Novel invertebrate‐killing compounds are required in agriculture and medicine to overcome resistance to existing treatments. Because insecticides and anthelmintics are discovered in phenotypic ...screens, a crucial step in the discovery process is determining the mode of action of hits. Visible whole‐organism symptoms are combined with molecular and physiological data to determine mode of action. However, manual symptomology is laborious and requires symptoms that are strong enough to see by eye. Here, we use high‐throughput imaging and quantitative phenotyping to measure Caenorhabditis elegans behavioral responses to compounds and train a classifier that predicts mode of action with an accuracy of 88% for a set of ten common modes of action. We also classify compounds within each mode of action to discover substructure that is not captured in broad mode‐of‐action labels. High‐throughput imaging and automated phenotyping could therefore accelerate mode‐of‐action discovery in invertebrate‐targeting compound development and help to refine mode‐of‐action categories.
Synopsis
A combination of imaging and machine learning is used to predict compound mode of action using the unique behavioural responses of the roundworm C. elegans to different pesticides and anthelmintics.
Insecticides affect phenotypes in multiple behavioural dimensions.
Compounds with the same mode of action have similar effects on behaviour.
Combining classifiers by voting enables mode of action prediction.
The approach allows mode of action deconvolution within classes.
A combination of imaging and machine learning is used to predict compound mode of action using the unique behavioural responses of the roundworm C. elegans to different pesticides and anthelmintics.
To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. ...elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals.
Knowledge of how drugs are metabolized and excreted is an essential component of understanding their fate within and among target and non-target organisms. Thiabendazole (TBZ) was the first ...benzimidazole (BZ) to be commercially available and remains one of the most important anthelmintic drugs for medical and veterinary use. We have characterized how Caenorhabditis elegans metabolizes and excretes TBZ. We have shown that TBZ directly binds to the nuclear hormone receptor (NHR)-176 and that this receptor is required for the induction by TBZ of the cytochrome P450 (CYP) encoded by cyp-35d1. Further, RNAi inhibition of cyp-35d1 in animals exposed to TBZ causes a reduction in the quantity of a hydroxylated TBZ metabolite and its glucose conjugate that is detected in C. elegans tissue by HPLC. This final metabolite is unique to nematodes and we also identify two P-glycoproteins (PGPs) necessary for its excretion. Finally, we have shown that inhibiting the metabolism we describe increases the susceptibility of C. elegans to TBZ in wild-type and in resistant genetic backgrounds.
Characterisation of the pathways by which xenobiotics are metabolised and excreted in both target and non-target organisms is crucial for the rational design of effective and specific novel bioactive ...molecules. Consequently, we have investigated the induced responses of the model nematode Caenorhabditis elegans to a variety of xenobiotics which represent a range of putative modes of action. The majority of genes that were specifically induced in preliminary microarray analyses encoded enzymes from Phase I and II metabolism, including cytochrome P450s, short chain dehydrogenases, UDP-glucuronosyl transferases and glutathione transferases. Changes in gene expression were confirmed by quantitative PCR and GFP induction in reporter strains driven by promoters for transcription of twelve induced enzymes was investigated. The particular complement of metabolic genes induced was found to be highly contingent on the xenobiotic applied. The known regulators of responses to applied chemicals ahr-1, hif-1, mdt-15 and nhr-8 were not required for any of these inducible responses and skn-1 regulated GFP expression from only two of the promoters. Reporter strains were used in conjunction with systematic RNAi screens to identify transcription factors which drive expression of these genes under xenobiotic exposure. These transcription factors appeared to regulate specific xenobiotic responses and have no reported phenotypes under standard conditions. Focussing on nhr-176 we demonstrate the role of this transcription factor in mediating the resistance to thiabendazole.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We investigated the metabolic capabilities of C. elegans using compounds whose metabolism has been well characterised in mammalian systems. We find that similar metabolites are produced in C. elegans ...as in mammals but that C. elegans is deficient in CYP1-like metabolism, as has been seen in other studies. We show that CYP-34A9, CYP-34A10 and CYP-36A1 are the principal enzymes responsible for the metabolism of tolbutamide in C. elegans. These are related to the mammalian enzymes that metabolise this compound but are not the closest homologs suggesting that sequence comparison alone will not predict functional conservation among cytochrome P450s. In mammals, metabolite production from amytryptiline and dextromethorphan is dependent on specific cytochrome P450s. However, in C. elegans we did not find evidence of similar specificity: the same metabolites were produced but in small amounts by numerous cytochrome P450s. We conclude that, while some aspects of cytochrome P450 mediated metabolism in C. elegans are similar to mammals, there are differences in the production of some metabolites and in the underlying genetics of metabolism.
A new rheological droplet interface bilayer (rheo-DIB) device is presented as a tool to apply shear stress on biological lipid membranes. Despite their exciting potential for affecting ...high-throughput membrane translocation studies, permeability assays conducted using DIBs have neglected the effect of the unstirred water layer (UWL). However as demonstrated in this study, neglecting this phenomenon can cause significant underestimates in membrane permeability measurements which in turn limits their ability to predict key processes such as drug translocation rates across lipid membranes. With the use of the rheo-DIB chip, the effective bilayer permeability can be modulated by applying shear stress to the droplet interfaces, inducing flow parallel to the DIB membranes. By analysing the relation between the effective membrane permeability and the applied stress, both the intrinsic membrane permeability and UWL thickness can be determined for the first time using this model membrane approach, thereby unlocking the potential of DIBs for undertaking diffusion assays. The results are also validated with numerical simulations.
Abstract
We report for the first time an autosomal recessive inborn error of de novo purine synthesis (DNPS)—PAICS deficiency. We investigated two siblings from the Faroe Islands born with multiple ...malformations resulting in early neonatal death. Genetic analysis of affected individuals revealed a homozygous missense mutation in PAICS (c.158A>G; p.Lys53Arg) that affects the structure of the catalytic site of the bifunctional enzyme phosphoribosylaminoimidazole carboxylase (AIRC, EC 4.1.1.21)/phosphoribosylaminoimidazole succinocarboxamide synthetase (SAICARS, EC 6.3.2.6) (PAICS). The mutation reduced the catalytic activity of PAICS in heterozygous carrier and patient skin fibroblasts to approximately 50 and 10% of control levels, respectively. The catalytic activity of the corresponding recombinant enzyme protein carrying the mutation p.Lys53Arg expressed and purified from E. coli was reduced to approximately 25% of the wild-type enzyme. Similar to other two known DNPS defects—adenylosuccinate lyase deficiency and AICA-ribosiduria—the PAICS mutation prevented purinosome formation in the patient’s skin fibroblasts, and this phenotype was corrected by transfection with the wild-type but not the mutated PAICS. Although aminoimidazole ribotide (AIR) and aminoimidazole riboside (AIr), the enzyme substrates that are predicted to accumulate in PAICS deficiency, were not detected in patient’s fibroblasts, the cytotoxic effect of AIr on various cell lines was demonstrated. PAICS deficiency is a newly described disease that enhances our understanding of the DNPS pathway and should be considered in the diagnosis of families with recurrent spontaneous abortion or early neonatal death.
For the past decade, droplet interface bilayers (DIBs) have had an increased prevalence in biomolecular and biophysical literature. However, much of the underlying physics of these platforms is ...poorly characterized. To further our understanding of these structures, lipid membrane tension on DIB membranes is measured by analysing the equilibrium shape of asymmetric DIBs. To this end, the morphology of DIBs is explored for the first time using confocal laser scanning fluorescence microscopy. The experimental results confirm that, in accordance with theory, the bilayer interface of a volume-asymmetric DIB is curved towards the smaller droplet and a lipid-asymmetric DIB is curved towards the droplet with the higher monolayer surface tension. Moreover, the DIB shape can be exploited to measure complex bilayer surface energies. In this study, the bilayer surface energy of DIBs composed of lipid mixtures of phosphatidylgylcerol (PG) and phosphatidylcholine are shown to increase linearly with PG concentrations up to 25%. The assumption that DIB bilayer area can be geometrically approximated as a spherical cap base is also tested, and it is discovered that the bilayer curvature is negligible for most practical symmetric or asymmetric DIB systems with respect to bilayer area.
Simple diffusion of molecular entities through a phospholipid bilayer, is a phenomenon of great importance to the pharmaceutical and agricultural industries. Current model lipid systems to probe this ...typically only employ fluorescence as a readout, thus limiting the range of assessable chemical matter that can be studied. We report a new technology platform, the UV-DIB, which facilitates label free measurement of small molecule translocation rates. This is based upon the coupling of droplet interface bilayer technology with implemented fiber optics to facilitate analysis
ultraviolet spectroscopy, in custom designed PMMA wells. To improve on current DIB technology, the platform was designed to be reusable, with a high sampling rate and a limit of UV detection in the low μM regime. We demonstrate the use of our system to quantify passive diffusion in a reproducible and rapid manner where the system was validated by investigating multiple permeants of varying physicochemical properties across a range of lipid interfaces, each demonstrating differing kinetics. Our system permits the interrogation of structural dependence on the permeation rate of a given compound. We present this ability from two structural perspectives, that of the membrane, and the permeant. We observed a reduction in permeability between pure DOPC and DPhPC interfaces, concurring with literature and demonstrating our ability to study the effects of lipid composition on permeability. In relation to the effects of permeant structure, our device facilitated the rank ordering of various compounds from the xanthine class of compounds, where the structure of each permeant differed by a single group alteration. We found that DIBs were stable up to 5% DMSO, a molecule often used to aid solubilisation of pharmaceutical and agrochemical compounds. The ability of our device to rank-order compounds with such minor structural differences provides a level of precision that is rarely seen in current, industrially applied technologies.
Understanding spatiotemporal variation in vital rates and population growth rates is a central aim of population ecology, and is critical to conservation of migratory species where different ...populations may spend the non-breeding season in sometimes widely separated areas. However, estimating those parameters and identifying the underlying drivers of variation for species migrating to remote areas is challenging. The thick-billed murre
Uria lomvia
is a colonial seabird with a pan-Arctic distribution. Previous studies have documented large-scale spatial variation in population growth in the species’ Atlantic range, with eastern populations declining and western populations being stable, and suggested that this variation was linked to conditions in the wintering areas. We analyse variation in breeding success and adult survival from colonies throughout the Atlantic range, construct region-specific stochastic population models, and compare model predictions to observed colony growth rates. Breeding success was uniformly high in Greenland and Iceland, lower and more variable in Spitsbergen, Bjørnøya and Hudson Bay, and low at Jan Mayen. Adult survival was average or high in all colonies during the 2010s. Observed colony growth rates were lower than modelled growth rates for most populations. This suggests that pre-breeding survival or breeding propensity must have been lower than assumed in many of these populations. Low pre-breeding survival could be linked to poor feeding conditions in fall and winter, possibly influenced by large-scale oceanographic variation. Our results support the idea that seabird populations not subject to breeder mortality due to anthropogenic causes are regulated mainly through variation in pre-breeding survival.
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