenic fires have considerably impacted indigenous vegetation as humans used fire extensively to clear forests. Few studies of vegetation flammability have been undertaken in NZ, and only one has compared the flammability of indigenous plants; this was a qualitative assessment derived from expert opinion. We addressed this knowledge gap by measuring the flammability of terminal shoots from a range of trees and shrubs found in NZ. We quantified shoot flammability of 60 indigenous and exotic species, and compared our experimentally derived ranking with expert opinion. The most flammable species was the invasive exotic shrub Ulex europaeus, followed by Eucalyptus viminalis, Pomaderris kumeraho, Dacrydium cupressinum, and Lophozonia menziesii. Our experimentally derived ranking was strongly correlated with expert opinion, lending support to both methods. Our results are useful to ecologists seeking to understand how fires have and will influence NZ’s ecosystems, and for fire managers identifying high-risk landscapes, and low flammability species for ‘green firebreaks’

Kepler-4B: A Hot Neptune-Like Planet of A G0 Star Near Main-Sequence Turnoff

Early time-series photometry from NASA's Kepler spacecraft has revealed a planet transiting the star we term Kepler-4, at R.A. = 19(h)02(m)27.(s)68, delta = +50 degrees 08'08 '' 7. The planet has an orbital period of 3.213 days and shows transits with a relative depth of 0.87 x 10(-3) and a duration of about 3.95 hr. Radial velocity (RV) measurements from the Keck High Resolution Echelle Spectrometer show a reflex Doppler signal of 9.3(-1.9)(+1.1) m s(-1), consistent with a low-eccentricity orbit with the phase expected from the transits. Various tests show no evidence for any companion star near enough to affect the light curve or the RVs for this system. From a transit-based estimate of the host star's mean density, combined with analysis of high-resolution spectra, we infer that the host star is near turnoff from the main sequence, with estimated mass and radius of 1.223(-0.091)(+0.053) M(circle dot) and 1.487(-0.084)(+0.071) R(circle dot).We estimate the planet mass and radius to be {M(P), R(P)} = {24.5 +/- 3.8 M(circle plus), 3.99 +/- 0.21 R(circle plus)}. The planet's density is near 1.9 g cm(-3); it is thus slightly denser and more massive than Neptune, but about the same size.W. M. Keck FoundationNASA's Science Mission DirectorateAstronom

Kepler-7b: A Transiting Planet with Unusually Low Density

We report the discovery and confirmation of Kepler-7b, a transiting planet with unusually low density. The mass is less than half that of Jupiter, Mp = 0.43 Mj, but the radius is fifty percent larger, Rp = 1.48 Rj. The resulting density, 0.17 g/cc, is the second lowest reported so far for an extrasolar planet. The orbital period is fairly long, P = 4.886 days, and the host star is not much hotter than the Sun, Teff = 6000 K. However, it is more massive and considerably larger than the sun, Mstar = 1.35 Msun and Rstar = 1.84 Rsun, and must be near the end of its life on the Main Sequence.Comment: 19 pages, 3 figure

A Transiting Hot Jupiter Orbiting a Metal-Rich Star

We announce the discovery of Kepler-6b, a transiting hot Jupiter orbiting a star with unusually high metallicity, [Fe/H] = +0.34 +/- 0.04. The planet's mass is about 2/3 that of Jupiter, Mp = 0.67 Mj, and the radius is thirty percent larger than that of Jupiter, Rp = 1.32 Rj, resulting in a density of 0.35 g/cc, a fairly typical value for such a planet. The orbital period is P = 3.235 days. The host star is both more massive than the Sun, Mstar = 1.21 Msun, and larger than the Sun, Rstar = 1.39 Rsun.Comment: 12 pages, 2 figures, submitted to the Astrophysical Journal Letter