Comparative Toxicity of Fumigants and a Phosphine Synergist Using a Novel Containment Chamber for the Safe Generation of Concentrated Phosphine Gas

BACKGROUND: With the phasing out of ozone-depleting substances in accordance with the United Nations Montreal Protocol, phosphine remains as the only economically viable fumigant for widespread use. However the development of high-level resistance in several pest insects threatens the future usage of phosphine; yet research into phosphine resistance mechanisms has been limited due to the potential for human poisoning in enclosed laboratory environments. PRINCIPAL FINDINGS: Here we describe a custom-designed chamber for safely containing phosphine gas generated from aluminium phosphide tablets. In an improvement on previous generation systems, this chamber can be completely sealed to control the escape of phosphine. The device has been utilised in a screening program with C. elegans that has identified a phosphine synergist, and quantified the efficacy of a new fumigant against that of phosphine. The phosphine-induced mortality at 20°C has been determined with an LC(50) of 732 ppm. This result was contrasted with the efficacy of a potential new botanical pesticide dimethyl disulphide, which for a 24 hour exposure at 20°C is 600 times more potent than phosphine (LC(50) 1.24 ppm). We also found that co-administration of the glutathione depletor diethyl maleate (DEM) with a sublethal dose of phosphine (70 ppm, <LC(5)), results in a doubling of mortality in C. elegans relative to DEM alone. CONCLUSIONS: The prohibitive danger associated with the generation, containment, and use of phosphine in a laboratory environment has now been substantially reduced by the implementation of our novel gas generation chamber. We have also identified a novel phosphine synergist, the glutathione depletor DEM, suggesting an effective pathway to be targeted in future synergist research; as well as quantifying the efficacy of a potential alternative to phosphine, dimethyl disulphide

The rph1 Gene Is a Common Contributor to the Evolution of Phosphine Resistance in Independent Field Isolates of Rhyzopertha Dominica

Phosphine is the only economically viable fumigant for routine control of insect pests of stored food products, but its continued use is now threatened by the world-wide emergence of high-level resistance in key pest species. Phosphine has a unique mode of action relative to well-characterised contact pesticides. Similarly, the selective pressures that lead to resistance against field sprays differ dramatically from those encountered during fumigation. The consequences of these differences have not been investigated adequately. We determine the genetic basis of phosphine resistance in Rhyzopertha dominica strains collected from New South Wales and South Australia and compare this with resistance in a previously characterised strain from Queensland. The resistance levels range from 225 and 100 times the baseline response of a sensitive reference strain. Moreover, molecular and phenotypic data indicate that high-level resistance was derived independently in each of the three widely separated geographical regions. Despite the independent origins, resistance was due to two interacting genes in each instance. Furthermore, complementation analysis reveals that all three strains contain an incompletely recessive resistance allele of the autosomal rph1 resistance gene. This is particularly noteworthy as a resistance allele at rph1 was previously proposed to be a necessary first step in the evolution of high-level resistance. Despite the capacity of phosphine to disrupt a wide range of enzymes and biological processes, it is remarkable that the initial step in the selection of resistance is so similar in isolated outbreaks

Laterally Orienting C. elegans Using Geometry at Microscale for High-Throughput Visual Screens in Neurodegeneration and Neuronal Development Studies

C. elegans is an excellent model system for studying neuroscience using genetics because of its relatively simple nervous system, sequenced genome, and the availability of a large number of transgenic and mutant strains. Recently, microfluidic devices have been used for high-throughput genetic screens, replacing traditional methods of manually handling C. elegans. However, the orientation of nematodes within microfluidic devices is random and often not conducive to inspection, hindering visual analysis and overall throughput. In addition, while previous studies have utilized methods to bias head and tail orientation, none of the existing techniques allow for orientation along the dorso-ventral body axis. Here, we present the design of a simple and robust method for passively orienting worms into lateral body positions in microfluidic devices to facilitate inspection of morphological features with specific dorso-ventral alignments. Using this technique, we can position animals into lateral orientations with up to 84% efficiency, compared to 21% using existing methods. We isolated six mutants with neuronal development or neurodegenerative defects, showing that our technology can be used for on-chip analysis and high-throughput visual screens

Evasion of IFN-γ Signaling by Francisella novicida Is Dependent upon Francisella Outer Membrane Protein C

Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918.The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components

Influence of an additional amino group on the potency of aminoadamantanes against influenza virus A. II - Synthesis of spiropiperazines and in vitro activity against influenza A H3N2 virus

Spiro[piperidine-2,2′-adamantane] 4 is one of the most potent synthetic anti-influenza A aminoadamantanes or other cage structure amines tested so far. Based on previous results Tataridis et al. (2007) [5h] which demonstrate the boost of in vitro potency by the presence of an additional amino group, we examined whether the incorporation of a second amino group into this heterocycle would increase the anti-influenza A virus activity. The new synthetic molecules 5-7 are capable of forming two hydrogen bonds within the receptor. We identified the diamino derivatives 5 and 6, which are active against influenza A H3N2 virus although less potent than amantadine and its equipotent spiropiperidine 4. © 2010 Elsevier Inc. All rights reserved

Marburg Virus VP40 Antagonizes Interferon Signaling in a Species-Specific Manner▿

Marburgviruses are zoonotic pathogens that cause lethal hemorrhagic fever in humans and nonhuman primates. However, they do not cause lethal disease in immunocompetent mice unless they are adapted to this species. The adaptation process can therefore provide insight into the specific virus-host interactions that determine virulence. In primate cells, the Lake Victoria marburgvirus Musoke strain (MARV) VP40 matrix protein antagonizes alpha/beta interferon (IFN-α/β) and IFN-γ signaling by inhibiting the activation of the cellular tyrosine kinase Jak1. Here, VP40 from the Ravn strain (RAVV VP40)—from a distinct Marburg virus clade—is demonstrated to also inhibit IFN signaling in human cells. However, neither MARV nor RAVV VP40 effectively inhibited IFN-signaling in mouse cells, as assessed by assays of the antiviral effects of IFN-α/β and the IFN-α/β-induced phosphorylation of Jak1, STAT1, and STAT2. In contrast, the VP40 from a mouse-adapted RAVV (maRAVV) did inhibit IFN signaling. Effective Jak1 inhibition correlated with the species from which the cells were derived and did not depend upon whether Jak1 was of human or mouse origin. Of the seven amino acid changes that accumulated in VP40 during mouse adaptation, two (V57A and T165A) are sufficient to allow efficient IFN signaling antagonism by RAVV VP40 in mouse cells. The same two changes also confer efficient IFN antagonist function upon MARV VP40 in mouse cells. The mouse-adaptive changes did not affect the budding of RAVV VP40 in mouse cells, suggesting that this second major function of VP40 did not undergo adaptation. These data identify an apparent determinant of RAVV host range and virulence and define specific genetic determinants of this function