Structure-function studies of insecticidal atracotoxins

University of Technology, Sydney. Faculty of Science.The k-atracotoxins (k -ACTXs, previously the Janus-faced atracotoxins ) are a family of five insect-selective excitatory peptide neurotoxins containing 36-37 residues with four disulfide bonds. Toxins from this family were isolated from the venom of the Blue Mountains funnel-web spider (Hadronyche versuta) and Toowooba funnel-web spider (Hadronyche infensa). The NMR solution structure and primary sequence of the prototypic member k -ATCT-Hv1c provided few clues as to the likely molecular target. In order to characterise the site of action and phylogenetic specificity of these toxins, whole-cell patch-clamp electrophysiology was employed using isolated DUM neurons from the American cockroach (Periplaneta americana). k -ACTX-Hv1c had no effect on the gating or kinetics of INa or ICa at concentrations up to 1 μM. However, at the same concentration, k -ATCT-Hv1c reduced Kv channel currents by 56 ± 7% (n = 5). Subsequent experiments in insect DUM neurons indicated that inhibition of the macroscopic IK was due to a block of calcium-activated Kv (KCa) channels, with an IC50 of 2.3 nM and 2.9 nM for peak and late IK(Ca) respectively (n = 5), and not ‘A-type’ or delayed-rectifier Kv channels. Insect selectivity was confirmed by a lack of activity on rat dorsal root ganglion (DRG) neuron global IK as well as IK(Ca) at doses up to 1 μM. k -ACTX-Hv1c is a selective insect KCa (BKCa) channel pore-blocker, not a gating modifier, as inhibition of insect IK(Ca) occurred in the absence of any voltage-dependent actions on channel activation. Specificity for the insect BKCa channel was validated by k -ACTX-Hv1c induced inhibition of IK(Ca) from the cloned insect KCa channel a-subunit (pSlo) expressed in HEK293 cells (IC50 of 240 nM). The 80-fold reduction in IC50, most likely indicates that k-ACTX-Hv1c interacts with the auxillary subunits that form part of the wild-type channel, in a manner similar to the BKCa blocker, charybdotoxin (ChTX), as previously reported. Phyletic selectivity of k-ACTX-Hv1c was confirmed by the 9776-fold increase in IC50 against mSlo channels. Interestingly k-ACTX-Hv1c, like ChTX, failed to potently block the dSlo channel with the IC50 >10 μM. Additional experiments on DUM neuron IK(Ca) using alanine mutants confirmed the pharmacophore of bioactive residues k-ACTX-Hv1c comprises Arg8, Pro9, Val29 and Tyr31, previously identified by acute toxicity tests in house flies (Musca domestica). Interestingly, the functionally critical Arg8 and Tyr31 residues align extremelyç well with the Lys-Phe/Tyr diad conserved amongst structurally dissimilar Kv channel toxins, providing a possible basis for targeting of the toxin to K+ channels. Using a panel of 8 mutants (R8E, R8Q, R8K, R8H, Y31W, Y31F, Y31L and Y31V) the mechanism of interaction was investigated further. The Arg8 residue appears to interact with the channel via hydrogen bonding from the s-guanido group to carbonyl groups on the extracellular surface of the channel, as evidenced by the high potency of the R8H mutant. The imidazole group of His is an adequate substitute for the s-guanido group of arginine. In contrast the R8E, R8Q and R8K had reduced potency indicating that the positive charge of the amino group of Arg does not directly interact with the target nor is the alkyl group of Arg critical for binding to the target. The critically important Tyr31 interacts with the channel via non-specific hydrophobic interactions as substitution for an aromatic ring (Y31F & Y31W) maintains the potency of the toxin. In contrast substitution to small less hydrophobic side chains (Y31V, Y31L and Y31A) reduced potency. It appears therefore that Tyr31 in conjunction with IIe2 and Val29, that lie at either side of the primary pharmacophore, appear to act as ‘gasket’ residues to exclude bulk solvent from disrupting the Arg8-channel interaction. This study has identified k-atracotoxins as potential lead compounds in the development of new biopesticides and validates insect BKCa channels as potential insecticide targets. The second part of this thesis was to determine the target site for the ‘hybrid’ toxin FW178 from the venom of the Blue Mountains funnel-web spider (H. versuta). FW178 is a unique toxin that shares little homology to other known atracotoxins. In order to identify the site of action of this toxin, whole-cell patch-clamp electrophysiology was employed using isolated DUM neurons from the American cockroach (Periplaneta americana). FW178 failed to inhibit insect INa , IK(DR) or IK(A) at doses up to 1 μM. However, further studies demonstrated that Ƞ-ACTX-Hv1a blocks voltage-gated calcium (CaV) channel currents in DUM neurons as well as K(Ca) channel currents carried by pSlo channels with IC50 values of 409 nM and 671 nM, respectively. FW178 therefore blocks cockroach CaV currents with approximately the same potency as Ѡ -ACTX-Hv1a, a known insect M-LVA and HVA CaV channel blocker, while it blocks cockroach pSlo channels with about a 4-fold lower potency than k-ACTX-Hv1c. Interestingly FW178 has an LD50 of 38 ± 3 pmol/g when injected into M. domestica as compared to the LD50 values for Ѡ -ACTX-Hv1a (86.5 ± 1.3 pmol/g) and Ѡ -ACTX-Hv1c (91 ± 5 pmol/g). This makes FW178 at least two-fold more potent than any other atracotoxin isolated from Australian funnel-web spiders. Despite this, FW178 only blocks cockroach CaV channels with a similar potency to k-ACTX-Hv1a, and blocks cockroach BK(Ca) with 4-fold less potency than k-ACTX-Hv1c. Therefore the striking potency of FW178 may result from a synergistic action to block insect CaV and BK(Ca) channels. Not surprising the pharmacophore of FW178 (refer section 4.3) contains elements of the pharmacophore of both Ѡ-ACTX-Hv1a and k-ACTX-Hv1c. Thus FW178 directly block insect K(Ca) channels, but the toxin also enhances this action by indirectly reducing current through these channels by block of the transient inward flow of calcium through CaV channels. Therefore FW178 represents the first known dual-target, self-synergizing toxin and is an excellent lead compound for the development of a novel insecticide

Isolation of δ-missulenatoxin-Mb1a, the major vertebrate-active spider δ-toxin from the venom of Missulena bradleyi (Actinopodidae)

The present study describes the isolation and pharmacological characterisation of the neurotoxin δ-missulenatoxin-Mb1a (δ-MSTX-Mb1a) from the venom of the male Australian eastern mouse spider, Missulena bradleyi. This toxin was isolated using reverse-phase high-performance liquid chromatography and was subsequently shown to cause an increase in resting tension, muscle fasciculation and a decrease in indirect twitch tension in a chick biventer cervicis nerve-muscle bioassay. Interestingly, these effects were neutralised by antivenom raised against the venom of the Sydney funnel-web spider Atrax robustus. Subsequent whole-cell patch-clamp electrophysiology on rat dorsal root ganglion neurones revealed that δ-MSTX-Mb1a caused a reduction in peak tetrodotoxin (TTX)-sensitive sodium current, a slowing of sodium current inactivation and a hyperpolarising shift in the voltage at half-maximal activation. In addition, δ-MSTX-Mb1a failed to affect TTX-resistant sodium currents. Subsequent Edman degradation revealed a 42-residue peptide with unusual N- and C-terminal cysteines and a cysteine triplet (Cys14-16). This toxin was highly homologous to a family of δ-atracotoxins (δ-ACTX) from Australian funnel-web spiders including conservation of all eight cysteine residues. In addition to actions on sodium channel gating and kinetics to δ-ACTX, δ-MSTX-Mb1a caused significant insect toxicity at doses up to 2000 pmol/g. δ-MSTX-Mb1a therefore provides evidence of a highly conserved spider δ-toxin from a phylogenetically distinct spider family that has not undergone significant modification. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies

The Effect of Insecticide Synergists on the Response of Scabies Mites to Pyrethroid Acaricides

Synergists are commonly used in combination with pesticides to suppress metabolism-based resistance and increase the efficacy of the agents. They are also useful as tools for laboratory investigation of specific resistance mechanisms based on their ability to inhibit specific metabolic pathways. To determine the role of metabolic degradation as a mechanism for acaricide resistance in human scabies, PBO (piperonyl butoxide), DEF (S,S,S-tributyl phosphorotrithioate) and DEM (diethyl maleate) were used with permethrin as synergists in a bioassay of mite killing. A statistically significant difference in survival time of permethrin-resistant Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to survival time of mites exposed to permethrin alone (p<0.0001). These results indicate the potential utility of synergists in reversing tolerance to pyrethroid-based acaricides (i.e. the addition of synergists to permethrin-containing topical acaricide cream commonly used to treat scabies). To further verify specific metabolic pathways being inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase activity in scabies mites. Results of in vitro enzyme inhibition experiments showed lower levels of esterase activity with DEF; lower levels of GST activity with DEM and lower levels of cytochrome monooxygenase activity with PBO. These findings indicate a metabolic mechanism as mediating pyrethroid resistance in scabies mites

The promoter of ZmMRP-1, a maize transfer cell-specific transcriptional activator, is induced at solute exchange surfaces and responds to transport demands

Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs. The GUS signal predominantly appeared in regions of active transport between source and sink tissues, including nematode-induced feeding structures and at sites of vascular connection between developing organs and the main plant vasculature. In those cases, promoter induction was associated with the initial developmental stages of transport structures. Significantly, transfer cells also differentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might differentiate in a similar way under the influence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most effective inducer

Transparency and Trust in Human-AI-Interaction: The Role of Model-Agnostic Explanations in Computer Vision-Based Decision Support

Computer Vision, and hence Artificial Intelligence-based extraction of information from images, has increasingly received attention over the last years, for instance in medical diagnostics. While the algorithms' complexity is a reason for their increased performance, it also leads to the "black box" problem, consequently decreasing trust towards AI. In this regard, "Explainable Artificial Intelligence" (XAI) allows to open that black box and to improve the degree of AI transparency. In this paper, we first discuss the theoretical impact of explainability on trust towards AI, followed by showcasing how the usage of XAI in a health-related setting can look like. More specifically, we show how XAI can be applied to understand why Computer Vision, based on deep learning, did or did not detect a disease (malaria) on image data (thin blood smear slide images). Furthermore, we investigate, how XAI can be used to compare the detection strategy of two different deep learning models often used for Computer Vision: Convolutional Neural Network and Multi-Layer Perceptron. Our empirical results show that i) the AI sometimes used questionable or irrelevant data features of an image to detect malaria (even if correctly predicted), and ii) that there may be significant discrepancies in how different deep learning models explain the same prediction. Our theoretical discussion highlights that XAI can support trust in Computer Vision systems, and AI systems in general, especially through an increased understandability and predictability

Enhanced text spacing improves reading performance in individuals with macular disease

The search by many investigators for a solution to the reading problems encountered by individuals with no central vision has been long and, to date, not very fruitful. Most textual manipulations, including font size, have led to only modest gains in reading speed. Previous work on spatial integrative properties of peripheral retina suggests that 'visual crowding' may be a major factor contributing to inefficient reading. Crowding refers to the fact that juxtaposed targets viewed eccentrically may be difficult to identify. The purpose of this study was to assess the combined effects of line spacing and word spacing on the ability of individuals with age-related macular degeneration (ARMD) to read short passages of text that were printed with either high (87.5%) or low contrast (17.5%) letters. Low contrast text was used to avoid potential ceiling effects and to mimic a possible reduction in letter contrast with light scatter from media opacities. For both low and high contrast text, the fastest reading speeds we measured were for passages of text with double line and double word spacing. In comparison with standard single spacing, double word/line spacing increased reading speed by approximately 26% with high contrast text (p < 0.001), and by 46% with low contrast text (p < 0.001). In addition, double line/word spacing more than halved the number of reading errors obtained with single spaced text. We compare our results with previous reading studies on ARMD patients, and conclude that crowding is detrimental to reading and that its effects can be reduced with enhanced text spacing. Spacing is particularly important when the contrast of the text is reduced, as may occur with intraocular light scatter or poor viewing conditions. We recommend that macular disease patients should employ double line spacing and double-character word spacing to maximize their reading efficiency. © 2013 Blackmore-Wright et al

Useful pharmacodynamic endpoints in children: selection, measurement, and next steps.

Pharmacodynamic (PD) endpoints are essential for establishing the benefit-to-risk ratio for therapeutic interventions in children and neonates. This article discusses the selection of an appropriate measure of response, the PD endpoint, which is a critical methodological step in designing pediatric efficacy and safety studies. We provide an overview of existing guidance on the choice of PD endpoints in pediatric clinical research. We identified several considerations relevant to the selection and measurement of PD endpoints in pediatric clinical trials, including the use of biomarkers, modeling, compliance, scoring systems, and validated measurement tools. To be useful, PD endpoints in children need to be clinically relevant, responsive to both treatment and/or disease progression, reproducible, and reliable. In most pediatric disease areas, this requires significant validation efforts. We propose a minimal set of criteria for useful PD endpoint selection and measurement. We conclude that, given the current heterogeneity of pediatric PD endpoint definitions and measurements, both across and within defined disease areas, there is an acute need for internationally agreed, validated, and condition-specific pediatric PD endpoints that consider the needs of all stakeholders, including healthcare providers, policy makers, patients, and families.Pediatric Research advance online publication, 11 April 2018; doi:10.1038/pr.2018.38

Prefrontal cortex activation and young driver behaviour: a fNIRS study

Road traffic accidents consistently show a significant over-representation for young, novice and particularly male drivers. This research examines the prefrontal cortex activation of young drivers and the changes in activation associated with manipulations of mental workload and inhibitory control. It also considers the explanation that a lack of prefrontal cortex maturation is a contributing factor to the higher accident risk in this young driver population. The prefrontal cortex is associated with a number of factors including mental workload and inhibitory control, both of which are also related to road traffic accidents. This experiment used functional near infrared spectroscopy to measure prefrontal cortex activity during five simulated driving tasks: one following task and four overtaking tasks at varying traffic densities which aimed to dissociate workload and inhibitory control. Age, experience and gender were controlled for throughout the experiment. The results showed that younger drivers had reduced prefrontal cortex activity compared to older drivers. When both mental workload and inhibitory control increased prefrontal cortex activity also increased, however when inhibitory control alone increased there were no changes in activity. Along with an increase in activity during overtaking manoeuvres, these results suggest that prefrontal cortex activation is more indicative of workload in the current task. There were no differences in the number of overtakes completed by younger and older drivers but males overtook significantly more than females. We conclude that prefrontal cortex activity is associated with the mental workload required for overtaking. We additionally suggest that the reduced activation in younger drivers may be related to a lack of prefrontal maturation which could contribute to the increased crash risk seen in this population