66 research outputs found

    Interactions of insecticidal spider peptide neurotoxins with insect voltage- and neurotransmitter-gated ion channels

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    University of Technology, Sydney. Faculty of Science.Two families of peptide neurotoxins that target insect large-conductance calcium-activated potassium channels (BKCa) have been isolated from the venom of two unrelated spiders. The ƙ-TRTX-Ec2 toxins are a family of three homologous peptides isolated from the African tarantula, Eucratoscelus longiceps and ƙ-HXTX-Hv1c is the prototypic member of a family of insect-selective neurotoxins isolated from the venom of the Blue Mountains funnel-web spider, Hadronyche versuta. This thesis describes the characterisation of these insecticidal toxins using voltage-clamp and current-clamp analysis of cockroach dorsal unpaired neurons utilising the whole-cell patch-clamp technique. The ability of these toxins to modulate the gating and kinetics of both voltage- and neurotransmitter-gated ion channels were assessed. Insect bioassays were also utilised to validate the insecticidal activities of various toxins that target KV channel subtypes in house crickets. The ƙ-TRTX-Ec2 family of toxins were found to be high affinity blockers of the insect BKCa channel while failing to modify voltage-gated sodium (NaV) and calcium (CaV) channels. ƙ-TRTX-Ec2a, -Ec2b and -Ec2c block cockroach BKCa channels with IC50 values of 3.7, 25.3 and 24.6 nM, respectively. Additionally, ƙ-TRTX-Ec2a was found to inhibit delayed-rectifier KV channel currents (IK(DR)), but only at significantly higher concentrations. ƙ-TRTX-Ec2 toxins induced voltage-independent channel block and are thus proposed to interact with the turret and/or loop region of the external vestibule of the insect BKCa channel. ƙ-HXTX-Hv1c has also been characterised to block the insect BKCa channel, while failing to modulate insect NaV and CaV channels. The unique insect-selective action of ƙ-HXTX-Hv1c involves a rare vicinal disulphide ring (Cys13-Cys14) that has been determined to act as part of the bioactive surface (pharmacophore) interacting with the molecular recognition site on the insect BKCa channel. However, despite the high affinity and selectivity for the BKCa channel it was discovered that the BKCa channel is unlikely to be the lethal target of ƙ-HXTX-Hv1c. Acute toxicity tests of classical non-phylum selective BKCa blockers such as paxilline, charybdotoxin and iberiotoxin did not induce acute toxicity in insects. Furthermore, while ƙ-HXTX-Hv1c was found to prolong action potential repolarisation, increase spontaneous firing frequency and reduce spike afterhyperpolarisation, these results were markedly reduced in the presence of the BKCa channel blocker iberiotoxin. Subsequent testing of cockroach KV channel currents revealed that ƙ-HXTX-Hv1c failed to modify sodium-activated or delayed-rectifier KV channel currents, but 1 ”M ƙ-HXTX-Hv1c did produce a 29% block of ‘A-type’ fast-transient KV channel currents (IK(A)). This suggests that ƙ-HXTX-Hv1c additionally targets insect KV1- or KV4-like channel subtypes. The lethal insecticidal action of 4-AP in crickets further supports an action of ƙ-HXTX-Hv1c to block IK(A). The results of co-application experiments revealed that ƙ-HXTX-Hv1c blocks the same channel as the non-phylum selective vertebrate KV4 channel toxin, ƙ-sparatoxin-Hv1b. However, it was found that ƙ-sparatoxin-Hv1b, either alone or in combination with iberiotoxin, was not insecticidal and thus the KV4 and BKCa channels are unlikely to be the lethal targets of ƙ-HXTX-Hv1c. To determine if the lethal target was a neurotransmitter-gated ion channel, the effects of ƙ-HXTX-Hv1c were investigated on chloride-gated GABAA (GABA-Cl) and glutamate (Glu-Cl) channel currents and nAChR channel currents. It was revealed that 1 ”M ƙ-HXTX-Hv1c failed to modify GABAA channel currents while causing only a moderate 21% increase in Glu-Cl channel currents. Alternately, it was found that ƙ-HXTX-Hv1c caused a concentration-dependent (EC50 183 nM) slowing of nicotinic acetylcholine receptor (nAChR) channel current decay and reversed channel desensitisation. In addition, ƙ-HXTX-Hv1c moderately increased nAChR sensitivity to nicotine. These findings are consistent with a positive allosteric modulation of insect nAChRs to slow receptor desensitisation. The nAChR is a validated insecticidal target for various agrochemical insecticides, including the allosteric modulator spinosyn A. Therefore it is believed that the lethal target of ƙ-HXTX-Hv1c is the insect nAChR, whose modulation would lead to an increase in neurotransmission consistent with the excitotoxic phenotype of the toxin. This action is possibly augmented by additional actions on BKCa and KV4 like channels to increase neuronal excitability

    Evaluation of chemical strategies for improving the stability and oral toxicity of insecticidal peptides

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    © 2018 by the authors. Spider venoms are a rich source of insecticidal peptide toxins. Their development as bioinsecticides has, however, been hampered due to concerns about potential lack of stability and oral bioactivity. We therefore systematically evaluated several synthetic strategies to increase the stability and oral potency of the potent insecticidal spider-venom peptide !-HXTX-Hv1a (Hv1a). Selective chemical replacement of disulfide bridges with diselenide bonds and N- to C-terminal cyclization were anticipated to improve Hv1a resistance to proteolytic digestion, and thereby its activity when delivered orally. We found that native Hv1a is orally active in blowflies, but 91-fold less potent than when administered by injection. Introduction of a single diselenide bond had no effect on the susceptibility to scrambling or the oral activity of Hv1a. N- to C-terminal cyclization of the peptide backbone did not significantly improve the potency of Hv1a when injected into blowflies and it led to a significant decrease in oral activity. We show that this is likely due to a dramatically reduced rate of translocation of cyclic Hv1a across the insect midgut, highlighting the importance of testing bioavailability in addition to toxin stability

    Geological archive of the onset of plate tectonics

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    © 2018 The Author(s) Published by the Royal Society. All rights reserved. Plate tectonics, involving a globally linked system of lateral motion of rigid surface plates, is a characteristic feature of our planet, but estimates of how long it has been the modus operandi of lithospheric formation and interactions range from the Hadean to the Neoproterozoic. In this paper, we review sedimentary, igneous and metamorphic proxies along with palaeomagnetic data to infer both the development of rigid lithospheric plates and their independent relative motion, and conclude that significant changes in Earth behaviour occurred in the mid- to late Archaean, between 3.2 Ga and 2.5 Ga. These data include: sedimentary rock associations inferred to have accumulated in passive continental margin settings, marking the onset of seafloor spreading; the oldest foreland basin deposits associated with lithospheric convergence; a change from thin, new continental crust of mafic composition to thicker crust of intermediate composition, increased crustal reworking and the emplacement of potassic and peraluminous granites, indicating stabilization of the lithosphere; replacement of dome and keel structures in granite-greenstone terranes, which relate to vertical tectonics, by linear thrust imbricated belts; the commencement of temporally paired systems of intermediate and high dT/dP gradients, with the former interpreted to represent subduction to collisional settings and the latter representing possible hinterland back-arc settings or ocean plateau environments. Palaeomagnetic data from the Kaapvaal and Pilbara cratons for the interval 2780-2710Ma and from the Superior, Kaapvaal and Kola-Karelia cratons for 2700-2440Ma suggest significant relative movements. We consider these changes in the behaviour and character of the lithosphere to be consistent with a gestational transition from a non-plate tectonic mode, arguably with localized subduction, to the onset of sustained plate tectonics

    IMPACT-Global Hip Fracture Audit: Nosocomial infection, risk prediction and prognostication, minimum reporting standards and global collaborative audit. Lessons from an international multicentre study of 7,090 patients conducted in 14 nations during the COVID-19 pandemic

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    Relative contributions of crust and mantle to generation of Campanian high-K calc-alkaline I-type granitoids in a subduction setting, with special reference to the Harsit Pluton, Eastern Turkey

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    We present elemental and Sr-Nd-Pb isotopic data for the magmatic suite (similar to 79 Ma) of the Harsit pluton, from the Eastern Pontides (NE Turkey), with the aim of determining its magma source and geodynamic evolution. The pluton comprises granite, granodiorite, tonalite and minor diorite (SiO(2) = 59.43-76.95 wt%), with only minor gabbroic diorite mafic microgranular enclaves in composition (SiO(2) = 54.95-56.32 wt%), and exhibits low Mg# (<46). All samples show a high-K calc-alkaline differentiation trend and I-type features. The chondrite-normalized REE patterns are fractionated [(La/Yb)(n) = 2.40-12.44] and display weak Eu anomalies (Eu/Eu* = 0.30-0.76). The rocks are characterized by enrichment of LILE and depletion of HFSE. The Harsit host rocks have weak concave-upward REE patterns, suggesting that amphibole and garnet played a significant role in their generation during magma segregation. The host rocks and their enclaves are isotopically indistinguishable. Sr-Nd isotopic data for all of the samples display I(Sr) = 0.70676-0.70708, epsilon(Nd)(79 Ma) = -4.4 to -3.3, with T(DM) = 1.09-1.36 Ga. The lead isotopic ratios are ((206)Pb/(204)pb) = 18.79-18.87, ((207)Pb/(204)Pb) = 15.59-15.61 and ((208)Pb/(204)Pb) = 38.71-38.83. These geochemical data rule out pure crustal-derived magma genesis in a post-collision extensional stage and suggest mixed-origin magma generation in a subduction setting. The melting that generated these high-K granitoidic rocks may have resulted from the upper Cretaceous subduction of the Izmir-Ankara-Erzincan oceanic slab beneath the Eurasian block in the region. The back-arc extensional events would have caused melting of the enriched subcontinental lithospheric mantle and formed mafic magma. The underplating of the lower crust by mafic magmas would have played a significant role in the generation of high-K magma. Thus, a thermal anomaly induced by underplated basic magma into a hot crust would have caused partial melting in the lower part of the crust. In this scenario, the lithospheric mantle-derived basaltic melt first mixed with granitic magma of crustal origin at depth. Then, the melts, which subsequently underwent a fractional crystallization and crustal assimilation processes, could ascend to shallower crustal levels to generate a variety of rock types ranging from diorite to granite. Sr-Nd isotope modeling shows that the generation of these magmas involved similar to 65-75% of the lower crustal-derived melt and similar to 25-35% of subcontinental lithospheric mantle. Further, geochemical data and the Ar-Ar plateau age on hornblende, combined with regional studies, imply that the Harsit pluton formed in a subduction setting and that the back-arc extensional period started by least similar to 79 Ma in the Eastern Pontides.Geochemistry & GeophysicsMineralogySCI(E)33ARTICLE4467-48716

    Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors

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    © 2017 Elsevier Ltd Îș-Hexatoxins (Îș-HXTXs) are a family of excitotoxic insect-selective neurotoxins from Australian funnel-web spiders that are lethal to a wide range of insects, but display no toxicity towards vertebrates. The prototypic Îș-HXTX-Hv1c selectively blocks native and expressed cockroach large-conductance calcium-activated potassium (BKCa or KCa1.1) channels, but not their mammalian orthologs. Despite this potent and selective action on insect KCa1.1 channels, we found that the classical KCa1.1 blockers paxilline, charybdotoxin and iberiotoxin, which all block insect KCa1.1 channels, are not lethal in crickets. We therefore used whole-cell patch-clamp analysis of cockroach dorsal unpaired median (DUM) neurons to study the effects of Îș-HXTX-Hv1c on sodium-activated (KNa), delayed-rectifier (KDR) and ‘A-type’ transient (KA) K+ channels. 1 ÎŒM Îș-HXTX-Hv1c failed to significantly inhibit cockroach KNa and KDR channels, but did cause a 30 ± 7% saturating inhibition of KA channel currents, possibly via a Kv4 (Shal-like) action. However, this modest action at such a high concentration of Îș-HXTX-Hv1c would indicate a different lethal target. Accordingly, we assessed the actions of Îș-HXTX-Hv1c on neurotransmitter-gated ion channels in cockroach DUM neurons. We found that Îș-HXTX-Hv1c failed to produce any major effects on GABAA or glutamate-Cl receptors but dramatically slowed nicotine-evoked ACh receptor (nAChR) current decay and reversed nAChR desensitization. These actions occurred without any alterations to nAChR current amplitude or the nicotine concentration-response curve, and are consistent with a positive allosteric modulation of nAChRs. Îș-HXTX-Hv1c therefore represents the first venom peptide that selectively modulates insect nAChRs with a mode of action similar to the excitotoxic insecticide spinosyn A. This article is part of the Special Issue entitled ‘Venom-derived Peptides as Pharmacological Tools.

    A novel family of insect-selective peptide neurotoxins targeting insect large-conductance calcium-activated K<sup>+</sup> channels isolated from the venom of the theraphosid spider Eucratoscelus constrictus

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    Spider venoms are actively being investigated as sources of novel insecticidal agents for biopesticide engineering. After screening 37 theraphosid spider venoms, a family of three new "short-loop" inhibitory cystine knot insecticidal toxins (Îș-TRTX-Ec2a, Îș-TRTX-Ec2b, and Îș-TRTX-Ec2c) were isolated and characterized from the venom of the African tarantula Eucratoscelus constrictus. Whole-cell patch-clamp recordings from cockroach dorsal unpaired median neurons revealed that, despite significant sequence homology with other theraphosid toxins, these 29-residue peptides lacked activity on insect voltage-activated sodium and calcium channels. It is noteworthy that Îș-TRTX-Ec2 toxins were all found to be high-affinity blockers of insect large-conductance calcium-activated K+ (BK Ca) channel currents with IC50 values of 3 to 25 nM. In addition, Îș-TRTX-Ec2a caused the inhibition of insect delayed-rectifier K+ currents, but only at significantly higher concentrations. Îș-TRTX-Ec2a and Îș-TRTX-Ec2b demonstrated insect-selective effects, whereas the homologous Îș-TRTX-Ec2c also resulted in neurotoxic signs in mice when injected intracerebroventricularly. Unlike other theraphosid toxins, Îș-TRTX-Ec2 toxins induce a voltage-independent channel block, and therefore, we propose that these toxins interact with the turret and/or loop region of the external entrance to the channel and do not project deeply into the pore of the channel. Furthermore, Îș-TRTX-Ec2a and Îș-TRTX-Ec2b differ from other theraphotoxins at the C terminus and positions 5 to 6, suggesting that these regions of the peptide contribute to the phyla selectivity and are involved in targeting BKCa channels. This study therefore establishes these toxins as tools for studying the role of BKCa channels in insects and lead compounds for the development of novel insecticides. Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics

    Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-Throughput automated patch clamp electrophysiology

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    KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-Type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-Throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3-4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-Throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant

    CONTINENTAL GEOTHERMS DURING THE ARCHAEAN

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    PRESSURE and temperature data from Archaean terrains have been used to test whether heat production during the Archaean at about three times the present rate resulted in correspondingly steeper continental geothermal gradients 1-6. The rocks considered here lie mostly in the range of medium pressure metamorphism, recording pressures of 7-13 kbar at temperatures of 600-900 °C (ref. 2). These pressures, with the underlying, normal, thickness of crust imply great thickening of continental crust during the Archaean. © 1979 Nature Publishing Group
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