Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Turning a Drug Target into a Drug Candidate: A New Paradigm for Neurological Drug Discovery?

The conventional paradigm for developing new treatments for disease mainly involves either the discovery of new drug targets, or finding new, improved drugs for old targets. However, an ion channel found only in invertebrates offers the potential of a completely new paradigm in which an established drug target can be re-engineered to serve as a new candidate therapeutic agent. The L-glutamate-gated chloride channels (GluCls) of invertebrates are absent from vertebrate genomes, offering the opportunity to introduce this exogenous, inhibitory, L-glutamate receptor into vertebrate neuronal circuits either as a tool with which to study neural networks, or a candidate therapy. Epileptic seizures can involve L-glutamate-induced hyper-excitation and toxicity. Variant GluCls, with their inhibitory responses to L-glutamate, when engineered into human neurons, might counter the excitotoxic effects of excess L-glutamate. In reviewing recent studies on model organisms, it appears that this approach might offer a new paradigm for the development of candidate therapeutics for epilepsy.

A novel Caenorhabditis elegans allele, smn-1(cb131), mimicking a mild form of spinal muscular atrophy, provides a convenient drug screening platform highlighting new and pre-approved compounds.

Spinal muscular atrophy (SMA), an autosomal recessive genetic disorder, is characterized by the selective degeneration of lower motor neurons, leading to muscle atrophy and, in the most severe cases, paralysis and death. Deletions and point mutations cause reduced levels of the widely expressed survival motor neuron (SMN) protein, which has been implicated in a range of cellular processes. The mechanisms underlying disease pathogenesis are unclear, and there is no effective treatment. Several animal models have been developed to study SMN function including the nematode, Caenorhabditis elegans, in which a large deletion in the gene homologous to SMN, smn-1, results in neuromuscular dysfunction and larval lethality. Although useful, this null mutant, smn-1(ok355), is not well suited to drug screening. We report the isolation and characterization of smn-1(cb131), a novel allele encoding a substitution in a highly conserved residue of exon 2, resembling a point mutation found in a patient with type IIIb SMA. The smn-1(cb131) animals display milder yet similar defects when compared with the smn-1 null mutant. Using an automated phenotyping system, mutants were shown to swim slower than wild-type animals. This phenotype was used to screen a library of 1040 chemical compounds for drugs that ameliorate the defect, highlighting six for subsequent testing. 4-aminopyridine, gaboxadol hydrochloride and N-acetylneuraminic acid all rescued at least one aspect of smn-1 phenotypic dysfunction. These findings may assist in accelerating the development of drugs for the treatment of SMA.

A Cys-loop mutation in the Caenorhabditis elegans nicotinic receptor subunit UNC-63 impairs but does not abolish channel function.

The nematode Caenorhabditis elegans is an established model organism for studying neurobiology. UNC-63 is a C. elegans nicotinic acetylcholine receptor (nAChR) α-subunit. It is an essential component of the levamisole-sensitive muscle nAChR (L-nAChR) and therefore plays an important role in cholinergic transmission at the nematode neuromuscular junction. Here, we show that worms with the unc-63(x26) allele, with its αC151Y mutation disrupting the Cys-loop, have deficient muscle function reflected by impaired swimming (thrashing). Single-channel recordings from cultured muscle cells from the mutant strain showed a 100-fold reduced frequency of opening events and shorter channel openings of L-nAChRs compared with those of wild-type worms. Anti-UNC-63 antibody staining in both cultured adult muscle and embryonic cells showed that L-nAChRs were expressed at similar levels in the mutant and wild-type cells, suggesting that the functional changes in the receptor, rather than changes in expression, are the predominant effect of the mutation. The kinetic changes mimic those reported in patients with fast-channel congenital myasthenic syndromes. We show that pyridostigmine bromide and 3,4-diaminopyridine, which are drugs used to treat fast-channel congenital myasthenic syndromes, partially rescued the motility defect seen in unc-63(x26). The C. elegans unc-63(x26) mutant may therefore offer a useful model to assist in the development of therapies for syndromes produced by altered function of human nAChRs.

Nicotinic acetylcholine receptor signalling: roles in Alzheimer's disease and amyloid neuroprotection.

Alzheimer's disease (AD), the major contributor to dementia in the elderly, involves accumulation in the brain of extracellular plaques containing the beta-amyloid protein (Abeta) and intracellular neurofibrillary tangles of hyperphosphorylated tau protein. AD is also characterized by a loss of neurons, particularly those expressing nicotinic acetylcholine receptors (nAChRs), thereby leading to a reduction in nAChR numbers. The Abeta(1-42) protein, which is toxic to neurons, is critical to the onset and progression of AD. The discovery of new drug therapies for AD is likely to be accelerated by an improved understanding of the mechanisms whereby Abeta causes neuronal death. We examine the evidence for a role in Abeta(1-42) toxicity of nAChRs; paradoxically, nAChRs can also protect neurons when activated by nicotinic ligands. Abeta peptides and nicotine differentially activate several intracellular signaling pathways, including the phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene homolog pathway, the extracellular signal-regulated kinase/mitogen-activated protein kinase, and JAK-2/STAT-3 pathways. These pathways control cell death or survival and the secretion of Abeta peptides. We propose that understanding the differential activation of these pathways by nicotine and/or Abeta(1-42) may offer the prospect of new routes to therapy for AD.

Automated phenotyping of mosquito larvae enables high-throughput screening for novel larvicides and offers potential for smartphone-based detection of larval insecticide resistance.

Pyrethroid-impregnated nets have contributed significantly to halving the burden of malaria but resistance threatens their future efficacy and the pipeline of new insecticides is short. Here we report that an invertebrate automated phenotyping platform (INVAPP), combined with the algorithm Paragon, provides a robust system for measuring larval motility in Anopheles gambiae (and An. coluzzi) as well as Aedes aegypti with the capacity for high-throughput screening for new larvicides. By this means, we reliably quantified both time- and concentration-dependent actions of chemical insecticides faster than using the WHO standard larval assay. We illustrate the effectiveness of the system using an established larvicide (temephos) and demonstrate its capacity for library-scale chemical screening using the Medicines for Malaria Venture (MMV) Pathogen Box library. As a proof-of-principle, this library screen identified a compound, subsequently confirmed to be tolfenpyrad, as an effective larvicide. We have also used the INVAPP / Paragon system to compare responses in larvae derived from WHO classified deltamethrin resistant and sensitive mosquitoes. We show how this approach to monitoring larval response to insecticides can be adapted for use with a smartphone camera application and therefore has potential for further development as a simple portable field-assay with associated real-time, geo-located information to identify hotspots.

Splice-variant- and stage-specific RNA editing of the Drosophila GABA receptor modulates agonist potency.

The molecular diversity of many gene products functioning in the nervous system is enhanced by alternative splicing and adenosine-to-inosine editing of pre-mRNA. Using RDL, a Drosophila melanogaster GABA-gated ion channel, we examined the functional impact of RNA editing at several sites along with alternative splicing of more than one exon. We show that alternative splicing and RNA editing have a combined influence on the potency of the neurotransmitter GABA, and the editing isoforms detected in vivo span the entire functional range of potencies seen for all possible edit variants expressed in Xenopus laevis oocytes. The extent of RNA editing is developmentally regulated and can also be linked to the choice of alternative exons. These results provide insights into how the rich diversity of signaling necessary for complex brain function can be achieved by relatively few genes.

Fast, automated measurement of nematode swimming (thrashing) without morphometry.

BACKGROUND: The "thrashing assay", in which nematodes are placed in liquid and the frequency of lateral swimming ("thrashing") movements estimated, is a well-established method for measuring motility in the genetic model organism Caenorhabditis elegans as well as in parasitic nematodes. It is used as an index of the effects of drugs, chemicals or mutations on motility and has proved useful in identifying mutants affecting behaviour. However, the method is laborious, subject to experimenter error, and therefore does not permit high-throughput applications. Existing automation methods usually involve analysis of worm shape, but this is computationally demanding and error-prone. Here we present a novel, robust and rapid method of automatically counting the thrashing frequency of worms that avoids morphometry but nonetheless gives a direct measure of thrashing frequency. Our method uses principal components analysis to remove the background, followed by computation of a covariance matrix of the remaining image frames from which the interval between statistically-similar frames is estimated. RESULTS: We tested the performance of our covariance method in measuring thrashing rates of worms using mutations that affect motility and found that it accurately substituted for laborious, manual measurements over a wide range of thrashing rates. The algorithm used also enabled us to determine a dose-dependent inhibition of thrashing frequency by the anthelmintic drug, levamisole, illustrating the suitability of the system for assaying the effects of drugs and chemicals on motility. Furthermore, the algorithm successfully measured the actions of levamisole on a parasitic nematode, Haemonchus contortus, which undergoes complex contorted shapes whilst swimming, without alterations in the code or of any parameters, indicating that it is applicable to different nematode species, including parasitic nematodes. Our method is capable of analyzing a 30 s movie in less than 30 s and can therefore be deployed in rapid screens. CONCLUSION: We demonstrate that a covariance-based method yields a fast, reliable, automated measurement of C. elegans motility which can replace the far more time-consuming, manual method. The absence of a morphometry step means that the method can be applied to any nematode that swims in liquid and, together with its speed, this simplicity lends itself to deployment in large-scale chemical and genetic screens.

Calcium signalling in mammalian cell lines expressing wild type and mutant human α1-Antitrypsin.

A possible role for calcium signalling in the autosomal dominant form of dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB), has been proposed, which may point towards a mechanism by which cells could sense and respond to the accumulation of mutant serpin polymers in the endoplasmic reticulum (ER). We therefore explored possible defects in Ca2+-signalling, which may contribute to the pathology associated with another serpinopathy, α1-antitrypsin (AAT) deficiency. Using CHO K1 cell lines stably expressing a wild type human AAT (MAAT) and a disease-causing polymer-forming variant (ZAAT) and the truncated variant (NHK AAT), we measured basal intracellular free Ca2+, its responses to thapsigargin (TG), an ER Ca2+-ATPase blocker, and store-operated Ca2+-entry (SOCE). Our fura2 based Ca2+ measurements detected no differences between these 3 parameters in cell lines expressing MAAT and cell lines expressing ZAAT and NHK AAT mutants. Thus, in our cell-based models of α1-antitrypsin (AAT) deficiency, unlike the case for FENIB, we were unable to detect defects in calcium signalling.

Strategies for automated analysis of C. elegans locomotion.

Automated analysis of C. elegans behaviour is a rapidly developing field, offering the possibility of behaviour-based, high-throughput drug screens and systematic phenotyping. Standard methods for parameterizing worm shapes and movements are emerging, and progress has been made towards overcoming the difficulties introduced by interactions between worms, as well as worm coiling and omega turning. Current methods have facilitated the identification of subtle phenotypes and the characterisation of roles of neurones in forward locomotion and chemotaxis, as well as the quantitative characterisation of behaviour choice and circadian patterns of activity. Given the speed with which C. elegans has been deployed in genetic screens and chemical screens, it is to be hoped that wormtrackers may eventually provide similar rapidity in assaying behavioural phenotypes. However, considerable progress must be made before this can be accomplished. In the case of genome-wide RNAi screens, for example, the presence in the worm genome of some 19,000 genes means that even the minimal user intervention in an automatic phenotyping system will be very costly. Nonetheless, recent advances have shown that drug actions on large numbers of worms can be tracked, raising hopes that high-throughput behavioural screens may soon be available.

Action potential shape change in an electrically coupled network during propagation: a computer simulation.

We applied compartmental computer modeling to test a model of spike shape change in the jellyfish, Polyorchis penicillatus, to determine whether adaptive spike shortening can be attributed to the inactivation properties of a potassium channel. We modeled the jellyfish outer nerve-ring as a continuous linear segment, using ion channel and membrane properties derived in earlier studies. The model supported action potentials that shortened as they propagated away from the site of initiation and this was found to be largely independent of potassium channel inactivation. Spike broadening near the site of initiation was found to be due to a depolarization plateau that collapsed as two spikes spread from the point of initiation. The lifetime of this plateau was found to depend critically on the inward current flux and the space constant of the membrane. These data suggest that the spike shape changes may be due not only to potassium channel inactivation, but also to the passive properties of the membrane.

Chemistry-to-gene screens in Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans is a genetic model organism linked to an impressive portfolio of fundamental discoveries in biology. This free-living nematode, which can be easily and inexpensively grown in the laboratory, is also a natural vehicle for screening for drugs that are active against nematode parasites. Here, we show that chemistry-to-gene screens using this animal model can define targets of antiparasitic drugs, identify novel candidate drug targets and contribute to the discovery of new drugs for treating human diseases.

Insect toxins - selective pharmacological tools and drug/chemical leads.

Insect toxins comprise a diverse array of chemicals ranging from small molecules, polyamines and peptide toxins. Many target nervous system and neuromuscular ion channels and so rapidly affect the behaviour of animals to which the toxin is applied or injected. Other modes of action have also been identified. Wasps, bees, flies, beetles and ants generate a rich arsenal of channel-active toxins, some of which offer selective pharmacological probes that target particular ion channels, while others act on more than one type of channel. Philanthotoxins from the digger wasp have been fruitful in adding to our understanding of ligand-gated ion channels both in the nervous system and at neuromuscular junctions. Fire ants produce the toxic alkaloid solenopsin, a molecule which has stimulated attempts to generate synthetic compounds with insecticidal activity. Apamin from bee venom targets calcium-activated potassium channels, which can in turn influence the release of neuropeptides. Melittin, another bee venom component, is a membrane-acting peptide. The saliva of the assassin bug contains toxins that target the voltage-gated calcium channels of their insect prey. Certain beetles produce diamphotoxin, a haemolytic peptide toxin with traditional use as an arrow poison and others generate leptinotarsin with similar properties. Mastoparan is a powerful peptide toxin present in the venom of wasps. Its toxic actions can be engineered out leaving a potent antimicrobial molecule of interest. In this short review we describe the actions of selected insect toxins and evaluate their potential as neuroactive pharmacological tools, candidate lead molecules for insect control and therapeutic candidates with potential antimicrobial, antiviral and anti-cancer applications.

The fungal alkaloid Okaramine-B activates an L-glutamate-gated chloride channel from Ixodes scapularis, a tick vector of Lyme disease.

A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50-400 nA currents in response to 100 µM L-glutamate. Responses to L-glutamate were concentration-dependent (pEC50 3.64 ±â€¯0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100 µM aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1 µM activated IscaGluCl1, whereas picrotoxinin (pIC50 6.20 ±â€¯0.04) and the phenylpyrazole fipronil (pIC50 6.90 ±â€¯0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC50 5.43 ±â€¯0.43) and may serve as a candidate lead compound for the development of new acaricides.

An automated high-throughput system for phenotypic screening of chemical libraries on C. elegans and parasitic nematodes.

Parasitic nematodes infect hundreds of millions of people and farmed livestock. Further, plant parasitic nematodes result in major crop damage. The pipeline of therapeutic compounds is limited and parasite resistance to the existing anthelmintic compounds is a global threat. We have developed an INVertebrate Automated Phenotyping Platform (INVAPP) for high-throughput, plate-based chemical screening, and an algorithm (Paragon) which allows screening for compounds that have an effect on motility and development of parasitic worms. We have validated its utility by determining the efficacy of a panel of known anthelmintics against model and parasitic nematodes: Caenorhabditis elegans, Haemonchus contortus, Teladorsagia circumcincta, and Trichuris muris. We then applied the system to screen the Pathogen Box chemical library in a blinded fashion and identified compounds already known to have anthelmintic or anti-parasitic activity, including tolfenpyrad, auranofin, and mebendazole; and 14 compounds previously undescribed as anthelmintics, including benzoxaborole and isoxazole chemotypes. This system offers an effective, high-throughput system for the discovery of novel anthelmintics.

The Abeta1-42M35C mutated amyloid peptide Abeta1-42 and the 25-35 fragment fail to mimic the subtype-specificity of actions on recombinant human nicotinic acetylcholine receptors (alpha7, alpha4beta2, alpha3beta4).

Alzheimer's disease (AD) is a neurodegenerative condition involving accumulation of the beta-amyloid peptide, Abeta1-42. Previously we have shown that amyloid peptides (Abeta1-42, Abeta1-40) have different actions on the three major brain nicotinic acetylcholine receptor (nAChR) subtypes (alpha7, alpha4beta2 and alpha3beta4). The methionine in position 35 of Abeta (M35) has been shown to be important in the toxicity of Abeta and the 25-35 fragment can mimic some of the actions of the Abeta1-42 peptide. However, the extent to which this mutant and the fragment mimic subtype selectivity is unknown. Two-electrode voltage-clamp electrophysiology has been used to study the actions on alpha7, alpha4beta2 and alpha3beta4 recombinant nAChRs expressed in Xenopus laevis oocytes of full length Abeta1-42, and Abeta peptide fragments, scrambled peptides, and the Abeta1-42 peptide containing mutations of the methionine in position 35. The Abeta25-35 fragment did not display subunit specificity. Abeta1-42 with an M35C mutation showed similar subtype-specificity to wild-type Abeta1-42. However, Abeta1-42 with an M35V substitution reduced the peak amplitude of ACh-induced currents recorded from alpha4beta2 nAChRs, but did not affect those recorded from alpha7 or alpha3beta4. These results indicate that the amino acid in position 35 of Abeta1-42 is an important determinant of the subtype-specificity of this peptide on human recombinant alpha7, alpha4beta2 and alpha3beta4 nAChRs and that the 25-35 fragment fails to mimic all of the actions of the full-length peptide.

Actions of snake neurotoxins on an insect nicotinic cholinergic synapse.

Here we examine the actions of six snake neurotoxins (alpha-cobratoxin from Naja naja siamensis, erabutoxin-a and b from Laticauda semifasciata; CM12 from N. haje annulifera, toxin III 4 from Notechis scutatus and a long toxin from N. haje) on nicotinic acetylcholine receptors in the cercal afferent, giant interneuron 2 synapse of the cockroach, Periplaneta americana. All toxins tested reduced responses to directly-applied ACh as well as EPSPs evoked by electrical stimulation of nerve XI with similar time courses, suggesting that their action is postsynaptic. Thus, these nicotinic receptors in a well-characterized insect synapse are sensitive to both long and short chain neurotoxins. This considerably expands the range of snake toxins that block insect nicotinic acetylcholine receptors and may enable further pharmacological distinctions between nAChR subtypes.

Modes of Action, Resistance and Toxicity of Insecticides Targeting Nicotinic Acetylcholine Receptors.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) of insects play a key role in fast excitatory neurotransmission. Several classes of insecticides target insect nAChRs, which are composed of subunit members of a family of multiple subunit encoding genes. Alternative splicing and RNA A-to-I editing can add further to receptor diversity. Native and recombinant receptors have been explored as sites of insecticide action using radioligands, electrophysiology and site-directed mutagenesis. METHODS: We have reviewed the properties of native and recombinant insect nAChRs, the challenges of functional recombinant insect nAChR expression, nAChR interactions with ligands acting at orthosteric and allosteric sites and in particular their interactions with insecticides. RESULTS: Actions on insect nAChRs of cartap, neonicotinoids, spinosyns, sulfoxamines, butenolides and mesoionic insecticides are reviewed and current knowledge of their modes of action are addressed. Mutations that add to our understanding of insecticide action and those leading to resistance are discussed. Co-crystallisation of neonicotinoids with the acetylcholine binding protein (AChBP), a surrogate for the nAChR ligand binding domain, has proved instructive. Toxicity issues relating to insecticides targeting nAChRs are also considered. CONCLUSION: An overview of insecticide classes targeting insect nAChRs has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native nAChRs remains poorly understood and functional expression still presents difficulties. These topics together with improved understanding of the precise sites of insecticide actions on insect nAChRs will be the subject of future research.

Mechanisms of Action, Resistance and Toxicity of Insecticides Targeting GABA Receptors.

BACKGROUND: γ-Aminobutyric acid (GABA) receptors play a central role in fast inhibitory neurotransmission in insects. Several classes of insecticides targeting insect GABA-gated chloride channels have been developed. The important resistant to dieldrin GABA receptor subunit (RDL) has been used to investigate insecticide sites of action using radioligands, electrophysiology and site-directed mutagenesis. Although this important subunit readily forms robust functional homomeric receptors when expressed, alternative splicing and RNA A-to-I editing can generate diverse forms of the receptor. METHODS: We have reviewed studies on native and recombinant insect GABA-gated chloride channels, their interactions with ligands acting at orthosteric and allosteric sites and their interactions with insecticides. Since some GABA receptor modulators act on L-glutamate-gated chloride channels, some comparisons are included. RESULTS: The actions on GABA-gated chloride channels of polychlorocycloalkanes, cyclodienes, macrocyclic lactones, phenylpyrazoles, isoxazolines, and metadiamides are described and the mechanisms of action of members of these insecticide classes are addressed. Mutations that lead to resistance are discussed as they can be important in developing field diagnostic tests. Toxicity issues relating to insecticides targeting GABA-gated chloride channels are also addressed. An overview of all major insecticide classes targeting insect GABA-gated chloride channels has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native GABA receptors remains unknown and studies to clarify this are needed. Also, the precise sites of action of the recently introduced isoxazolines and meta-diamides will be of interest to pursue.

Structure and function of two-pore-domain K+ channels: contributions from genetic model organisms.

K+ channels that possess two pore domains in each channel subunit are common in many animal tissues. Such channels are generated from large families of subunits and are implicated in several functions, including temperature sensation, responses to ischaemia, K+ homeostasis and setting the resting potential of the cell. Their activity can be modulated by polyunsaturated fatty acids, pH and oxygen, and some are candidate targets of volatile anaesthetics. However, despite their potential as targets for novel drugs for human health, comparatively little is known about the molecular basis of their diverse physiological and pharmacological properties. Genetic model organisms have considerable potential for improving our understanding of these channels. In this article, we review the contributions of some of these genetic model organisms to recent advances in our knowledge of two-pore-domain K+ channels.