Glutaraldehyde inactivation of exotic animal viruses in swine heart tissue.

Glutaraldehyde, 0.2%, in a 1:100 (wt/vol) ratio, inactivated four animal viruses (foot-and-mouth disease, swine vesicular disease, African swine fever, hog cholera) in swine heart tissues during 11-day exposures at 22 to 26 degrees C.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Temperature response of denitrification rate and greenhouse gas production in agricultural river marginal wetland soils

Soils are predicted to exhibit significant feedback to global warming via the temperature response of greenhouse gas (GHG) production. However, the temperature response of hydromorphic wetland soils is complicated by confounding factors such as oxygen (O₂), nitrate () and soil carbon (C). We examined the effect of a temperature gradient (2–25 °C) on denitrification rates and net nitrous oxide (N₂O), methane (CH₄) production and heterotrophic respiration in mineral (Eutric cambisol and Fluvisol) and organic (Histosol) soil types in a river marginal landscape of the Tamar catchment, Devon, UK, under non‐flooded and flooded with enriched conditions. It was hypothesized that the temperature response is dependent on interactions with ‐enriched flooding, and the physicochemical conditions of these soil types. Denitrification rate (mean, 746 ± 97.3 μg m^?2 h^?1), net N₂O production (mean, 180 ± 26.6 μg m^?2 h^?1) and net CH₄ production (mean, 1065 ± 183 μg m^?2 h^?1) were highest in the organic Histosol, with higher organic matter, ammonium and moisture, and lower concentrations. Heterotrophic respiration (mean, 127 ± 4.6 mg m^?2 h^?1) was not significantly different between soil types and dominated total GHG (CO₂eq) production in all soil types. Generally, the temperature responses of denitrification rate and net N₂O production were exponential, whilst net CH₄ production was unresponsive, possibly due to substrate limitation, and heterotrophic respiration was exponential but limited in summer at higher temperatures. Flooding with increased denitrification rate, net N₂O production and heterotrophic respiration, but a reduction in net CH₄ production suggests inhibition of methanogenesis by or N₂O produced from denitrification. Implications for management and policy are that warming and flood events may promote microbial interactions in soil between distinct microbial communities and increase denitrification of excess with N₂O production contributing to no more than 50% of increases in total GHG production.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons

Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.     

Eight new microsatellite markers in Atlantic cod (Gadus morhua L.) derived from an enriched genomic library

One hundred and fifty random clones from an enriched genomic library of Atlantic cod were sequenced. Primer pairs were designed for 15 microsatellites containing perfect di‐, tri‐, tetra‐ and hexanucleotide repeats and characterized in 96 unrelated fish. Eight markers were successfully amplified, with the number of alleles ranging from two to nine per locus and observed heterozygosity ranging from 0.341 to 0.977. Loci Gmo‐G13 and Gmo‐G14 had a significant excess of homozygotes. All loci conformed to the Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

Y chromosome lineage- and village-specific genes on chromosomes 1p22 and 6q27 control visceral leishmaniasis in Sudan

Familial clustering and ethnic differences suggest that visceral leishmaniasis caused by Leishmania donovani is under genetic control. A recent genome scan provided evidence for a major susceptibility gene on Chromosome 22q12 in the Aringa ethnic group in Sudan. We now report a genome-wide scan using 69 families with 173 affected relatives from two villages occupied by the related Masalit ethnic group. A primary ten-centimorgan scan followed by refined mapping provided evidence for major loci at 1p22 (LOD score 5.65; nominal p = 1.72 × 10⁻⁷; empirical p < 1 × 10⁻⁵; λ_S = 5.1) and 6q27 (LOD score 3.74; nominal p = 1.68 × 10⁻⁵; empirical p < 1 × 10⁻⁴; λ_S = 2.3) that were Y chromosome–lineage and village-specific. Neither village supported a visceral leishmaniasis susceptibility gene on 22q12. The results suggest strong lineage-specific genes due to founder effect and consanguinity in these recently immigrant populations. These chance events in ethnically uniform African populations provide a powerful resource in the search for genes and mechanisms that regulate this complex disease.