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.

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.

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.

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.

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.

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.

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.

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.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Automated, high-throughput, motility analysis in Caenorhabditis elegans and parasitic nematodes: Applications in the search for new anthelmintics.

The scale of the damage worldwide to human health, animal health and agricultural crops resulting from parasitic nematodes, together with the paucity of treatments and the threat of developing resistance to the limited set of widely-deployed chemical tools, underlines the urgent need to develop novel drugs and chemicals to control nematode parasites. Robust chemical screens which can be automated are a key part of that discovery process. Hitherto, the successful automation of nematode behaviours has been a bottleneck in the chemical discovery process. As the measurement of nematode motility can provide a direct scalar readout of the activity of the neuromuscular system and an indirect measure of the health of the animal, this omission is acute. Motility offers a useful assay for high-throughput, phenotypic drug/chemical screening and several recent developments have helped realise, at least in part, the potential of nematode-based drug screening. Here we review the challenges encountered in automating nematode motility and some important developments in the application of machine vision, statistical imaging and tracking approaches which enable the automated characterisation of nematode movement. Such developments facilitate automated screening for new drugs and chemicals aimed at controlling human and animal nematode parasites (anthelmintics) and plant nematode parasites (nematicides).

Glutamate-gated chloride channels of Haemonchus contortus restore drug sensitivity to ivermectin resistant Caenorhabditis elegans.

Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance.

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.

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.

Proteins interacting with nicotinic acetylcholine receptors: expanding functional and therapeutic horizons.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that carry out the fast actions of the neurotransmitter acetylcholine (ACh). Over the past 30 years, it has become clear that the activity of nAChRs is dependent on their interaction with a host of proteins, and the number of these that have been identified has increased considerably with recent large-scale proteomic analyses. This review focuses on these interacting proteins, discussing how they regulate a wide range of functions including receptor assembly, and trafficking to and from the cell surface, as well as how they directly modulate functional characteristics such as sensitivity and the degree of response to ACh. Mutations giving rise to disease states highlight the importance of these interacting proteins. Here, we consider their potential as future therapeutic targets for treating diseases associated with altered nAChR function.

Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone.

The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

Alternative splicing of the Anopheles gambiae nicotinic acetylcholine receptor, Agamalphabeta9, generates both alpha and beta subunits.

Nicotinic acetylcholine receptors (nAChRs) are the members of the cys-loop ligand-gated ion channel superfamily and are formed by five subunits arranged around a central ion channel. Each subunit is encoded by a separate gene and is classified as either alpha or non-alpha depending on the presence or absence, respectively, of two adjacent cysteine residues which are important for acetylcholine binding. Here, we report for the first time a single nAChR gene encoding both alpha and non-alpha subunits. Specifically, alternative splicing of the Anopheles gambiae nAChR subunit, previously called Agamalpha9 and renamed here Agamalphabeta9, generates two variants, one possessing the two cysteines (denoted Agamalphabeta9(alpha)) and the other lacking the cysteine doublet (Agamalphabeta9(beta)). Attempts to heterologously express functional nAChRs consisting of the Agamalphabeta9 splice variants in Xenopus laevis oocytes were unsuccessful. Our findings further characterise a potential target to control the malaria mosquito as well as provide insights into the diversification of nAChRs.

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.

Comparative pharmacology and computational modelling yield insights into allosteric modulation of human alpha7 nicotinic acetylcholine receptors.

The human alpha7 nicotinic acetylcholine receptor (nAChR) subunit and its Caenorhabditis elegans homolog, ACR-16, can generate functional recombinant homomeric receptors when expressed in Xenopus laevis oocytes. Both nAChRs express robustly in the presence of the co-injected chaperone, RIC-3, and show striking differences in the actions of a type I positive allosteric modulator (PAM), ivermectin (IVM). Type I PAMs are characterised by an increase in amplitude only of the response to acetylcholine (ACh), whereas type II PAMs exhibit, in addition, changes in time-course/desensitization of the ACh response. The type I PAMs, ivermectin, 5-hydroxyindole (5-HI), NS-1738 and genistein and the type II PAM, PNU-120596, are all active on human alpha7 but are without PAM activity on ACR-16, where they attenuate the amplitude of the ACh response. We used the published structure of avermectin B1a to generate a model of IVM, which was then docked into the candidate transmembrane allosteric binding site on alpha7 and ACR-16 in an attempt to gain insights into the observed differences in IVM actions. The new pharmacological findings and computational approaches being developed may inform the design of novel PAM drugs targeting major neurological disorders.

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.